Difference between revisions of "Team:Rotterdam HR/Notebook"

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<html>
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<head>
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<meta name="viewport" content="width=device-width, initial-scale=1">
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<link rel="stylesheet" href="https://2018.igem.org/Template:Rotterdam_HR/css/main?action=raw&ctype=text/css">
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<style>
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.menu #notebook, .menu #project-category {
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color: goldenrod;
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}
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</style>
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</head>
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<body>
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<style>
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.header::before {
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background: black url("https://static.igem.org/mediawiki/2018/3/3e/T--Rotterdam_HR--Notebook-banner.jpeg") no-repeat left;
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  background-size: cover;
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}
 +
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@media screen and (max-width: 720px) {
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.header::before {
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background: black url("https://static.igem.org/mediawiki/2018/7/77/T--Rotterdam_HR--Notebook-banner_small.jpeg") no-repeat left;
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background-size: cover;
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}
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}
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</style>
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<div class="header">
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  <div>
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<div id="icon"></div>
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<script> $('#icon').load("https://2018.igem.org/Template:Rotterdam_HR/icon/notebook?action=raw&ctype=text/html"); </script>
 +
    <h1 style="background: none;">Notebook</h1>
 +
  </div>
 +
</div>
 +
<div class="page page-notebook">
 +
<div id="navbar" style="position: -webkit-sticky;position: sticky;top: -2px; width: 100%; z-index: 100; margin-bottom: 100px;"></div>
 
<style>  
 
<style>  
 
table {
 
table {
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</div>
 
</div>
 
<div class="attendees">Rens Boeser</div>
 
<div class="attendees">Rens Boeser</div>
−
<div class="description"><p>Created the first prototype for the team page</p></div>
+
<div class="description">
 +
<p>Created the first prototype for the team page</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
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</div>
 
</div>
 
<div class="attendees">Rens Boeser</div>
 
<div class="attendees">Rens Boeser</div>
−
<div class="description"><p>Social media created</p></div>
+
<div class="description">
 +
<p>Social media created</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
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</div>
 
</div>
 
<div class="attendees">Rens Boeser</div>
 
<div class="attendees">Rens Boeser</div>
−
<div class="description"><p>Social media buttons same color and fixed iGEM style issues</p></div>
+
<div class="description">
 +
<p>Social media buttons same color and fixed iGEM style issues</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
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</div>
 
</div>
 
<div class="attendees">Rens Boeser</div>
 
<div class="attendees">Rens Boeser</div>
−
<div class="description"><p>Multiple week summary styles created for the notes</p></div>
+
<div class="description">
 +
<p>Multiple week summary styles created for the notes</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
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</div>
 
</div>
 
<div class="attendees">Rens Boeser</div>
 
<div class="attendees">Rens Boeser</div>
−
<div class="description"><p>Added the header with mobile compatibility als resized logos and readded them to the header</p></div>
+
<div class="description">
 +
<p>Added the header with mobile compatibility als resized logos and readded them to the header</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
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</div>
 
</div>
 
<div class="attendees">Rens Boeser</div>
 
<div class="attendees">Rens Boeser</div>
−
<div class="description"><p>Added template header and footer with 'under construction' content</p></div>
+
<div class="description">
 +
<p>Added template header and footer with 'under construction' content</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
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</div>
 
</div>
 
<div class="attendees">Rens Boeser</div>
 
<div class="attendees">Rens Boeser</div>
−
<div class="description"><p>Small fixes and menu adjustments</p></div>
+
<div class="description">
 +
<p>Small fixes and menu adjustments</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
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</div>
 
</div>
 
<div class="attendees">Rens Boeser</div>
 
<div class="attendees">Rens Boeser</div>
−
<div class="description"><p>Added a PI list with the secondary PI</p></div>
+
<div class="description">
 +
<p>Added a PI list with the secondary PI</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
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</div>
 
</div>
 
<div class="attendees">Rens Boeser</div>
 
<div class="attendees">Rens Boeser</div>
−
<div class="description"><p>Designed and added content holders to the index Page</p></div>
+
<div class="description">
 +
<p>Designed and added content holders to the index Page</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
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</div>
 
</div>
 
<div class="attendees">Rens Boeser</div>
 
<div class="attendees">Rens Boeser</div>
−
<div class="description"><p>Added myself to the teammember Page and did some bugfixing</p></div>
+
<div class="description">
 +
<p>Added myself to the teammember Page and did some bugfixing</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
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</div>
 
</div>
 
<div class="attendees">Rens Boeser</div>
 
<div class="attendees">Rens Boeser</div>
−
<div class="description"><p>Started working on a better looking menubar</p></div>
+
<div class="description">
 +
<p>Started working on a better looking menubar</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
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</div>
 
</div>
 
<div class="attendees">Rens Boeser</div>
 
<div class="attendees">Rens Boeser</div>
−
<div class="description"><p>Added the ability to add sub-menus to items in the menu bar. Integration into the website and added indication for which Page you are on</p></div>
+
<div class="description">
 +
<p>Added the ability to add sub-menus to items in the menu bar. Integration into the website and added indication for which Page you are on</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
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</div>
 
</div>
 
<div class="attendees">Rens Boeser</div>
 
<div class="attendees">Rens Boeser</div>
−
<div class="description"><p>Updated all Pages to the new style with an under construction sign</p></div>
+
<div class="description">
 +
<p>Updated all Pages to the new style with an under construction sign</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
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</div>
 
</div>
 
<div class="attendees">Rens Boeser</div>
 
<div class="attendees">Rens Boeser</div>
−
<div class="description"><p>Added a way to recognise Pages under construction using the menubar</p></div>
+
<div class="description">
 +
<p>Added a way to recognise Pages under construction using the menubar</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
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</div>
 
</div>
 
<div class="attendees">Rens Boeser</div>
 
<div class="attendees">Rens Boeser</div>
−
<div class="description"><p>Updated the content containers so that they are responsive</p></div>
+
<div class="description">
 +
<p>Updated the content containers so that they are responsive</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
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</div>
 
</div>
 
<div class="attendees">Tom van Dijk | Mike Vrieswijk</div>
 
<div class="attendees">Tom van Dijk | Mike Vrieswijk</div>
−
<div class="description"><p>The first draft of the architecture design for the temperature controller was made.</p></div>
+
<div class="description">
 +
<p>The first draft of the architecture design for the temperature controller was made.</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
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</div>
 
</div>
 
<div class="attendees">Rens Boeser</div>
 
<div class="attendees">Rens Boeser</div>
−
<div class="description"><p>Content added to the human practices page</p></div>
+
<div class="description">
 +
<p>Content added to the human practices page</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
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</div>
 
</div>
 
<div class="attendees">Rens Boeser</div>
 
<div class="attendees">Rens Boeser</div>
−
<div class="description"><p>Content created and added to the main page</p></div>
+
<div class="description">
 +
<p>Content created and added to the main page</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
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</div>
 
</div>
 
<div class="attendees">Tom van Dijk | Mike Vrieswijk</div>
 
<div class="attendees">Tom van Dijk | Mike Vrieswijk</div>
−
<div class="description"><p>Today a few changes have been made to the architecture design of the temperature controller. The choice was made to use a premade power supply as opposed to a self-made one, because this was considering the time left to finish this device the best choice. A few alterations were made to make the architecture design better readable.</p></div>
+
<div class="description">
 +
<p>Today a few changes have been made to the architecture design of the temperature controller. The choice was made to use a premade power supply as opposed to a self-made one, because this was considering the time left to finish this device the best choice. A few alterations were made to make the architecture design better readable.</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
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</div>
 
</div>
 
<div class="attendees">Tom van Dijk | Mike Vrieswijk</div>
 
<div class="attendees">Tom van Dijk | Mike Vrieswijk</div>
−
<div class="description"><p>A 3d drawing was made for the casing that will hold the temperature controller. The first prototype was printed, but was determent to be inadequate due to the lack of enough space for the cooler block.</p></div>
+
<div class="description">
 +
<p>A 3d drawing was made for the casing that will hold the temperature controller. The first prototype was printed, but was determent to be inadequate due to the lack of enough space for the cooler block.</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
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</div>
 
</div>
 
<div class="attendees">Rens Boeser</div>
 
<div class="attendees">Rens Boeser</div>
−
<div class="description"><p>Added the remaining instructors to the team</p></div>
+
<div class="description">
 +
<p>Added the remaining instructors to the team</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
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</div>
 
</div>
 
<div class="attendees">Tom van Dijk | Mike Vrieswijk</div>
 
<div class="attendees">Tom van Dijk | Mike Vrieswijk</div>
−
<div class="description"><p>In the drawing space was added to fit the cooler block for the temperature controller. Slits were added to the drawing for the nuts to slide into. The altered drawing was printed. The newly printed parts, cooler block, fan and Peltier element were assembled. Everything fit together.</p></div>
+
<div class="description">
 +
<p>In the drawing space was added to fit the cooler block for the temperature controller. Slits were added to the drawing for the nuts to slide into. The altered drawing was printed. The newly printed parts, cooler block, fan and Peltier element were assembled. Everything fit together.</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
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</div>
 
</div>
 
<div class="attendees">Dustin van der Meulen</div>
 
<div class="attendees">Dustin van der Meulen</div>
−
<div class="description"><p>To start testing the production of CooA, we firstly resuspended and transformed the BioBricks from the kit witch we wanted to use (Table 1). This has been done using the iGEM Kit plate instructions (Protocol 1, step 1 - 3) and the NEB 10 be&#769;ta transformation protocol adapted New England Biolabs (Protocol 2), using bought cells with a reported transformation efficienty of 1-3 10^9 cfu/&mu;g pUC19. Inucbation was at 37&deg;C.<br>
+
<div class="description">
 +
<p>To start testing the production of CooA, we firstly resuspended and transformed the BioBricks from the kit witch we wanted to use (Table 1). This has been done using the iGEM Kit plate instructions (Protocol 1, step 1 - 3) and the NEB 10 be&#769;ta transformation protocol adapted New England Biolabs (Protocol 2), using bought cells with a reported transformation efficienty of 1-3 10^9 cfu/&mu;g pUC19. Inucbation was at 37&deg;C.<br>
 
<br>
 
<br>
 
Table 1 : Resuspended BioBricks from the iGEM 2018 Distribution kit and their uses.
 
Table 1 : Resuspended BioBricks from the iGEM 2018 Distribution kit and their uses.
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</tbody>
 
</tbody>
 
</table>
 
</table>
−
</p></div>
+
</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
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</div>
 
</div>
 
<div class="attendees">Tom van Dijk | Mike Vrieswijk</div>
 
<div class="attendees">Tom van Dijk | Mike Vrieswijk</div>
−
<div class="description"><p>A short test run was done with the newly assembled cooler. The test concluded that the temperature controller could cool a water droplet to the point of freezing and heat it back up to its boiling point.</p></div>
+
<div class="description">
 +
<p>A short test run was done with the newly assembled cooler. The test concluded that the temperature controller could cool a water droplet to the point of freezing and heat it back up to its boiling point.</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
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</div>
 
</div>
 
<div class="attendees">Dustin van der Meulen</div>
 
<div class="attendees">Dustin van der Meulen</div>
−
<div class="description"><p><p>After making a 20 mL overnight culture of our NEB 10 be&#769;ta cells containing our BioBricks, we performed a mini prep (Protocol 3). Using our nanodrop spectrophotometer&nbsp;&nbsp;which gave us the following DNA concentrations:<br>
+
<div class="description">
 +
<p><p>After making a 20 mL overnight culture of our NEB 10 be&#769;ta cells containing our BioBricks, we performed a mini prep (Protocol 3). Using our nanodrop spectrophotometer&nbsp;&nbsp;which gave us the following DNA concentrations:<br>
 
&nbsp;</p>
 
&nbsp;</p>
  
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After assessign our results, in the future we will perform the ethanol carry-over steps.<br>
 
After assessign our results, in the future we will perform the ethanol carry-over steps.<br>
 
<br>
 
<br>
−
Also from the previously mentioned overnight culture 1 mL has been used to make Glycerol Stock (Protocol 4)</p></div>
+
Also from the previously mentioned overnight culture 1 mL has been used to make Glycerol Stock (Protocol 4)</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
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</div>
 
</div>
 
<div class="attendees">Tom van Dijk | Mike Vrieswijk</div>
 
<div class="attendees">Tom van Dijk | Mike Vrieswijk</div>
−
<div class="description"><p>The choice to use a computer PSU as a power supply was made. The choice was made on the grounds, that computer PSU have a standard 12, 5 and 3.3V output. This is beneficial because these are the voltages necessary for the selected components.</p></div>
+
<div class="description">
 +
<p>The choice to use a computer PSU as a power supply was made. The choice was made on the grounds, that computer PSU have a standard 12, 5 and 3.3V output. This is beneficial because these are the voltages necessary for the selected components.</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
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</div>
 
</div>
 
<div class="attendees">Dustin van der Meulen</div>
 
<div class="attendees">Dustin van der Meulen</div>
−
<div class="description"><p><p>Today we performed a digestion, dephosphorilation and ligation (following protocols 4 and 5) of a few promotor and RBS BioBricks. Because of administrative compliations only the variants with J23113 and a RBS could be made.</p>
+
<div class="description">
−
</p></div>
+
<p><p>Today we performed a digestion, dephosphorilation and ligation (following protocols 4 and 5) of a few promotor and RBS BioBricks. Because of administrative compliations only the variants with J23113 and a RBS could be made.</p>
 +
</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
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</div>
 
</div>
 
<div class="attendees">Tom van Dijk | Mike Vrieswijk</div>
 
<div class="attendees">Tom van Dijk | Mike Vrieswijk</div>
−
<div class="description"><p>Changes were made to the architecture design of the temperature controller. Different voltage regulators were selected to fit the newly chosen power supply. To better readability of the architecture design a legend was added.</p></div>
+
<div class="description">
 +
<p>Changes were made to the architecture design of the temperature controller. Different voltage regulators were selected to fit the newly chosen power supply. To better readability of the architecture design a legend was added.</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
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</div>
 
</div>
 
<div class="attendees">Tom van Dijk | Mike Vrieswijk</div>
 
<div class="attendees">Tom van Dijk | Mike Vrieswijk</div>
−
<div class="description"><p>Custom footprints were made for multiple components and added to the parts library in the PCB design software.</p></div>
+
<div class="description">
 +
<p>Custom footprints were made for multiple components and added to the parts library in the PCB design software.</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
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</div>
 
</div>
 
<div class="attendees">Tom van Dijk | Mike Vrieswijk</div>
 
<div class="attendees">Tom van Dijk | Mike Vrieswijk</div>
−
<div class="description"><p>The provisional electronic circuit design for the temperature controller was made.</p></div>
+
<div class="description">
 +
<p>The provisional electronic circuit design for the temperature controller was made.</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
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</div>
 
</div>
 
<div class="attendees">Tom van Dijk | Mike Vrieswijk</div>
 
<div class="attendees">Tom van Dijk | Mike Vrieswijk</div>
−
<div class="description"><p>The electronic circuit design for the temperature controller was finished. Measuring point were added to the electronic circuit design.</p></div>
+
<div class="description">
 +
<p>The electronic circuit design for the temperature controller was finished. Measuring point were added to the electronic circuit design.</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
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</div>
 
</div>
 
<div class="attendees">Elise Grootscholten | Loraine Nelson | Paul Reusink</div>
 
<div class="attendees">Elise Grootscholten | Loraine Nelson | Paul Reusink</div>
−
<div class="description"><p>To have some basic biobricks ready, we stocked them by doing a plasmid isolation and freeze the DNA. </p></div>
+
<div class="description">
 +
<p>To have some basic biobricks ready, we stocked them by doing a plasmid isolation and freeze the DNA. </p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
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</div>
 
</div>
 
<div class="attendees">Elise Grootscholten | Loraine Nelson | Paul Reusink</div>
 
<div class="attendees">Elise Grootscholten | Loraine Nelson | Paul Reusink</div>
−
<div class="description"><p>To start on a basic construct we did a digestion and ligation with the parts we isolated earlier. To prevent original constructs we did a defosphorylation after the digestion. We controlled the digestion by doing a gelelktroforeses. </p></div>
+
<div class="description">
 +
<p>To start on a basic construct we did a digestion and ligation with the parts we isolated earlier. To prevent original constructs we did a defosphorylation after the digestion. We controlled the digestion by doing a gelelktroforeses. </p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
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</div>
 
</div>
 
<div class="attendees">Tom van Dijk | Mike Vrieswijk</div>
 
<div class="attendees">Tom van Dijk | Mike Vrieswijk</div>
−
<div class="description"><p>Started working on the board layout for the PCB of the temperature controller.</p></div>
+
<div class="description">
 +
<p>Started working on the board layout for the PCB of the temperature controller.</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
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</div>
 
</div>
 
<div class="attendees">Tom van Dijk | Randall de Waard</div>
 
<div class="attendees">Tom van Dijk | Randall de Waard</div>
−
<div class="description"><p>We continued with the ligated DNA by transformating it to Neb10Beta. This was plated onto agarplates and incubated for 1 day. </p></div>
+
<div class="description">
 +
<p>We continued with the ligated DNA by transformating it to Neb10Beta. This was plated onto agarplates and incubated for 1 day. </p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
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</div>
 
</div>
 
<div class="attendees">Tom van Dijk | Mike Vrieswijk</div>
 
<div class="attendees">Tom van Dijk | Mike Vrieswijk</div>
−
<div class="description"><p>Further work was done to the board layout for the temperature controller.</p></div>
+
<div class="description">
 +
<p>Further work was done to the board layout for the temperature controller.</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
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</div>
 
</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
−
<div class="description"><p>The results of 7/12/2018 were collected, in action of this we decided to plate some ligated and transferred DNA from 7/12/2018 again. This we did because there wasn't a clear grow of colonies on the plates.</p></div>
+
<div class="description">
 +
<p>The results of 7/12/2018 were collected, in action of this we decided to plate some ligated and transferred DNA from 7/12/2018 again. This we did because there wasn't a clear grow of colonies on the plates.</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
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</div>
 
</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
−
<div class="description"><p>We tried to let bacteria grow on paper, therefore we used our own buisnesscards. We let the buisnesscards absorb some LB-medium and put them inside petridishes. </p></div>
+
<div class="description">
 +
<p>We tried to let bacteria grow on paper, therefore we used our own buisnesscards. We let the buisnesscards absorb some LB-medium and put them inside petridishes. </p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
Line 2,008: Line 2,159:
 
</div>
 
</div>
 
<div class="attendees">Tom van Dijk | Mike Vrieswijk</div>
 
<div class="attendees">Tom van Dijk | Mike Vrieswijk</div>
−
<div class="description"><p>Work has been done on the board layout for the temperature controller. The board layout is now finished.</p></div>
+
<div class="description">
 +
<p>Work has been done on the board layout for the temperature controller. The board layout is now finished.</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
Line 2,049: Line 2,203:
 
</div>
 
</div>
 
<div class="attendees">Mei Ju Goemans | Elise Grootscholten | Dustin van der Meulen | Randall de Waard</div>
 
<div class="attendees">Mei Ju Goemans | Elise Grootscholten | Dustin van der Meulen | Randall de Waard</div>
−
<div class="description"><p>To know if the ligation was succesfull we will controll the DNA, therefor we ented the bacteria's with DNA in LB-medium to grow overnight. We also plated the used bacteria's onto new plates to use for further experiments. </p></div>
+
<div class="description">
 +
<p>To know if the ligation was succesfull we will controll the DNA, therefor we ented the bacteria's with DNA in LB-medium to grow overnight. We also plated the used bacteria's onto new plates to use for further experiments. </p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
Line 2,090: Line 2,247:
 
</div>
 
</div>
 
<div class="attendees">Mei Ju Goemans | Elise Grootscholten | Dustin van der Meulen | Randall de Waard</div>
 
<div class="attendees">Mei Ju Goemans | Elise Grootscholten | Dustin van der Meulen | Randall de Waard</div>
−
<div class="description"><p>We did a blue white screening on our buisnesscards to see if bacteria had grew on them. We transferred the buisnesscards to new petrydishes and added ITPG and X-gal to the petrydishes. </p></div>
+
<div class="description">
 +
<p>We did a blue white screening on our buisnesscards to see if bacteria had grew on them. We transferred the buisnesscards to new petrydishes and added ITPG and X-gal to the petrydishes. </p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
Line 2,131: Line 2,291:
 
</div>
 
</div>
 
<div class="attendees">Rens Boeser</div>
 
<div class="attendees">Rens Boeser</div>
−
<div class="description"><p>Frontend design for the notebook</p></div>
+
<div class="description">
 +
<p>Frontend design for the notebook</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
Line 2,172: Line 2,335:
 
</div>
 
</div>
 
<div class="attendees">Rens Boeser | Dustin van der Meulen | Loraine Nelson  | Suzanne Romeijn | Randall de Waard</div>
 
<div class="attendees">Rens Boeser | Dustin van der Meulen | Loraine Nelson  | Suzanne Romeijn | Randall de Waard</div>
−
<div class="description"><p>The resulst of 7/16/2018 showed that the DNA didnt ligate as planned.  To be able to digest enough DNA we did a plasmid isolation again. We digested this DNA and froze it for further use. </p></div>
+
<div class="description">
 +
<p>The resulst of 7/16/2018 showed that the DNA didnt ligate as planned.  To be able to digest enough DNA we did a plasmid isolation again. We digested this DNA and froze it for further use. </p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
Line 2,213: Line 2,379:
 
</div>
 
</div>
 
<div class="attendees">Dustin van der Meulen | Loraine Nelson | Suzanne Romeijn | Mike Vrieswijk | Randall de Waard</div>
 
<div class="attendees">Dustin van der Meulen | Loraine Nelson | Suzanne Romeijn | Mike Vrieswijk | Randall de Waard</div>
−
<div class="description"><p>We did another round of plasmid isolation for stocking. </p></div>
+
<div class="description">
 +
<p>We did another round of plasmid isolation for stocking. </p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
Line 2,254: Line 2,423:
 
</div>
 
</div>
 
<div class="attendees">Rens Boeser</div>
 
<div class="attendees">Rens Boeser</div>
−
<div class="description"><p>Designed a way to follow experiments that take up multiple days and eventually designed a notebook entry page and made it a responsive page</p></div>
+
<div class="description">
 +
<p>Designed a way to follow experiments that take up multiple days and eventually designed a notebook entry page and made it a responsive page</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
Line 2,295: Line 2,467:
 
</div>
 
</div>
 
<div class="attendees">Dustin van der Meulen | Mei Ju Goemans| Randall de Waard </div>
 
<div class="attendees">Dustin van der Meulen | Mei Ju Goemans| Randall de Waard </div>
−
<div class="description"><p>We digested all DNA we have in stock to test for our basic construct. We tested the DNA by doing a gelelktroforeses. </p></div>
+
<div class="description">
 +
<p>We digested all DNA we have in stock to test for our basic construct. We tested the DNA by doing a gelelktroforeses. </p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
Line 2,336: Line 2,511:
 
</div>
 
</div>
 
<div class="attendees">Rens Boeser</div>
 
<div class="attendees">Rens Boeser</div>
−
<div class="description"><p>Created a one page notebook. Made the notebook entry page more suited for the entry length</p></div>
+
<div class="description">
 +
<p>Created a one page notebook. Made the notebook entry page more suited for the entry length</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
Line 2,377: Line 2,555:
 
</div>
 
</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
−
<div class="description"><p><p>Transformations of the&nbsp;biobricks K133071, K173003 and I13453 are performed. K133071 will produce CO2 if there's pyruvate present and K173003 will produce CO2 if urea is present. I13453 is a promotor which will work if there's arabinose present.&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;&nbsp;<br>
+
<div class="description">
 +
<p><p>Transformations of the&nbsp;biobricks K133071, K173003 and I13453 are performed. K133071 will produce CO2 if there's pyruvate present and K173003 will produce CO2 if urea is present. I13453 is a promotor which will work if there's arabinose present.&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;&nbsp;<br>
 
The biobricks were transformed first into electrocometent cells and later&nbsp;into chemical&nbsp;competent cells from the strain NEb10Beta. After transformations the culture was plated on agar plates with&nbsp;antibiotics. The first transformations didn't work out, but the second did, because then the right competent cells were used.&nbsp;</p>
 
The biobricks were transformed first into electrocometent cells and later&nbsp;into chemical&nbsp;competent cells from the strain NEb10Beta. After transformations the culture was plated on agar plates with&nbsp;antibiotics. The first transformations didn't work out, but the second did, because then the right competent cells were used.&nbsp;</p>
−
</p></div>
+
</p>
 +
<p class="experiment"><p><span style="font-size: 14px;"><strong>Experiment 1: Transformation biobricks</strong></span><br>
 +
<br>
 +
Transformation of the biobricks K133071, K173003 and I13453 in NEB10B�ta.<br>
 +
<br>
 +
<span style="font-size:14px;"><strong>Materials&nbsp;</strong></span></p>
 +
 
 +
<p>- Biobricks (Bba_K133071,&nbsp;Bba_K173003 and Bba_I3453)&nbsp;<br>
 +
- Nuclease free water<br>
 +
- LB- agar<br>
 +
- Variable Volume Pipettes&nbsp;<br>
 +
- Sterile pipetpoints<br>
 +
- Chemically competent cells ( NEB10Beta, 1x10^9)&nbsp;<br>
 +
- Heater (42�C) &nbsp;<br>
 +
- Incubator + shaker (37�C and 250 rpm)&nbsp;<br>
 +
- Eppendorf tubes (1,5 and 2 ml)&nbsp;<br>
 +
- Stable outgrow medium for NEB10Beta or SOC- outgrow medium&nbsp;<br>
 +
-Chloramphenicol (40 mg/mL)<br>
 +
<br>
 +
<span style="font-size:14px;"><strong><span style="font-family:arial,helvetica,sans-serif;">Methods</span></strong></span><br>
 +
-K173003 --&gt; pSB1C3 backbone, plate 6, well 15N<br>
 +
-K133071 --&gt; pSB1C3 backbone, plate 6, wel 21A<br>
 +
-I13453 --&gt; pSB1C3 backbone, plate 3, well 19O</p>
 +
 
 +
<p>To be able to use the DNA from the biobricks, they were first diluted in Nuclease free water. This was done by adding 10ul of nuclease free water to the DNA and incubated for 5 minutes at room temperature (18-24�C). After incubation the DNA is transferred to Eppendorf tubes&nbsp;and put on ice.&nbsp;<br>
 +
<br>
 +
For the transformation 1 ul of the DNA was added&nbsp;to 50 ul&nbsp;chemically competent cells strain&nbsp;NEB10Beta. The mixture was incubated for 30 minutes on ice. The mixture got a heat shock by putting the tube&nbsp;in a heater (42�C) for 30 seconds. After the heat shock the tube was immediately put on ice for 5 minutes.&nbsp;<br>
 +
For good grow of the cells, 950 ul of outgrow medium was added to the mixture. After mixing the cells with outgrow medium, the culture was incubated for 1 hour at 37�C and 250 rpm.&nbsp;&nbsp;<br>
 +
<br>
 +
After incubation the culture was plated onto LB-agar plates. For specific growth of bacteria there was antibiotics added to the plates. The antibiotics that were used, depended on the plasmid. The biobricks used in this experiment were resistent to chloramphenicol (Can). We worked with a work concentration of 35 ug/ml for chloramphenicol. For good results we plated 100 ul undiluted culture onto a plate and centrifuged the rest of the culture. The supernatant was discarded and the pellet was resuspended and plated onto a LB-agar plate. The plates were then incubated by 37�C.&nbsp;<br>
 +
<br>
 +
<strong>Results</strong><br>
 +
The first transformation was done in electrocompetent cells instead of chemocompetent cells so the first transformation did not succeed.</p>
 +
 
 +
<p>&nbsp;</p>
 +
 
 +
<p><strong>Conclusion</strong><br>
 +
<br>
 +
The second transformation from the biobricks was successful. The bacteria with the biobricks plasmids will be stored by 4�C and can be used for further experiments.&nbsp;<br>
 +
The CFU from the&nbsp;negative controle will be plated on a plate with antibiotics to make sure there isn't a plasmid in the bacteria. The outgrow medium will be sterilized by filter&nbsp;again.&nbsp;<br>
 +
&nbsp;</p>
 +
</p>
 +
</div>
 
</div>
 
</div>
  
Line 2,420: Line 2,641:
 
</div>
 
</div>
 
<div class="attendees">Rens Boeser | Mei Ju Goemans | Elise Grootscholten | Dustin van der Meulen | Loraine Nelson | Suzanne Romeijn | Randall de Waard</div>
 
<div class="attendees">Rens Boeser | Mei Ju Goemans | Elise Grootscholten | Dustin van der Meulen | Loraine Nelson | Suzanne Romeijn | Randall de Waard</div>
−
<div class="description"><p>New biobricks, used to make an ATP sensor, were transformed into NEB10Beta. The culture was than plated onto agar and incubated for 1 day.  </p></div>
+
<div class="description">
 +
<p>New biobricks, used to make an ATP sensor, were transformed into NEB10Beta. The culture was than plated onto agar and incubated for 1 day.  </p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
Line 2,461: Line 2,685:
 
</div>
 
</div>
 
<div class="attendees">Rens Boeser | Mei Ju Goemans | Elise Grootscholten | Dustin van der Meulen | Loraine Nelson | Suzanne Romeijn | Randall de Waard</div>
 
<div class="attendees">Rens Boeser | Mei Ju Goemans | Elise Grootscholten | Dustin van der Meulen | Loraine Nelson | Suzanne Romeijn | Randall de Waard</div>
−
<div class="description"><p>New biobricks, which produces gasses, were transformed into NEB10Beta. The culture was than plated onto agar and incubated for 1 day.  </p></div>
+
<div class="description">
 +
<p>New biobricks, which produces gasses, were transformed into NEB10Beta. The culture was than plated onto agar and incubated for 1 day.  </p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
Line 2,502: Line 2,729:
 
</div>
 
</div>
 
<div class="attendees">Rens Boeser</div>
 
<div class="attendees">Rens Boeser</div>
−
<div class="description"><p>Started working on an automatic notebook creator</p></div>
+
<div class="description">
 +
<p>Started working on an automatic notebook creator</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
Line 2,543: Line 2,773:
 
</div>
 
</div>
 
<div class="attendees">Loraine Nelson | Elise Grootscholten | Paul Reusink</div>
 
<div class="attendees">Loraine Nelson | Elise Grootscholten | Paul Reusink</div>
−
<div class="description"><p><p>Today we did a transformation with&nbsp;new biobricks. The biobricks were built into chemically competent cells from the strain NEb10Beta. After transformation the culture was plated on agar plates with&nbsp;antibiotics. The results were checked the next day.&nbsp;</p>
+
<div class="description">
−
</p></div>
+
<p><p>Today we did a transformation with&nbsp;new biobricks. The biobricks were built into chemically competent cells from the strain NEb10Beta. After transformation the culture was plated on agar plates with&nbsp;antibiotics. The results were checked the next day.&nbsp;</p>
 +
</p>
 +
<p class="experiment">Materials:&nbsp;
 +
<p>- Biobricks (Bba-K1499004 and Bba- K1614019)&nbsp;<br>
 +
- Nuclease free water<br>
 +
- LB- agar<br>
 +
- Variable Volume Pipettes&nbsp;<br>
 +
- Sterile pipetpoints<br>
 +
- Chemically competent cells ( NEB10Beta, 1x10^9)&nbsp;<br>
 +
- Heater (42�C) &nbsp;<br>
 +
- Incubator + shaker (37�C and 250 rpm)&nbsp;<br>
 +
- Eppendorf tubes (1,5 and 2 ml)&nbsp;<br>
 +
- Stable outgrow medium for NEB10Beta or SOC- outgrow medium&nbsp;<br>
 +
<br>
 +
Method:&nbsp;<br>
 +
<br>
 +
To be able to use the DNA from the biobricks, they were first diluted in Nuclease free water. This was done by adding 10ul of nuclease free water to the DNA and incubated for 5 minutes at room temperature (18-24�C). After incubation the DNA is transferred to Eppendorf tubes&nbsp;and put on ice.&nbsp;<br>
 +
<br>
 +
For the transformation 1 ul of the DNA was added&nbsp;to 50 ul&nbsp;chemically competent cells strain&nbsp;NEB10Beta. The mixture was incubated for 30 minutes on ice. The mixture got a heat shock by putting the tube&nbsp;in a heater (42�C) for 30 seconds. After the heat shock the tube was immediately put on ice for 5 minutes.&nbsp;<br>
 +
For good grow of the cells, 950 ul of outgrow medium was added to the mixture. After mixing the cells with outgrow medium, the culture was incubated for 1 hour at 37�C and 250 rpm.&nbsp;&nbsp;<br>
 +
<br>
 +
After incubation the culture was plated onto LB-agar plates. For specific growth of bacteria there was antibiotics added to the plates. The antibiotics that were used, depended on the plasmid. The biobricks used in this experiment were resistent to chloramphenicol (Can). We worked with a work concentration of 35 ug/ml for chloramphenicol. For good results we plated 100 ul undiluted culture onto a plate and centrifuged the rest of the culture. The supernatant was discarded and the pellet was resuspended and plated onto a LB-agar plate. The plates were then incubated by 37�C.&nbsp;<br>
 +
<br>
 +
Results:<br>
 +
Tabel 1 shows the results of the plates which were incubated.&nbsp;</p>
 +
 
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 427px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 107px;">Biobricks:&nbsp;</td>
 +
<td style="width: 44px;">Antibiotics:&nbsp;</td>
 +
<td style="width: 86px;">Dilution:&nbsp;</td>
 +
<td style="width: 73px;">Colonies:</td>
 +
<td style="width: 101px;">CFU:&nbsp;</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 107px;">Bba-K1614019</td>
 +
<td style="width: 44px;">&nbsp;Can&nbsp;</td>
 +
<td style="width: 86px;">0x&nbsp;</td>
 +
<td style="width: 73px;">109</td>
 +
<td style="width: 101px;">1*10^3 /ml</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 107px;">Bba-K1614019</td>
 +
<td style="width: 44px;">Can</td>
 +
<td style="width: 86px;">Centrifuged</td>
 +
<td style="width: 73px;">&gt;300</td>
 +
<td style="width: 101px;">&nbsp; &nbsp;-</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 107px;">Bba-K1499004&nbsp;&nbsp;&nbsp;&nbsp;</td>
 +
<td style="width: 44px;">Can</td>
 +
<td style="width: 86px;">0x</td>
 +
<td style="width: 73px;">33</td>
 +
<td style="width: 101px;">3*10^2 /ml</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 107px;">Bba-K1499004&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</td>
 +
<td style="width: 44px;">Can</td>
 +
<td style="width: 86px;">Centrifuged</td>
 +
<td style="width: 73px;">228</td>
 +
<td style="width: 101px;">&nbsp; &nbsp;-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 107px;">Positive controle: Bba- K1499004</td>
 +
<td style="width: 44px;">None</td>
 +
<td style="width: 86px;">0x</td>
 +
<td style="width: 73px;">&gt; 300</td>
 +
<td style="width: 101px;">&nbsp; &nbsp;-</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 107px;">Negative controle: outgrow medium</td>
 +
<td style="width: 44px;">none</td>
 +
<td style="width: 86px;">0x</td>
 +
<td style="width: 73px;">18</td>
 +
<td style="width: 101px;">&nbsp; &nbsp;-&nbsp;</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
 
 +
<p>Tabel 1: results from the transformation with the biobricks Bba-K1614019 and Bba-K1499004.&nbsp;<br>
 +
<br>
 +
The plates show CFU which can be used for further experiments. &nbsp;The negative controle also has CFU, this shows that the outgrow medium was infected and not sterile.&nbsp;<br>
 +
The plates are stored by 4�C.&nbsp;<br>
 +
<br>
 +
Conclusion:&nbsp;<br>
 +
<br>
 +
The transformation from the biobricks was successful. The bacteria with the biobricks plasmids will be stored by 4�C and can be used for further experiments.&nbsp;<br>
 +
The CFU from the&nbsp;negative controle will be plated on a plate with antibiotics to make sure there isn't a plasmid in the bacteria. The outgrow medium will be sterilized by filter&nbsp;again.&nbsp;<br>
 +
<br>
 +
&nbsp;</p>
 +
</p>
 +
</div>
 
</div>
 
</div>
  
Line 2,585: Line 2,907:
 
</div>
 
</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
−
<div class="description"><p><p>To be able to always get the necessary biobricks, there have been&nbsp;made&nbsp;glycerolstocks of the transformed biobricks K133071, K173003 and I13453. The glycerolstocks are stored at -80 degrees Celsius. If needed, they can be retrieved from this storage to use for experiments.&nbsp;</p>
+
<div class="description">
−
</p></div>
+
<p><p>To be able to always get the necessary biobricks, there have been&nbsp;made&nbsp;glycerolstocks of the transformed biobricks K133071, K173003 and I13453. The glycerolstocks are stored at -80 degrees Celsius. If needed, they can be retrieved from this storage to use for experiments.&nbsp;</p>
 +
</p>
 +
<p class="experiment"><p><span style="font-size: 14px;"><strong>Experiment 2: Glycerolstocks</strong></span><br>
 +
<br>
 +
Making glycerol stocks of the biobricks K133071, K173003, I13453.&nbsp;<br>
 +
<br>
 +
<span style="font-size:14px;"><strong>Materials:&nbsp;</strong></span><br>
 +
- 87% glycerol&nbsp;<br>
 +
- culture with biobrick plasmids<br>
 +
- LB-medium<br>
 +
- Variable Volume Pipette<br>
 +
- Sterile pipetpoints<br>
 +
<br>
 +
<strong><span style="font-size:14px;">Methods:&nbsp;</span></strong><br>
 +
The colonies from the transformation were used to make an overnight culture. The culture was made by putting a colony in LB-medium and then the culture was&nbsp;incubated for a night at 37�C. &nbsp;<br>
 +
<br>
 +
The next day the cultures had grown close and were ready for a glycerol stock. For making glycerol stocks there was added 250 ul 87% glycerol to 1 ml culture. This was frozen in -80�C. &nbsp;<br>
 +
<br>
 +
The stocks were stored at -80�C in drawer 3, tower 1, drawer&nbsp;</p>
 +
</p>
 +
</div>
 
</div>
 
</div>
  
Line 2,627: Line 2,969:
 
</div>
 
</div>
 
<div class="attendees">Rens Boeser</div>
 
<div class="attendees">Rens Boeser</div>
−
<div class="description"><p>Automatic generation of entries completed</p></div>
+
<div class="description">
 +
<p>Automatic generation of entries completed</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
Line 2,668: Line 3,013:
 
</div>
 
</div>
 
<div class="attendees">Rens Boeser</div>
 
<div class="attendees">Rens Boeser</div>
−
<div class="description"><p>Used the Google drive REST API to download the entries from drive</p></div>
+
<div class="description">
 +
<p>Used the Google drive REST API to download the entries from drive</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
Line 2,709: Line 3,057:
 
</div>
 
</div>
 
<div class="attendees">Rens Boeser</div>
 
<div class="attendees">Rens Boeser</div>
−
<div class="description"><p>Used the igemwiki API to update pages using software</p></div>
+
<div class="description">
 +
<p>Used the igemwiki API to update pages using software</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
Line 2,750: Line 3,101:
 
</div>
 
</div>
 
<div class="attendees">Loraine Nelson | Elise Grootscholten | Paul Reusink</div>
 
<div class="attendees">Loraine Nelson | Elise Grootscholten | Paul Reusink</div>
−
<div class="description"><p><p>To be able to always get the necessary biobricks, we made an glycerol stock after every transformation. The glycerol stocks are stored at -80 degrees Celsius. If needed, they can retrieved from this storage to use for experiments.&nbsp;</p>
+
<div class="description">
−
</p></div>
+
<p><p>To be able to always get the necessary biobricks, we made an glycerol stock after every transformation. The glycerol stocks are stored at -80 degrees Celsius. If needed, they can retrieved from this storage to use for experiments.&nbsp;</p>
 +
</p>
 +
<p class="experiment"><p>Making glycerol stocks of the biobricks.&nbsp;<br>
 +
<br>
 +
Materials:&nbsp;<br>
 +
- 87% glycerol&nbsp;<br>
 +
- culture with biobrick plasmids<br>
 +
- LB-medium<br>
 +
- Variable Volume Pipette<br>
 +
- Sterile pipetpoints<br>
 +
<br>
 +
<br>
 +
Method:&nbsp;<br>
 +
<br>
 +
The colonies from the transformation were used to make an overnight culture. The culture was made by putting a colony in LB-medium and then the culture was&nbsp;incubated for a night at 37�C. &nbsp;<br>
 +
<br>
 +
The next day the cultures had grown close and were ready for a glycerol stock. For making glycerol stocks there was added 250 ul 87% glycerol to 1 ml culture. This was frozen in -80�C. &nbsp;<br>
 +
<br>
 +
The stocks were stored at -80�C in drawer 3, tower 1, drawer&nbsp;</p>
 +
</p>
 +
</div>
 
</div>
 
</div>
  
Line 2,792: Line 3,163:
 
</div>
 
</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
−
<div class="description"><p><p>For further experiments there is&nbsp;isolated DNA needed of the biobricks J23100, K133071, K173003 and I134353. The DNA is isolated out of the bacteria with the help of a plasmid purifaction kit. After isolation this DNA can be used for digestion and ligation or other experiments.&nbsp;<span style="font-size:12px;">J23100 (from the glycerolstock): 325,59 ng/ul, K173003: 217,06 ng/ul, K133071: 186,79 ng/ul,&nbsp;</span>I13453: 88,18 ng/ul</p>
+
<div class="description">
−
</p></div>
+
<p><p>For further experiments there is&nbsp;isolated DNA needed of the biobricks J23100, K133071, K173003 and I134353. The DNA is isolated out of the bacteria with the help of a plasmid purifaction kit. After isolation this DNA can be used for digestion and ligation or other experiments.&nbsp;<span style="font-size:12px;">J23100 (from the glycerolstock): 325,59 ng/ul, K173003: 217,06 ng/ul, K133071: 186,79 ng/ul,&nbsp;</span>I13453: 88,18 ng/ul</p>
 +
</p>
 +
<p class="experiment"><p><span style="font-size:14px;"><strong>Experiment 3: Minipreps</strong></span><br>
 +
<br>
 +
<span style="font-size:12px;">Minipreps of the following biobricks in NEB10B�ta:</span><br>
 +
-J23100: Constitutive promoter<br>
 +
-K133071: Urea --&gt; ammonia + CO2<br>
 +
-K173003: Pyruvate --&gt; acetaldehyde + CO2<br>
 +
-I134353: Promoter (AraC protein binds with arabinose)<br>
 +
<br>
 +
<strong><span style="font-size:14px;">Materials:&nbsp;</span></strong><br>
 +
- Mini prep, Plasmid Purification Kit, Machery Michels&nbsp;<br>
 +
- Eppendorf cups<br>
 +
- Variable Volume Pipets<br>
 +
- sterile pipet points<br>
 +
-Chloramphenicol (40 mg/mL)<br>
 +
<br>
 +
<span style="font-size:14px;"><strong>Method:&nbsp;</strong></span><br>
 +
<span style="font-size:14px;"><strong>Miniprep protocol...??</strong></span><br>
 +
After the transformation we made an overnight culture from the colonies. The colonies were anted into 20 ml LB and incubated overnight at&nbsp;37�C and 150 rpm. The culture was mini prepped the next day. For isolation, 5,4 ml was added step wise in a 2ml Eppendorf cup. The culture was then centrifuged at &gt;12000x g for 30 seconds. The supernatant was deposed, and the pellet was resupended with A1 buffer and vortexed. To the resuspended culture was <strong><span style="font-size:14px;">then.....???<br>
 +
<br>
 +
Results</span></strong><br>
 +
<br>
 +
<span style="font-size:12px;">J23100: 325,59 ng/ul<br>
 +
K173003: 217,06 ng/ul<br>
 +
K133071: 186,79 ng/ul</span><br>
 +
I13453: 88,18 ng/ul</p>
 +
</p>
 +
</div>
 
</div>
 
</div>
  
Line 2,834: Line 3,233:
 
</div>
 
</div>
 
<div class="attendees">Rens Boeser</div>
 
<div class="attendees">Rens Boeser</div>
−
<div class="description"><p>Connected the page uploader to the notebook generator</p></div>
+
<div class="description">
 +
<p>Connected the page uploader to the notebook generator</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
Line 2,875: Line 3,277:
 
</div>
 
</div>
 
<div class="attendees">Rens Boeser</div>
 
<div class="attendees">Rens Boeser</div>
−
<div class="description"><p>Created a program that uploads all updated pages</p></div>
+
<div class="description">
 +
<p>Created a program that uploads all updated pages</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
Line 2,916: Line 3,321:
 
</div>
 
</div>
 
<div class="attendees">Rens Boeser</div>
 
<div class="attendees">Rens Boeser</div>
−
<div class="description"><p>Built a page builder that automatically adds the header and footer before uploading</p></div>
+
<div class="description">
 +
<p>Built a page builder that automatically adds the header and footer before uploading</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
Line 2,957: Line 3,365:
 
</div>
 
</div>
 
<div class="attendees">Rens Boeser</div>
 
<div class="attendees">Rens Boeser</div>
−
<div class="description"><p>Redone the google API handler for reusability and readability and made the google API handler generic</p></div>
+
<div class="description">
 +
<p>Redone the google API handler for reusability and readability and made the google API handler generic</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
Line 2,998: Line 3,409:
 
</div>
 
</div>
 
<div class="attendees">Rens Boeser</div>
 
<div class="attendees">Rens Boeser</div>
−
<div class="description"><p>revamped the main program</p></div>
+
<div class="description">
 +
<p>revamped the main program</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
Line 3,039: Line 3,453:
 
</div>
 
</div>
 
<div class="attendees">Rens Boeser</div>
 
<div class="attendees">Rens Boeser</div>
−
<div class="description"><p>Finished redoing the application for generating and uploading pages</p></div>
+
<div class="description">
 +
<p>Finished redoing the application for generating and uploading pages</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
Line 3,080: Line 3,497:
 
</div>
 
</div>
 
<div class="attendees">Rens Boeser</div>
 
<div class="attendees">Rens Boeser</div>
−
<div class="description"><p>Added month categorization to the page</p></div>
+
<div class="description">
 +
<p>Added month categorization to the page</p>
 +
<p class="experiment"></p>
 +
</div>
 
</div>
 
</div>
  
Line 3,121: Line 3,541:
 
</div>
 
</div>
 
<div class="attendees">Loraine Nelson | Elise Grootscholten | Paul Reusink</div>
 
<div class="attendees">Loraine Nelson | Elise Grootscholten | Paul Reusink</div>
−
<div class="description"><p><p>For further experiments we needed isolated DNA. The DNA is isolated out of the bacteria with the help of a plasmid purifaction kit. After isolation this DNA can be used for digestion and ligation or other experiments.</p>
+
<div class="description">
−
</p></div>
+
<p><p>For further experiments we needed isolated DNA. The DNA is isolated out of the bacteria with the help of a plasmid purifaction kit. After isolation this DNA can be used for digestion and ligation or other experiments.</p>
 +
</p>
 +
<p class="experiment"><p>Materials:&nbsp;<br>
 +
- Mini prep, Plasmid Purification Kit, Machery Michels&nbsp;<br>
 +
- Eppendorf cups<br>
 +
- Variable Volume Pipets<br>
 +
- sterile pipet points<br>
 +
<br>
 +
<br>
 +
Method:&nbsp;<br>
 +
After the transformation we made an overnight culture from the colonies. The colonies were anted into 20 ml LB and incubated overnight at&nbsp;<span style="display: inline !important; float: none; background-color: transparent; color: rgb(51, 51, 51); font-family: Arial,Helvetica,sans-serif; font-size: 12px; font-style: normal; font-variant: normal; font-weight: 400; letter-spacing: normal; orphans: 2; text-align: left; text-decoration: none; text-indent: 0px; text-transform: none; -webkit-text-stroke-width: 0px; white-space: normal; word-spacing: 0px;">37�C and 150 rpm. The culture was mini prepped the next day. For isolation, 5,4 ml was added step wise in a 2ml Eppendorf cup. The culture was then centrifuged at &gt;12000x g for 30 seconds. The supernatant was deposed, and the pellet was resupended with A1 buffer and vortexed. To the resuspended culture was then.&nbsp;</span></p>
 +
</p>
 +
</div>
 
</div>
 
</div>
  
Line 3,163: Line 3,595:
 
</div>
 
</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
−
<div class="description"><p><p>The biobricks&nbsp;J23100, K133071, K173003 and I134353 were&nbsp;sucessfully digested after the second time. After the&nbsp;digestions the&nbsp;biobricks K133071 and K173003&nbsp;were dephosphorylated and ligated with the inserts J23100 and I13453. This was done in the original backbone of K133071 and K173003, and not another control backbone. To know&nbsp;if the biobricks were right ligated this was done by testing practically.&nbsp;See the experiments:&nbsp;<em>Testing gas production.</em>&nbsp;</p>
+
<div class="description">
−
</p></div>
+
<p><p>The biobricks&nbsp;J23100, K133071, K173003 and I134353 were&nbsp;sucessfully digested after the second time. After the&nbsp;digestions the&nbsp;biobricks K133071 and K173003&nbsp;were dephosphorylated and ligated with the inserts J23100 and I13453. This was done in the original backbone of K133071 and K173003, and not another control backbone. To know&nbsp;if the biobricks were right ligated this was done by testing practically.&nbsp;See the experiments:&nbsp;<em>Testing gas production.</em>&nbsp;</p>
 +
</p>
 +
<p class="experiment"><p><span style="font-size: 14px;"><strong>Experiment 4: Digestion, Defosforylation and&nbsp;ligation</strong></span><br>
 +
<span style="font-size:14px;"><strong>Digestions</strong></span><br>
 +
<span style="font-size:12px;">30-07-18 and 08-08-18<br>
 +
<br>
 +
Biobricks: J23100, K133071, K173003 and I13453</span><br>
 +
<br>
 +
Used&nbsp;pipette scheme (Table 1) to prepare the DNA to be cut with multiple combinations of EcoRI-HF, SpeI-HF, PstI and XbaI.</p>
 +
 
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 500px;">
 +
<tbody>
 +
<tr>
 +
<td>DNA</td>
 +
<td>~ 2 &mu;g</td>
 +
</tr>
 +
<tr>
 +
<td>NEBbuffer 2.1</td>
 +
<td>5 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>Restriction enzyme 1 (1 U/&mu;L)</td>
 +
<td>2 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>Restriction enzyme 2 (1 U/&mu;L)</td>
 +
<td>2 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>Nuclease free water</td>
 +
<td>Fill to 100 &mu;L</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<em>(Table 1 ; Double Digest Pipette Scheme using :&nbsp;<a href="https://nebcloner.neb.com/#!/redigest"><u><font color="#0066cc">https://nebcloner.neb.com/#!/redigest</font></u></a>, and doubling the amount)</em><br>
 +
<br>
 +
<br>
 +
<strong>Biobricks:</strong><br>
 +
J23100: Constitutive promotor<br>
 +
K133071: Urea --&gt; ammonia + CO2<br>
 +
K173003: Pyruvate --&gt; acetaldehyde + CO2<br>
 +
I134353: Promotor (AraC protein binds with arabinose)<br>
 +
<br>
 +
With this we started a digest of the following BioBricks using the following Restriction enzyme couples (Table 2)<br>
 +
&nbsp;
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 571px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 87px;"><strong>Number</strong></td>
 +
<td style="width: 162px;"><strong>BioBrick BBa_ code</strong></td>
 +
<td style="width: 191px;"><strong>Restriction enzyme 1</strong></td>
 +
<td style="width: 198px;"><strong>Restriction enzyme 2</strong></td>
 +
</tr>
 +
<tr>
 +
<td style="width: 87px;">9</td>
 +
<td style="width: 162px;">J23100 (35 bp)</td>
 +
<td style="width: 191px;">EcoRI</td>
 +
<td style="width: 198px;">SpeI</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 87px;">11</td>
 +
<td style="width: 162px;">K133071 (1707 bp)</td>
 +
<td style="width: 191px;">EcoRI</td>
 +
<td style="width: 198px;">XbaI</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 87px;">12</td>
 +
<td style="width: 162px;">K173003 (3052 bp)</td>
 +
<td style="width: 191px;">EcoRI</td>
 +
<td style="width: 198px;">XbaI</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 87px;">10</td>
 +
<td style="width: 162px;">I13453 (130 bp)</td>
 +
<td style="width: 191px;">EcoRI</td>
 +
<td style="width: 198px;">SpeI</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
 
 +
<p><em>Table 2: Biobricks and restriction enzymes</em><br>
 +
<br>
 +
Digestion was started on the 30th of July, ~16:00, 37&deg;C and 185 RPM.<br>
 +
Digestion was stopped on the 31st of July, ~10:00.<br>
 +
<br>
 +
This has been done in tandem with the corresponding part from&nbsp;<a href="/members/experiments/browser/#view=experiment&amp;nodeID=313406"><u><font color="#0066cc">Cloning Mulitple BioBricks to Assess CooA Production</font></u></a>&nbsp;Part X&nbsp;</p>
 +
 
 +
<p><br>
 +
<br>
 +
<strong><span style="font-size:14px;">Dephosphorylation</span></strong><br>
 +
<span style="font-size:12px;">10-08-18</span><br>
 +
<br>
 +
K133071 and K173003 were dephosphoralized:<br>
 +
&nbsp;</p>
 +
 
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 313px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 128px;"><strong>Volume</strong></td>
 +
<td style="width: 172px;"><strong>Compound</strong></td>
 +
</tr>
 +
<tr>
 +
<td style="width: 128px;"><span style="font-size:12px;"><span style="font-family:arial,helvetica,sans-serif;">25&nbsp;&mu;L</span></span></td>
 +
<td style="width: 172px;">Vector DNA (<span style="font-size:12px;"><span style="font-family:arial,helvetica,sans-serif;">0,5&nbsp;&mu;g)</span></span></td>
 +
</tr>
 +
<tr>
 +
<td style="width: 128px;"><span style="font-size:12px;"><span style="font-family:arial,helvetica,sans-serif;">3&nbsp;&mu;L</span></span></td>
 +
<td style="width: 172px;"><span style="font-size:12px;"><span style="font-family:arial,helvetica,sans-serif;">phosphatase buffer (10x)</span></span></td>
 +
</tr>
 +
<tr>
 +
<td style="width: 128px;"><span style="font-size:12px;"><span style="font-family:arial,helvetica,sans-serif;">1&nbsp;&mu;L</span></span></td>
 +
<td style="width: 172px;"><span style="font-size:12px;"><span style="font-family:arial,helvetica,sans-serif;">milli Q</span></span></td>
 +
</tr>
 +
<tr>
 +
<td style="width: 128px;"><span style="font-size:12px;"><span style="font-family:arial,helvetica,sans-serif;">1&nbsp;&mu;L</span></span></td>
 +
<td style="width: 172px;"><span style="font-size:12px;"><span style="font-family:arial,helvetica,sans-serif;">phosphatase (1 U/&mu;L)</span></span></td>
 +
</tr>
 +
<tr>
 +
<td style="width: 128px;"><span style="font-size:12px;"><span style="font-family:arial,helvetica,sans-serif;">30&nbsp;&mu;L</span></span></td>
 +
<td style="width: 172px;"><span style="font-size:12px;"><span style="font-family:arial,helvetica,sans-serif;">Total</span></span></td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
 
 +
<div class="step" data-stepid="362681">
 +
<div class="stepBody"><em>Table 3: Pipetting scheme</em><br>
 +
<br>
 +
The new DNA concentration of the reaction mix is 0,5&nbsp;&mu;g/30&nbsp;&mu;L<br>
 +
The reaction mix was incubated for 10 minutes at 37&nbsp;&deg;C<br>
 +
The phosphatase was inactivated for 2 minutes at 75&nbsp;&deg;C</div>
 +
</div>
 +
 
 +
<div class="step" data-stepid="362684">
 +
<div class="stepHeader">
 +
<div class="stepTitle">&nbsp;</div>
 +
 
 +
<div class="stepMenu">
 +
<ul>
 +
<li class="btnEditSave" style="display: none;">&nbsp;</li>
 +
<li class="btnMoveStep" style="display: none;">&nbsp;</li>
 +
<li class="btnRemoveStep warning" style="display: none;">&nbsp;</li>
 +
</ul>
 +
</div>
 +
</div>
 +
 
 +
<div class="stepBody">
 +
<div class="stepContents">For the ligation mix 100 ng of vector DNA was used from the dephosphorylation reaction mix.<br>
 +
The following vector-insert combinations were ligated:</div>
 +
</div>
 +
</div>
 +
 
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 403px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 119px;"><strong>Vector</strong></td>
 +
<td style="width: 132px;"><strong>Insert 1</strong></td>
 +
<td style="width: 134px;"><strong>Insert 2</strong></td>
 +
</tr>
 +
<tr>
 +
<td style="width: 119px;">K133071</td>
 +
<td style="width: 132px;">J23100</td>
 +
<td style="width: 134px;">I13453</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 119px;">K173003</td>
 +
<td style="width: 132px;">J23100</td>
 +
<td style="width: 134px;">I13453</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
 
 +
<div class="stepMenu">
 +
<ul>
 +
<li class="btnEditSave" style="display: none;">&nbsp;</li>
 +
<li class="btnMoveStep" style="display: none;">&nbsp;</li>
 +
<li class="btnRemoveStep warning" style="display: none;">&nbsp;</li>
 +
</ul>
 +
</div>
 +
<em>Table 4: Biobrick combination</em><br>
 +
<br>
 +
The original plasmids of the vector and inserts that were digested are about the same size in bp and the ratio vector DNA-insert DNA is determined to be 1:3. Therefore, the amount of insert DNA that was used for the ligation was&nbsp;140 ng (rounded up to 7&nbsp;&mu;L of the digestion reaction mix). Because there was only 5&nbsp;&mu;L left of the K352001 insert DNA, the total 5&nbsp;&mu;L were used and an extra 2&nbsp;&mu;L of milli Q water were added to the pre-ligation mix.<br>
 +
<br>
 +
Pre-Ligationmix:
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 313px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 128px;"><strong>Volume</strong></td>
 +
<td style="width: 172px;"><strong>Compound</strong></td>
 +
</tr>
 +
<tr>
 +
<td style="width: 128px;"><span style="font-size:12px;"><span style="font-family:arial,helvetica,sans-serif;">6 &mu;L</span></span></td>
 +
<td style="width: 172px;">Vector DNA (<span style="font-size:12px;"><span style="font-family:arial,helvetica,sans-serif;">0,5&nbsp;&mu;g/30 &mu;L)</span></span></td>
 +
</tr>
 +
<tr>
 +
<td style="width: 128px;"><span style="font-size:12px;"><span style="font-family:arial,helvetica,sans-serif;">7 &mu;L</span></span></td>
 +
<td style="width: 172px;"><font face="arial, helvetica, sans-serif">Insert DNA (2&nbsp;</font><span style="font-size:12px;"><span style="font-family:arial,helvetica,sans-serif;">&mu;g/100&nbsp;&mu;L)</span></span></td>
 +
</tr>
 +
<tr>
 +
<td style="width: 128px;"><span style="font-size:12px;"><span style="font-family:arial,helvetica,sans-serif;">4 &mu;L</span></span></td>
 +
<td style="width: 172px;">Delutionbuffer (5x)</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 128px;"><span style="font-size:12px;"><span style="font-family:arial,helvetica,sans-serif;">3 &mu;L</span></span></td>
 +
<td style="width: 172px;">Milli Q</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 128px;"><span style="font-size:12px;"><span style="font-family:arial,helvetica,sans-serif;">20&nbsp;&mu;L</span></span></td>
 +
<td style="width: 172px;"><span style="font-size:12px;"><span style="font-family:arial,helvetica,sans-serif;">Total</span></span></td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<em>Table 5: Pre-ligationmix pipette scheme</em><br>
 +
<br>
 +
Ligationmix:<br>
 +
incubation at room-temperature for 20 minutes.
 +
<div class="stepBody">
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 313px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 128px;"><strong>Volume</strong></td>
 +
<td style="width: 172px;"><strong>Compound</strong></td>
 +
</tr>
 +
<tr>
 +
<td style="width: 128px;"><span style="font-size:12px;"><span style="font-family:arial,helvetica,sans-serif;">10 &mu;L</span></span></td>
 +
<td style="width: 172px;">Pre-ligationmix</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 128px;"><span style="font-size:12px;"><span style="font-family:arial,helvetica,sans-serif;">2 &mu;L</span></span></td>
 +
<td style="width: 172px;">10x T4 ligationbuffer</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 128px;"><span style="font-size:12px;"><span style="font-family:arial,helvetica,sans-serif;">1 &mu;L</span></span></td>
 +
<td style="width: 172px;">T4 DNA ligase (1U/<span style="font-size:12px;"><span style="font-family:arial,helvetica,sans-serif;">&mu;L)</span></span></td>
 +
</tr>
 +
<tr>
 +
<td style="width: 128px;"><span style="font-size:12px;"><span style="font-family:arial,helvetica,sans-serif;">8 &mu;L</span></span></td>
 +
<td style="width: 172px;">Milli Q</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 128px;"><span style="font-size:12px;"><span style="font-family:arial,helvetica,sans-serif;">20&nbsp;&mu;L</span></span></td>
 +
<td style="width: 172px;"><span style="font-size:12px;"><span style="font-family:arial,helvetica,sans-serif;">Total</span></span></td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<em>Table 6: Ligationmix pipette scheme</em><br>
 +
&nbsp;</div>
 +
 
 +
<div class="stepBody">
 +
<div class="step" data-stepid="362684">
 +
<div class="stepBody">
 +
<div class="stepContents">All the vectors contain the CamR gene. 30 plates were poured with Cam (14 for the transformed cells (100&mu;L), 14 for the transformed cells (900&nbsp;&mu;L centrifuged and resuspended in 100&nbsp;&mu;L and 1 positive and 1 negative control)</div>
 +
</div>
 +
</div>
 +
</div>
 +
 
 +
<p>&nbsp;</p>
 +
</p>
 +
</div>
 
</div>
 
</div>
  
Line 3,205: Line 3,894:
 
</div>
 
</div>
 
<div class="attendees">Randall de Waard | Elise Grootscholten</div>
 
<div class="attendees">Randall de Waard | Elise Grootscholten</div>
−
<div class="description"><p><p><span style="font-size:14px;">Testing different amounts of urea and sodiumpyruvate to know which concentrations the bacteria survive.</span></p>
+
<div class="description">
−
</p></div>
+
<p><p><span style="font-size:14px;">Testing different amounts of urea and sodiumpyruvate to know which concentrations the bacteria survive.</span></p>
 +
</p>
 +
<p class="experiment"><p><span style="font-size: 16px;"><strong>Experiment 1:&nbsp;Testing different concentrations of Urea and Sodium pyruvate</strong></span><br>
 +
<span style="font-size:14px;"><strong>08-08-18</strong></span><br>
 +
<br>
 +
<span style="font-size:14px;">Testing different amountsof urea and sodiumpyruvate to know which concentrations the bacteria survive.<br>
 +
<br>
 +
-First let the LB agarose heat up (95 degrees Celcius)<br>
 +
-Second, weigh urea and sodium pyruvate in beakers (see table 1)</span></p>
 +
 
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 404px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 122px;"><span style="font-size:14px;"><strong>What</strong></span></td>
 +
<td style="width: 113px;"><span style="font-size:14px;"><strong>How much</strong></span></td>
 +
<td style="width: 153px;"><span style="font-size:14px;"><strong>Solution</strong></span></td>
 +
</tr>
 +
<tr>
 +
<td style="width: 122px;"><span style="font-size:14px;">Urea</span></td>
 +
<td style="width: 113px;"><span style="font-size:14px;">1 g</span></td>
 +
<td style="width: 153px;"><span style="font-size:14px;">1 mL purified water</span></td>
 +
</tr>
 +
<tr>
 +
<td style="width: 122px;"><span style="font-size:14px;">Urea</span></td>
 +
<td style="width: 113px;"><span style="font-size:14px;">2,4 g</span></td>
 +
<td style="width: 153px;"><span style="font-size:14px;">2 mL purified water</span></td>
 +
</tr>
 +
<tr>
 +
<td style="width: 122px;"><span style="font-size:14px;">Urea</span></td>
 +
<td style="width: 113px;"><span style="font-size:14px;">4,8 g</span></td>
 +
<td style="width: 153px;"><span style="font-size:14px;">4 mL purified water</span></td>
 +
</tr>
 +
<tr>
 +
<td style="width: 122px;"><span style="font-size:14px;">Sodium pyruvate </span></td>
 +
<td style="width: 113px;"><span style="font-size:14px;">5,7 mL</span></td>
 +
<td style="width: 153px;"><span style="font-size:14px;">None</span></td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
 
 +
<p><span style="font-size:14px;"><em>Table 1: weighing urea and sodium pyruvate for LB agarose plates.</em><br>
 +
-Purify these substances, so it is free of bacteria. Work&nbsp;beneath&nbsp;the flame and pour it in to plastic 50 mL tubes.<br>
 +
-Pour the LB agarose in the plastic tubes when it's about 45 degrees Celcius and mix gently.<br>
 +
-Pour the LB agarose mix in a petridish and wait until it's dry. Do this for all the plastic tubes.<br>
 +
-Also fill 2 petridishes with LB agarose without other substances (positive and negative control).<br>
 +
-prick a BL21(DE3) bacteria and put it in a 100 mL erlenmeyer with 20 mL culture medium.<br>
 +
<br>
 +
<strong>09-08-18</strong><br>
 +
<br>
 +
Pipette 100 ul overnight culture on each plate,&nbsp;except on&nbsp;the negative control,&nbsp;and divide it.&nbsp;(24h 37 degrees Celcius)</span><br>
 +
<br>
 +
&nbsp;</p>
 +
 
 +
<p><span style="font-size:14px;"><strong>Results</strong></span><br>
 +
&nbsp;</p>
 +
 
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 526px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 123px;"><span style="font-size:14px;"><strong>What</strong></span></td>
 +
<td style="width: 95px;"><span style="font-size:14px;"><strong>How much</strong></span></td>
 +
<td style="width: 170px;"><span style="font-size:14px;"><strong>Solution</strong></span></td>
 +
<td style="width: 117px;"><span style="font-size:14px;"><strong>Result</strong></span></td>
 +
</tr>
 +
<tr>
 +
<td style="width: 123px;"><span style="font-size:14px;">Urea</span></td>
 +
<td style="width: 95px;"><span style="font-size:14px;">1 g</span></td>
 +
<td style="width: 170px;"><span style="font-size:14px;">1 mL purified water</span></td>
 +
<td style="width: 117px;"><span style="font-size:14px;">Grow</span></td>
 +
</tr>
 +
<tr>
 +
<td style="width: 123px;"><span style="font-size:14px;">Urea</span></td>
 +
<td style="width: 95px;"><span style="font-size:14px;">2,4 g</span></td>
 +
<td style="width: 170px;"><span style="font-size:14px;">2 mL purified water</span></td>
 +
<td style="width: 117px;"><span style="font-size:14px;">No grow</span></td>
 +
</tr>
 +
<tr>
 +
<td style="width: 123px;"><span style="font-size:14px;">Urea</span></td>
 +
<td style="width: 95px;"><span style="font-size:14px;">4,8 g</span></td>
 +
<td style="width: 170px;"><span style="font-size:14px;">4 mL purified water</span></td>
 +
<td style="width: 117px;"><span style="font-size:14px;">No grow</span></td>
 +
</tr>
 +
<tr>
 +
<td style="width: 123px;"><span style="font-size:14px;">Sodium pyruvate</span></td>
 +
<td style="width: 95px;"><span style="font-size:14px;">5,7 mL</span></td>
 +
<td style="width: 170px;"><span style="font-size:14px;">-</span></td>
 +
<td style="width: 117px;"><span style="font-size:14px;">Grow</span></td>
 +
</tr>
 +
<tr>
 +
<td style="width: 123px;"><span style="font-size:14px;">Positive control</span></td>
 +
<td style="width: 95px;"><span style="font-size:14px;">-</span></td>
 +
<td style="width: 170px;"><span style="font-size:14px;">-</span></td>
 +
<td style="width: 117px;"><span style="font-size:14px;">Grow</span></td>
 +
</tr>
 +
<tr>
 +
<td style="width: 123px;"><span style="font-size:14px;">Negative control</span></td>
 +
<td style="width: 95px;"><span style="font-size:14px;">-</span></td>
 +
<td style="width: 170px;"><span style="font-size:14px;">-</span></td>
 +
<td style="width: 117px;"><span style="font-size:14px;">No grow</span></td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
 
 +
<p><span style="font-size:14px;"><em>Table 1: Results experiment 1</em><br>
 +
<br>
 +
<strong>Conclusion</strong><br>
 +
<br>
 +
More tests will be done with urea and sodiumpyruvate because with 1 g urea the bacteria live and with 2,4 g urea the bacteria die.<br>
 +
The bacteria survive 5,7 mL sodiumpyruvate so we will test with higher concentrations.</span><br>
 +
&nbsp;</p>
 +
</p>
 +
</div>
 
</div>
 
</div>
  
Line 3,247: Line 4,047:
 
</div>
 
</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
−
<div class="description"><p><p>Transformations of the biobricks (K133071 + J23100), (K13071 + I1345), (K173003 + J23100) and (K173003 + I13453) in NEB10B�ta.&nbsp;There was no grow except for the biobrick combination K133071 + J23100.</p>
+
<div class="description">
−
</p></div>
+
<p><p>Transformations of the biobricks (K133071 + J23100), (K13071 + I1345), (K173003 + J23100) and (K173003 + I13453) in NEB10B�ta.&nbsp;There was no grow except for the biobrick combination K133071 + J23100.</p>
 +
</p>
 +
<p class="experiment"><p><span style="font-size: 16px;"><strong>Experiment 5: Transformation</strong></span></p>
 +
 
 +
<p><span style="color:#000000;"><span style="font-size:12px;">13-08-2018<br>
 +
Transformations of the ligations of experiment 4</span></span><br>
 +
<br>
 +
<strong style="color: rgb(0, 0, 0); font-family: arial, helvetica, sans-serif;">Materials</strong></p>
 +
 
 +
<p><span style="font-family:arial,helvetica,sans-serif;"><span style="color:#000000;">-Chloramphenicol (40 mg/mL)<br>
 +
-LB-agar<br>
 +
-Ligations<br>
 +
-Heat block,42 degrees Celcius<br>
 +
-Shaker, 37 degrees Celsius&nbsp;<br>
 +
-Chemo competent cells NEB10b�ta (New England Biolabs)</span></span><br>
 +
<br>
 +
<strong>Methods</strong><br>
 +
<br>
 +
10 ul ligationmix is used for every transformation</p>
 +
 
 +
<h1><span style="font-size: 16px;">Transformation Protocol</span><br>
 +
<span style="font-size: 14px;"><span style="color: rgb(0, 0, 0);">Overview</span></span></h1>
 +
 
 +
<p><span style="font-size: 14px;"><span style="color: rgb(0, 0, 0);"><span style="font-family: arial,helvetica,sans-serif;">Quick Ligation products may be transformed by many different methods. The following protocol is recommended by New England Biolabs.</span></span></span></p>
 +
 
 +
<section class="paragraph  ">
 +
<h2><span style="font-size: 14px;">Protocol</span></h2>
 +
 
 +
<ol>
 +
<li>Thaw competent cells on ice.</li>
 +
<li>Chill approximately 5 ng (2 &mu;l) of the ligation mixture in a 1.5 ml microcentrifuge tube.</li>
 +
<li>Add 50 &mu;l of competent cells to the DNA. Mix gently by pipetting up and down or flicking the tube 4�5 times to mix the cells and DNA. Do not vortex.</li>
 +
<li>Place the mixture on ice for 30 minutes. Do not mix.</li>
 +
<li>Heat shock at 42&deg;C for 30 seconds*. Do not mix.</li>
 +
<li>Add 950 &mu;l of room temperature media* to the tube.</li>
 +
<li>Place tube at 37&deg;C for 60 minutes. Shake vigorously (250 rpm) or rotate.</li>
 +
<li>Warm selection plates to 37&deg;C.</li>
 +
<li>Spread 50�100 &mu;l of the cells and ligation mixture onto the plates.</li>
 +
<li>Incubate overnight at 37&deg;C.
 +
<p>* Please note: For the duration and temperature of the heat shock step as well as for the media to be used during the recovery period, please follow the recommendations provided by the competent cells� manufacturer.</p>
 +
</li>
 +
</ol>
 +
</section>
 +
 
 +
<p><br>
 +
Instead of 2 ul of the ligation mix, use 10 ul ligation mix.</p>
 +
</p>
 +
</div>
 
</div>
 
</div>
  
Line 3,289: Line 4,136:
 
</div>
 
</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
−
<div class="description"><p><p>A colony PCR is done for the NEB10B�ta E.coli cells with expected&nbsp;the biobrick combination of K133071 with J23100. Nevertheless, on a gel the difference with and without promotor couldn't be seen. So there must be another way of proving the right biobricks are there.</p>
+
<div class="description">
−
</p></div>
+
<p><p>A colony PCR is done for the NEB10B�ta E.coli cells with expected&nbsp;the biobrick combination of K133071 with J23100. Nevertheless, on a gel the difference with and without promotor couldn't be seen. So there must be another way of proving the right biobricks are there.</p>
 +
</p>
 +
<p class="experiment"><p><span style="font-size: 14px;"><strong>Expermiment 6: Colony PCR K133071 + J23100</strong></span><br>
 +
<br>
 +
Mastermix</p>
 +
 
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 470px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 163px;"><strong>What</strong></td>
 +
<td style="width: 147px;"><strong>1 reaction</strong></td>
 +
<td style="width: 146px;"><strong>30 reactions</strong></td>
 +
</tr>
 +
<tr>
 +
<td style="width: 163px;">Taq buffer(10x)</td>
 +
<td style="width: 147px;">5 ul</td>
 +
<td style="width: 146px;">150 ul</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 163px;">10 mM dntp</td>
 +
<td style="width: 147px;">1 ul</td>
 +
<td style="width: 146px;">30 ul</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 163px;">10 uM Forward primer</td>
 +
<td style="width: 147px;">1 ul</td>
 +
<td style="width: 146px;">30 ul</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 163px;">10 uM Reverse primer</td>
 +
<td style="width: 147px;">1 ul</td>
 +
<td style="width: 146px;">30 ul</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 163px;">Taq polymerase</td>
 +
<td style="width: 147px;">0,25 ul</td>
 +
<td style="width: 146px;">7,5 ul</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 163px;">Nucease free hydrogen</td>
 +
<td style="width: 147px;">41,75 ul</td>
 +
<td style="width: 146px;">1252,5 ul</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 163px;"><strong>Total</strong></td>
 +
<td style="width: 147px;"><strong>50 ul</strong></td>
 +
<td style="width: 146px;"><strong>1500 ul</strong></td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
 
 +
<p>&nbsp;</p>
 +
</p>
 +
</div>
 
</div>
 
</div>
  
Line 3,331: Line 4,231:
 
</div>
 
</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
−
<div class="description"><p>The ligations and transformations of the biobricks K173003 + J23100, K173003 + I13453, and K133071 + I13453 has been performed again.&nbsp;There was a lot of grow on the agar plates. To know&nbsp;if the biobricks were right ligated this was done by testing practically.&nbsp;See the experiments:&nbsp;<em>Testing gas production.</em>&nbsp;</p></div>
+
<div class="description">
 +
<p>The ligations and transformations of the biobricks K173003 + J23100, K173003 + I13453, and K133071 + I13453 have been performed again.&nbsp;There was a lot of grow on the agar plates. To know&nbsp;if the biobricks were right ligated this was done by testing practically.&nbsp;See the experiments:&nbsp;<em>Testing gas production.</em>&nbsp;</p>
 +
<p class="experiment"><p><span style="font-size: 14px;"><strong>Experiment 7: Ligation of biobricks</strong></span><br>
 +
K173003 + J23100<br>
 +
K173003 + I13453<br>
 +
K133071 + I13453<br>
 +
&nbsp;</p>
 +
 
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 300px;">
 +
<thead>
 +
<tr>
 +
<th scope="col" style="width: 79px;">Volume</th>
 +
<th scope="col" style="width: 213px;">Compound</th>
 +
</tr>
 +
</thead>
 +
<tbody>
 +
<tr>
 +
<td style="width: 67px;">6&nbsp;&mu;L</td>
 +
<td style="width: 201px;">vector DNA (0,5&nbsp;&mu;g/30&nbsp;&mu;L) (100 ng)</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 67px;">14&nbsp;&mu;L</td>
 +
<td style="width: 201px;">insert DNA (2&nbsp;&mu;g/100 &mu;L) (280 ng)</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 67px;">4&nbsp;&mu;L</td>
 +
<td style="width: 201px;">dilutionbuffer (5x)</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 67px;">1&nbsp;&mu;L</td>
 +
<td style="width: 201px;">milli Q water</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 67px;">25&nbsp;&mu;L</td>
 +
<td style="width: 201px;">Total</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
 
 +
<p><br>
 +
Use 10 mL<br>
 +
<br>
 +
Ligationmix:</p>
 +
 
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 300px;">
 +
<thead>
 +
<tr>
 +
<th scope="col" style="width: 79px;">Volume</th>
 +
<th scope="col" style="width: 213px;">Compound</th>
 +
</tr>
 +
</thead>
 +
<tbody>
 +
<tr>
 +
<td style="width: 67px;">10&nbsp;&mu;L</td>
 +
<td style="width: 201px;">pre-ligationmix&nbsp;</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 67px;">2&nbsp;&mu;L</td>
 +
<td style="width: 201px;">10x T4 ligationbuffer</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 67px;">1&nbsp;&mu;L</td>
 +
<td style="width: 201px;">T4 DNA ligase (1U/&mu;L)</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 67px;">8&nbsp;&mu;L</td>
 +
<td style="width: 201px;">milli Q water</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 67px;">20&nbsp;&mu;L</td>
 +
<td style="width: 201px;">Total</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
 
 +
<p>incubation at room-temperature for 20 minutes.<br>
 +
Store at -20 degrees Celcius</p>
 +
</p>
 +
</div>
 
</div>
 
</div>
  
Line 3,372: Line 4,350:
 
</div>
 
</div>
 
<div class="attendees">Elise Grootscholten | Paul Reusink</div>
 
<div class="attendees">Elise Grootscholten | Paul Reusink</div>
−
<div class="description"><p><p>Making a set up for the gas production testing and testing it with NEB10B�ta with the expected biobricks in it (K133071 + J23100) and a negative control.</p>
+
<div class="description">
−
</p></div>
+
<p><p>Making a set up for the gas production testing and testing it with NEB10B�ta with the expected biobricks in it (K133071 + J23100) and a negative control.</p>
 +
</p>
 +
<p class="experiment"><p><span style="font-size: 14px;"><strong>Experiment 1: Gasproduction testing</strong></span><br>
 +
<br>
 +
<strong>15-08-18</strong><br>
 +
Making a set up for the gas production testing and testing it with NEB10B�ta with the expected biobricks in it (K133071 + J23100) and a negative control.<br>
 +
<br>
 +
<strong>Materials</strong><br>
 +
-15 mL tubes<br>
 +
-Little glass tubes<br>
 +
-Cocktail prickers<br>
 +
-Culture medium<br>
 +
-Urea<br>
 +
-Chloramphenicol (40 mg/mL)<br>
 +
<br>
 +
<br>
 +
<strong>Methods</strong><br>
 +
Stick tape on the little tube and cover the little tubes with&nbsp;tin foil. This can now enter the autoclave.<br>
 +
Make overnight cultures (20 mL)of the desired colonies in culture medium with 17,5 ul/ 20mL Chloramphenicol.<br>
 +
<br>
 +
After a day, prepare the culture medium for the tests.<br>
 +
Put 1 g urea and 17,5 ul Chloramphenicol in 20 mL culture medium.<br>
 +
<br>
 +
Pipette&nbsp;10 mL of the overnight culture in the 15 mL tube en centrifuge&nbsp;5000 rpm fot 5 minutes. Throw the supernatant away.<br>
 +
Fill the 15 mL tube and the little tube to the edge with culture medium. Work sterile.<br>
 +
Put a paper on the little tube and turn it around.<br>
 +
Then hold it above the 15 mL tube and pull the paper away and let the little tube fall into the 15 mL tube.<br>
 +
Try to do this so there's no gas in the little tube.<br>
 +
Now wait until gas is produced.<br>
 +
<br>
 +
<strong>16-08-18</strong><br>
 +
Making a set up for the gas production testing and testing it with NEB10B�ta with the expected biobricks in it (K133071 + J23100).<br>
 +
20 different colonies are tested.<br>
 +
<br>
 +
<strong>Materials</strong><br>
 +
Use the same materials as above.<br>
 +
<br>
 +
<strong>Methods</strong><br>
 +
Use the protocol above.<br>
 +
Make 700 mL culture medium with 35&nbsp;g urea and 612,5 ul&nbsp;Chloramphenicol&nbsp;(40 mg/mL). This cannot be done sterile.<br>
 +
The culture medium can be used for the 20 different colonies with expected biobricks: K133071 + J23100.</p>
 +
 
 +
<p>&nbsp;<br>
 +
<br>
 +
<br>
 +
Results<br>
 +
<br>
 +
15-08-18<br>
 +
Gas was let into the little tubes so it was difficult to see whether there was gasproduction or not. Mainly in the negative control this was&nbsp;<br>
 +
<br>
 +
&nbsp;</p>
 +
</p>
 +
</div>
 
</div>
 
</div>
  
Line 3,414: Line 4,444:
 
</div>
 
</div>
 
<div class="attendees">Elise Grootscholten | Paul Reusink</div>
 
<div class="attendees">Elise Grootscholten | Paul Reusink</div>
−
<div class="description"><p><p>Making a set up for the gas production testing and testing it with NEB10B�ta with the expected biobricks in it (K133071 + I13453), (K173003 + J23100), (K173003 + I13453)&nbsp;and a negative control.</p>
+
<div class="description">
−
</p></div>
+
<p><p>Making a set up for the gas production testing and testing it with NEB10B�ta with the expected biobricks in it (K133071 + I13453), (K173003 + J23100), (K173003 + I13453)&nbsp;and a negative control.</p>
 +
</p>
 +
<p class="experiment"><p><span style="font-size: 14px;"><strong>Experiment 2: Gasproduction testing </strong></span><br>
 +
<strong>20-08-18</strong><br>
 +
<br>
 +
Making a set up for the gas production testing and testing it with NEB10B�ta with the expected biobricks in it (K133071 + I13453), (K173003 + J23100), (K173003 + I13453)&nbsp;and a negative control<br>
 +
<br>
 +
<strong>Materials</strong><br>
 +
See materials Experiment 1<br>
 +
<br>
 +
<strong>Methods</strong><br>
 +
See methods Experiment 1<br>
 +
850 mL culture medium is made with 21,5 g LB for 42 erlenmeyers (also for CooA production).<br>
 +
Overnight cultures are made of 10 colonies each of K173003&nbsp;+ I13453 and K173003 + J23100.<br>
 +
<br>
 +
<strong>21-08-18</strong><br>
 +
<br>
 +
K173003 + J23100 are tested (see methods experiment 1)<br>
 +
Overnight cultures are made of&nbsp; K133071 + I13453 and K173003 + I13453 are tested later.<br>
 +
<br>
 +
<strong>Results</strong><br>
 +
Colonies 9 and 10 are chosen use for further tests.</p>
 +
</p>
 +
</div>
 
</div>
 
</div>
  
Line 3,456: Line 4,509:
 
</div>
 
</div>
 
<div class="attendees">Elise Grootscholten | Paul Reusink</div>
 
<div class="attendees">Elise Grootscholten | Paul Reusink</div>
−
<div class="description"><p><p>Gasproduction testing for the biobricks (K173003 + I13453), (K133071 + I13453) and negative controls (B0015 and K133071 without urea and arabinose).<br>
+
<div class="description">
 +
<p><p>Gasproduction testing for the biobricks (K173003 + I13453), (K133071 + I13453) and negative controls (B0015 and K133071 without urea and arabinose).<br>
 
K173003 + I13453 is tested with sodiumpyruvate and arabinose for gasproduction and&nbsp;K133071 + I13453&nbsp;is tested with urea and arabinose for gasproduction.<br>
 
K173003 + I13453 is tested with sodiumpyruvate and arabinose for gasproduction and&nbsp;K133071 + I13453&nbsp;is tested with urea and arabinose for gasproduction.<br>
 
The negative control also produces a little bit gas.</p>
 
The negative control also produces a little bit gas.</p>
−
</p></div>
+
</p>
 +
<p class="experiment"><p><span style="font-size:14px;"><strong>Experiment 3: Gasproduction testing</strong></span><br>
 +
<br>
 +
Gasproduction testing for the biobricks K173003 + I13453,&nbsp;K133071 + I13453 and negative controls.<br>
 +
K173003 + I13453 is tested with sodiumpyruvate and arabinose for gasproduction and&nbsp;K133071 + I13453&nbsp;is tested with urea and arabinose for gasproduction.<br>
 +
<br>
 +
<strong>Materials</strong><br>
 +
See experiment 1 and 2.<br>
 +
<br>
 +
<strong>Methods</strong><br>
 +
See experiment 1 and 2.<br>
 +
375 mL culture medium with CAM (already in it) + 18,75&nbsp;g urea + 0,5 ul arabinose.<br>
 +
340 mL culture medium with CAM (already in it) + 51 mL natriumpyruvate (20 mM) + 0,5 ul arabinose.<br>
 +
<br>
 +
<br>
 +
<strong>Results and conclusion</strong><br>
 +
Colonies 5 and 7 of the biobricks K173003 + I13453 (pyruvate)&nbsp; and number 4 of the biobricks K133071 + I13453 (urea).<br>
 +
In both the negative controls there's been gas produced. Futher gasproduction test will be done.</p>
 +
</p>
 +
</div>
 
</div>
 
</div>
  
Line 3,500: Line 4,573:
 
</div>
 
</div>
 
<div class="attendees">Elise Grootscholten | Paul Reusink</div>
 
<div class="attendees">Elise Grootscholten | Paul Reusink</div>
−
<div class="description"><p><p>Testing the gasproduction&nbsp;of the colonies&nbsp;9 and 10 of biobricks (K173003 + J23100), colonies 5 and 7 of biobricks (K173003 + I13453) and colony 4 of&nbsp;biobricks (K133071 + I13453) with and without centrifuging the bacteria. There is also a negative control&nbsp;(J04450 pSB1K3) with Kanamycine. The negative contol started with a lot of gas inside the tube. We can not see wether there is produced more after a day or not. This have to be tested later.</p>
+
<div class="description">
−
</p></div>
+
<p><p>Testing the gasproduction&nbsp;of the colonies&nbsp;9 and 10 of biobricks (K173003 + J23100), colonies 5 and 7 of biobricks (K173003 + I13453) and colony 4 of&nbsp;biobricks (K133071 + I13453) with and without centrifuging the bacteria. There is also a negative control&nbsp;(J04450 pSB1K3) with Kanamycine. The negative contol started with a lot of gas inside the tube. We can not see wether there is produced more after a day or not. This have to be tested later.</p>
 +
</p>
 +
<p class="experiment"><p><span style="font-size:14px;"><strong>Experiment 4: Gasproduction testing&nbsp;</strong></span><br>
 +
24-08-18<br>
 +
<br>
 +
Testing the gasproduction with and without centrifuging the bacteria.<br>
 +
<br>
 +
<strong>methods</strong><br>
 +
See methods experiment 1<br>
 +
2 negative controls<br>
 +
colonies 9 and 10 of biobricks&nbsp;K173003 + J23100.<br>
 +
Colonies 5 and 7 of biobricks&nbsp;K173003 + I13453<br>
 +
Colony 4 of&nbsp;biobricks K133071 + I13453<br>
 +
<br>
 +
Culture medium:<br>
 +
150 mL: Sodiumpyruvate (20 mM) 22,5 mL +&nbsp;Chloramphenicol (40 mg/mL) 131 ul<br>
 +
150 mL: Sodiumpyruvate (20mM) 22,5 mL +&nbsp;&nbsp;Chloramphenicol (40 mg/mL) 131 ul + Arabinose (300 mg/mL) 0,5 ul<br>
 +
80 mL: Urea 4 g +&nbsp;Chloramphenicol (40 mg/mL) 70 ul +&nbsp;Arabinose (300 mg/mL) 0,5 ul<br>
 +
80 mL: Kanamycine (40 mg/mL) 70 ul<br>
 +
<br>
 +
&nbsp;</p>
 +
</p>
 +
</div>
 
</div>
 
</div>
  
Line 3,542: Line 4,637:
 
</div>
 
</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
−
<div class="description"><p><p>Minipreps are made of colonies 11 and 19 of biobrick combination&nbsp;K133071 + J23100.&nbsp;Results: 11. 270,57 ng/ul&nbsp;19. 253,59 ng/ul.</p>
+
<div class="description">
−
</p></div>
+
<p><p>Minipreps are made of colonies 11 and 19 of biobrick combination&nbsp;K133071 + J23100.&nbsp;Results: 11. 270,57 ng/ul&nbsp;19. 253,59 ng/ul.</p>
 +
</p>
 +
<p class="experiment"><p><span style="font-size:14px;"><strong>Minipreps</strong></span><br>
 +
21-08-18<br>
 +
<br>
 +
Minipreps of colonies 11 and 19 (see Testing gasproduction experiment 1)<br>
 +
<br>
 +
<strong>Materials</strong><br>
 +
See experiment 3<br>
 +
<br>
 +
<strong>Methods&nbsp;</strong><br>
 +
See experiment 3<br>
 +
<br>
 +
<strong>Results</strong><br>
 +
<br>
 +
11. K133071 + J23100: 270,57 ng/ul<br>
 +
19. K133071 + J23100: 253,59 ng/ul</p>
 +
</p>
 +
</div>
 
</div>
 
</div>
  
Line 3,584: Line 4,697:
 
</div>
 
</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
−
<div class="description"><p><p>A miniprep of the biobrick K352002 is made. The concentration is 69,77 ng/ul.</p>
+
<div class="description">
−
</p></div>
+
<p><p>A miniprep of the biobrick K352002 is made. The concentration is 69,77 ng/ul.</p>
 +
</p>
 +
<p class="experiment"><p><strong>Experiment 9: Minipreps</strong><br>
 +
28-08-18<br>
 +
<br>
 +
<strong>Materials</strong><br>
 +
See experiment 3<br>
 +
<br>
 +
<strong>Methods</strong><br>
 +
See experiment 3<br>
 +
Make minipreps of the biobricks K352002, K352003<br>
 +
<br>
 +
<strong>Results</strong><br>
 +
See figure 1<br>
 +
<br>
 +
29-08-18<br>
 +
<strong>Materials</strong><br>
 +
See experiment 3<br>
 +
<br>
 +
<strong>Methods</strong><br>
 +
See experiment 3<br>
 +
Make minipreps of the biobrick&nbsp;K352002<br>
 +
<br>
 +
<strong>Results</strong><br>
 +
See figure 2<br>
 +
<br>
 +
30-08-18<br>
 +
<strong>Materials</strong><br>
 +
See experiment 3<br>
 +
<br>
 +
<strong>Methods</strong><br>
 +
See experiment 3<br>
 +
Make minipreps of the biobricks K133116 and K173013<br>
 +
<br>
 +
<strong>Results</strong><br>
 +
See image experiment 9</p>
 +
</p>
 +
</div>
 
</div>
 
</div>
  
Line 3,626: Line 4,776:
 
</div>
 
</div>
 
<div class="attendees">Randall de Waard | Elise Grootscholten</div>
 
<div class="attendees">Randall de Waard | Elise Grootscholten</div>
−
<div class="description"><p><p><span style="font-size:14px;">Testing different amounts of urea and sodiumpyruvate to know which concentrations the bacteria survive.</span></p>
+
<div class="description">
−
</p></div>
+
<p><p><span style="font-size:14px;">Testing different amounts of urea and sodiumpyruvate to know which concentrations the bacteria survive.</span></p>
 +
</p>
 +
<p class="experiment"><p><span style="font-size: 16px;"><strong>Experiment 2:&nbsp;</strong></span><strong><span style="font-size:16px;">Testing different concentrations of Urea and Sodium pyruvate</span></strong><br>
 +
<strong><span style="font-size:16px;">13-08-18</span></strong><br>
 +
<br>
 +
<br>
 +
<span style="font-size:14px;">Same testing like experiment 1 but with different concentrations.</span></p>
 +
 
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 420px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 163px;"><span style="font-size: 14px;"><strong>What</strong></span></td>
 +
<td style="width: 84px;"><span style="font-size: 14px;"><strong>How much</strong></span></td>
 +
<td style="width: 157px;"><span style="font-size: 14px;"><strong>Solution</strong></span></td>
 +
</tr>
 +
<tr>
 +
<td style="width: 163px;"><span style="font-size: 14px;">Urea</span></td>
 +
<td style="width: 84px;"><span style="font-size: 14px;">1,2 g</span></td>
 +
<td style="width: 157px;"><span style="font-size: 14px;">1 mL purified water</span></td>
 +
</tr>
 +
<tr>
 +
<td style="width: 163px;"><span style="font-size: 14px;">Urea</span></td>
 +
<td style="width: 84px;"><span style="font-size: 14px;">1,6 g</span></td>
 +
<td style="width: 157px;"><span style="font-size: 14px;">2 mL purified water</span></td>
 +
</tr>
 +
<tr>
 +
<td style="width: 163px;"><span style="font-size: 14px;">Sodium pyruvate (10 mM)</span></td>
 +
<td style="width: 84px;"><span style="font-size: 14px;">10 mL</span></td>
 +
<td style="width: 157px;"><span style="font-size: 14px;">None</span></td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<br>
 +
<br>
 +
<span style="font-size:14px;"><strong>Results</strong></span><br>
 +
&nbsp;
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 564px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 198px;"><span style="font-size:14px;"><strong>What</strong></span></td>
 +
<td style="width: 93px;"><span style="font-size:14px;"><strong>How much</strong></span></td>
 +
<td style="width: 158px;"><span style="font-size:14px;"><strong>Solution</strong></span></td>
 +
<td style="width: 93px;"><strong><span style="font-size:14px;">Result</span></strong></td>
 +
</tr>
 +
<tr>
 +
<td style="width: 198px;"><span style="font-size:14px;">Urea</span></td>
 +
<td style="width: 93px;"><span style="font-size:14px;">1,2 g</span></td>
 +
<td style="width: 158px;"><span style="font-size:14px;">1 mL purified water</span></td>
 +
<td style="width: 93px;">Little grow</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 198px;"><span style="font-size:14px;">Urea</span></td>
 +
<td style="width: 93px;"><span style="font-size:14px;">1,6 g</span></td>
 +
<td style="width: 158px;"><span style="font-size:14px;">2 mL purified water</span></td>
 +
<td style="width: 93px;">No grow</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 198px;"><span style="font-size:14px;">Sodium pyruvate (10 mM)</span></td>
 +
<td style="width: 93px;"><span style="font-size:14px;">10 mL</span></td>
 +
<td style="width: 158px;"><span style="font-size: 14px;">-</span></td>
 +
<td style="width: 93px;">Grow</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 198px;"><span style="font-size:14px;">Positive control</span></td>
 +
<td style="width: 93px;"><span style="font-size:14px;">-</span></td>
 +
<td style="width: 158px;"><span style="font-size:14px;">-</span></td>
 +
<td style="width: 93px;">Grow</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 198px;"><span style="font-size:14px;">Negative control</span></td>
 +
<td style="width: 93px;"><span style="font-size:14px;">-</span></td>
 +
<td style="width: 158px;"><span style="font-size:14px;">-</span></td>
 +
<td style="width: 93px;">No Grow</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<em>Table 2: Results experiment 2</em><br>
 +
<br>
 +
<span style="font-size:14px;"><strong>Conclusion</strong><br>
 +
<br>
 +
For urea is 1 g used/20 mL and for sodiumpyruvate is 6 mL (10mM) or 3 mL (20 mM) used.</span><br>
 +
&nbsp;</p>
 +
</div>
 
</div>
 
</div>
  
Line 3,668: Line 4,900:
 
</div>
 
</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
−
<div class="description"><p><p>Digestions, gelelectrophoresis, dephosphorylations and ligations of different biobrick combinations. These biobrick combinations are 4 different promotors (J23100, I13453, K352002, K352003)&nbsp; with 4 different gasproduction biobricks (k173003, K173013, K133071, K133116). J23100 is about 1 kb to long. The rest seems likely to be right digested. The ligations will be transformed in NEB10b�ta and digested again as control.</p>
+
<div class="description">
−
</p></div>
+
<p><p>Digestions, gelelectrophoresis, dephosphorylations and ligations of different biobrick combinations. These biobrick combinations are 4 different promoters (J23100, I13453, K352002, K352003)&nbsp; with 4 different gasproduction biobricks (k173003, K173013, K133071, K133116). J23100 is about 1 kb to long. The rest seems likely to be right digested. The ligations will be transformed in NEB10b�ta and digested again as control.</p>
 +
</p>
 +
<p class="experiment"><p><span style="font-size:14px;"><strong>Experiment 10: Digestion, defosforylation and ligation</strong></span><br>
 +
<br>
 +
<strong>Materials</strong><br>
 +
See experiment...<br>
 +
<br>
 +
<strong>Methods</strong><br>
 +
See experiment ..</p>
 +
 
 +
<p>&nbsp;</p>
 +
 
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 346px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 221px;">DNA (J23100 325,59 ng/&mu;l)&nbsp;~ 2 &mu;g</td>
 +
<td style="width: 114px;">6,1</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Cutsmart (10x)</td>
 +
<td style="width: 114px;">5 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Restriction enzyme 1 (1 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Restriction enzyme 2 (1 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Nuclease free water</td>
 +
<td style="width: 114px;">Fill to 50 &mu;L</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
 
 +
<p><em>Table 1: Digestion scheme 1</em><br>
 +
&nbsp;</p>
 +
 
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 346px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 221px;">DNA (J23100 325,59 ng/&mu;l)&nbsp;~ 2 &mu;g</td>
 +
<td style="width: 114px;">6,1</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Cutsmart (10x)</td>
 +
<td style="width: 114px;">5 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Restriction enzyme 1 (1 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Nuclease free water</td>
 +
<td style="width: 114px;">Fill to 50 &mu;L</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<em>Table 2: Digestion scheme 2</em><br>
 +
&nbsp;
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 346px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 221px;">DNA (J23100 325,59 ng/&mu;l)&nbsp;~ 2 &mu;g</td>
 +
<td style="width: 114px;">6,1</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Cutsmart (10x)</td>
 +
<td style="width: 114px;">5 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Restriction enzyme 1 (1 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Nuclease free water</td>
 +
<td style="width: 114px;">Fill to 50 &mu;L</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<em>Table 3: Digestion scheme 3</em><br>
 +
&nbsp;
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 351px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 222px;">DNA (K352003 180,65 ng/&mu;l)&nbsp;~ 2 &mu;g</td>
 +
<td style="width: 117px;">11,1&nbsp;&mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 222px;">Cutsmart (10x)</td>
 +
<td style="width: 117px;">5 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 222px;">Restriction enzyme 1 (1 U/&mu;L)</td>
 +
<td style="width: 117px;">2 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 222px;">Restriction enzyme 2 (1 U/&mu;L)</td>
 +
<td style="width: 117px;">2 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 222px;">Nuclease free water</td>
 +
<td style="width: 117px;">Fill to 50 &mu;L</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<em>Table 4: Digestion scheme 4</em><br>
 +
&nbsp;
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 351px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 222px;">DNA (K352002 69,77 ng/&mu;l)&nbsp;~ 2 &mu;g</td>
 +
<td style="width: 117px;">28,7 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 222px;">Cutsmart (10x)</td>
 +
<td style="width: 117px;">5 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 222px;">Restriction enzyme 1 (1 U/&mu;L)</td>
 +
<td style="width: 117px;">2 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 222px;">Restriction enzyme 2 (1 U/&mu;L)</td>
 +
<td style="width: 117px;">2 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 222px;">Nuclease free water</td>
 +
<td style="width: 117px;">Fill to 50 &mu;L</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<em>Table 5: Digestion scheme 5</em><br>
 +
&nbsp;
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 352px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 224px;">DNA (K173003 217,06 ng/&mu;L)&nbsp;~ 2 &mu;g</td>
 +
<td style="width: 116px;">9,2&nbsp;&mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 224px;">Cutsmart (10x)</td>
 +
<td style="width: 116px;">5 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 224px;">Restriction enzyme 1 (1 U/&mu;L)</td>
 +
<td style="width: 116px;">2 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 224px;">Restriction enzyme 2 (1 U/&mu;L)</td>
 +
<td style="width: 116px;">2 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 224px;">Nuclease free water</td>
 +
<td style="width: 116px;">Fill to 50 &mu;L</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<em>Table 6: Digestion scheme 6</em><br>
 +
&nbsp;
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 359px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 227px;">DNA (I13453 88,18 ng/&mu;L)&nbsp;~ 2 &mu;g</td>
 +
<td style="width: 121px;">22,7&nbsp;&mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 227px;">Cutsmart (10x)</td>
 +
<td style="width: 121px;">5 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 227px;">Restriction enzyme 1 (1 U/&mu;L)</td>
 +
<td style="width: 121px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 227px;">Restriction enzyme 2 (1 U/&mu;L)</td>
 +
<td style="width: 121px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 227px;">Nuclease free water</td>
 +
<td style="width: 121px;">Fill to 50 &mu;L</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
&nbsp;<em>Table 7: Digestion scheme 7</em><br>
 +
&nbsp;
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 362px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 228px;">DNA (K133071 186,79&nbsp;ng/&mu;L)&nbsp;~ 2 &mu;g</td>
 +
<td style="width: 122px;">10,7&nbsp;&mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 228px;">Cutsmart (10x)</td>
 +
<td style="width: 122px;">5 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 228px;">Restriction enzyme 1 (1 U/&mu;L)</td>
 +
<td style="width: 122px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 228px;">Restriction enzyme 2 (1 U/&mu;L)</td>
 +
<td style="width: 122px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 228px;">Nuclease free water</td>
 +
<td style="width: 122px;">Fill to 50 &mu;L</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
&nbsp;<em>Table 8: Digestion scheme 8</em><br>
 +
&nbsp;
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 358px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 228px;">DNA (pSB1K3 163,93 ng/&mu;L)&nbsp;~ 2 &mu;g</td>
 +
<td style="width: 118px;">&nbsp;12,2 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 228px;">Cutsmart (10x)</td>
 +
<td style="width: 118px;">5 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 228px;">Restriction enzyme 1 (1 U/&mu;L)</td>
 +
<td style="width: 118px;">2 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 228px;">Restriction enzyme 2 (1 U/&mu;L)</td>
 +
<td style="width: 118px;">2 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 228px;">Nuclease free water</td>
 +
<td style="width: 118px;">Fill to 50 &mu;L</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<em>Table 9: Digestion scheme 9</em><br>
 +
&nbsp;
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 357px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 222px;">DNA (K173013 345,1 ng/&mu;l)&nbsp;~ 2 &mu;g</td>
 +
<td style="width: 123px;">&nbsp;5,8 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 222px;">Cutsmart (10x)</td>
 +
<td style="width: 123px;">5 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 222px;">Restriction enzyme 1 (1 U/&mu;L)</td>
 +
<td style="width: 123px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 222px;">Restriction enzyme 2 (1 U/&mu;L)</td>
 +
<td style="width: 123px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 222px;">Nuclease free water</td>
 +
<td style="width: 123px;">Fill to 50 &mu;L</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
&nbsp;<em>Table 10: Digestion scheme 10</em>
 +
 
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 357px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 222px;">DNA (K133116 194,1 ng/&mu;l)&nbsp;~ 2 &mu;g</td>
 +
<td style="width: 123px;">10,3&nbsp;&mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 222px;">Cutsmart (10x)</td>
 +
<td style="width: 123px;">5 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 222px;">Restriction enzyme 1 (1 U/&mu;L)</td>
 +
<td style="width: 123px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 222px;">Restriction enzyme 2 (1 U/&mu;L)</td>
 +
<td style="width: 123px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 222px;">Nuclease free water</td>
 +
<td style="width: 123px;">Fill to 50 &mu;L</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<em>Table 11: Digestion scheme 11</em><br>
 +
&nbsp;
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 471px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 124px;"><strong>Biobrick</strong></td>
 +
<td style="width: 158px;"><strong>Restriction-enzyme 1</strong></td>
 +
<td style="width: 172px;"><strong>Restriction enzyme 2</strong></td>
 +
</tr>
 +
<tr>
 +
<td style="width: 124px;">K173003</td>
 +
<td style="width: 158px;">XbaI</td>
 +
<td style="width: 172px;">PstI-HF</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 124px;">K173013</td>
 +
<td style="width: 158px;">XbaI</td>
 +
<td style="width: 172px;">PstI-HF</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 124px;">K133071</td>
 +
<td style="width: 158px;">XbaI</td>
 +
<td style="width: 172px;">PstI-HF</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 124px;">K133116</td>
 +
<td style="width: 158px;">XbaI</td>
 +
<td style="width: 172px;">PstI-HF</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 124px;">J23100</td>
 +
<td style="width: 158px;">EcoRI-HF</td>
 +
<td style="width: 172px;">SpeI-HF</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 124px;">I13453</td>
 +
<td style="width: 158px;">EcoRI-HF</td>
 +
<td style="width: 172px;">SpeI-HF</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 124px;">K352002</td>
 +
<td style="width: 158px;">EcoRI-HF</td>
 +
<td style="width: 172px;">SpeI-HF</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 124px;">K352003</td>
 +
<td style="width: 158px;">EcoRI-HF</td>
 +
<td style="width: 172px;">SpeI-HF</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 124px;">pSB1K3</td>
 +
<td style="width: 158px;">EcoRI-HF</td>
 +
<td style="width: 172px;">PstI-HF</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<em>Table 12: Digestion scheme used enzymes</em><br>
 +
<br>
 +
<strong>Gelelectrophoresis</strong>
 +
 
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 357px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 69px;"><strong>Number</strong></td>
 +
<td style="width: 170px;"><strong>What</strong></td>
 +
<td style="width: 101px;"><strong>Amount</strong></td>
 +
</tr>
 +
<tr>
 +
<td style="width: 69px;">1.</td>
 +
<td style="width: 170px;">Ladder</td>
 +
<td style="width: 101px;">5&nbsp;&mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 69px;">2.</td>
 +
<td style="width: 170px;">J23100 EcoRI-HF+ SPeI-HF</td>
 +
<td style="width: 101px;">5&nbsp;&mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 69px;">3.</td>
 +
<td style="width: 170px;">J23100 EcoRI-HF</td>
 +
<td style="width: 101px;">5&nbsp;&mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 69px;">4.</td>
 +
<td style="width: 170px;">J23100 SpeI-HF</td>
 +
<td style="width: 101px;">5&nbsp;&mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 69px;">5.</td>
 +
<td style="width: 170px;">pSB1K3 EcoRI-HF + PstI-HF</td>
 +
<td style="width: 101px;">5&nbsp;&mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 69px;">6</td>
 +
<td style="width: 170px;">K173013 XbaI + PstI-HF</td>
 +
<td style="width: 101px;">5&nbsp;&mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 69px;">7.</td>
 +
<td style="width: 170px;">K173003 XbaI + PstI-HF</td>
 +
<td style="width: 101px;">5&nbsp;&mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 69px;">8.</td>
 +
<td style="width: 170px;">K133071 XbaI + PstI-HF</td>
 +
<td style="width: 101px;">5&nbsp;&mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 69px;">9.</td>
 +
<td style="width: 170px;">K133116 XbaI + PstI-HF</td>
 +
<td style="width: 101px;">5&nbsp;&mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 69px;">10.</td>
 +
<td style="width: 170px;">K352002 EcoRI-HF + SpeI-HF</td>
 +
<td style="width: 101px;">5&nbsp;&mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 69px;">11.</td>
 +
<td style="width: 170px;">K352003 EcoRI-HF + SpeI-HF</td>
 +
<td style="width: 101px;">5&nbsp;&mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 69px;">12.</td>
 +
<td style="width: 170px;">I13453 EcoRI-HF + SpeI-HF</td>
 +
<td style="width: 101px;">5&nbsp;&mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 69px;">13.</td>
 +
<td style="width: 170px;">Ladder&nbsp;</td>
 +
<td style="width: 101px;">5&nbsp;&mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 69px;">14.</td>
 +
<td style="width: 170px;">Ladder</td>
 +
<td style="width: 101px;">10 &mu;L</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<em>Table 13: Gelelectrophoresis order of digestions</em><br>
 +
<br>
 +
<strong>dephosphorylation</strong>
 +
 
 +
<p>&nbsp;</p>
 +
 
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 219px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 58px;"><strong>Volume</strong></td>
 +
<td style="width: 149px;"><strong>Compound</strong></td>
 +
</tr>
 +
<tr>
 +
<td style="width: 58px;">12,5 &mu;L</td>
 +
<td style="width: 149px;">Vector DNA (0,5&nbsp;&mu;g)</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 58px;">2 &mu;L</td>
 +
<td style="width: 149px;">phosphatase buffer (10x)</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 58px;">4,5 &mu;L</td>
 +
<td style="width: 149px;">milli Q</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 58px;">1&nbsp;&mu;L</td>
 +
<td style="width: 149px;">phosphatase (1 U/&mu;L)</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 58px;">20 &mu;L</td>
 +
<td style="width: 149px;">Total</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<em>Table 14: Pipetting scheme&nbsp;dephosphorylation</em><br>
 +
&nbsp;
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 219px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 58px;"><strong>Volume</strong></td>
 +
<td style="width: 149px;"><strong>Compound</strong></td>
 +
</tr>
 +
<tr>
 +
<td style="width: 58px;">37,5 &mu;L</td>
 +
<td style="width: 149px;">Vector DNA (0,5&nbsp;&mu;g)</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 58px;">6 &mu;L</td>
 +
<td style="width: 149px;">phosphatase buffer (10x)</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 58px;">13,5 &mu;L</td>
 +
<td style="width: 149px;">milli Q</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 58px;">3&mu;L</td>
 +
<td style="width: 149px;">phosphatase (1 U/&mu;L)</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 58px;">60 &mu;L</td>
 +
<td style="width: 149px;">Total</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<em>Table 15: Pipetting scheme dephosphorylation</em><br>
 +
<br>
 +
<strong>Pre-Ligationmix:</strong>
 +
 
 +
<table border="1" cellpadding="1" cellspacing="1">
 +
<tbody>
 +
<tr>
 +
<td><strong>Volume</strong></td>
 +
<td><strong>Compound</strong></td>
 +
</tr>
 +
<tr>
 +
<td>7,5 &mu;L</td>
 +
<td>Insert DNA (2 &mu;g/50 &mu;L) 300 ng</td>
 +
</tr>
 +
<tr>
 +
<td>7,5 &mu;L</td>
 +
<td><font face="arial, helvetica, sans-serif">Insert DNA (2&nbsp;</font>&mu;g/50 &mu;L) 300 ng</td>
 +
</tr>
 +
<tr>
 +
<td>4&nbsp;&mu;L</td>
 +
<td>Backbone (0,5&nbsp;&mu;g/ 20&nbsp;&mu;L) 100 ng</td>
 +
</tr>
 +
<tr>
 +
<td>5 &mu;L</td>
 +
<td>Delutionbuffer (5x)</td>
 +
</tr>
 +
<tr>
 +
<td>1 &mu;L</td>
 +
<td>Milli Q</td>
 +
</tr>
 +
<tr>
 +
<td>25 &mu;L</td>
 +
<td>Total</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<em>Table 16: Pre-ligationmix pipette scheme</em><br>
 +
<br>
 +
<strong>Ligationmix:</strong>
 +
 
 +
<table border="1" cellpadding="1" cellspacing="1">
 +
<tbody>
 +
<tr>
 +
<td><strong>Volume</strong></td>
 +
<td><strong>Compound</strong></td>
 +
</tr>
 +
<tr>
 +
<td>10 &mu;L</td>
 +
<td>Pre-ligationmix</td>
 +
</tr>
 +
<tr>
 +
<td>2 &mu;L</td>
 +
<td>10x T4 ligationbuffer</td>
 +
</tr>
 +
<tr>
 +
<td>1 &mu;L</td>
 +
<td>T4 DNA ligase (1U/&mu;L)</td>
 +
</tr>
 +
<tr>
 +
<td>8 &mu;L</td>
 +
<td>Milli Q</td>
 +
</tr>
 +
<tr>
 +
<td>20&nbsp;&mu;L</td>
 +
<td>Total</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<em>Table 17: Ligationmix pipette scheme</em><br>
 +
<br>
 +
Ligation started at 15.45 (31-08-18) at room temperature.<br>
 +
end ligation around 11.00 (01-09-18)<br>
 +
Store at -20 degrees Celcius.<br>
 +
&nbsp;</p>
 +
</div>
 
</div>
 
</div>
  
Line 3,710: Line 5,509:
 
</div>
 
</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
−
<div class="description"><p><p>Transformations of the biobrick&nbsp;combinations in NEB10b�ta. The transformations are plated on kanamycine agar plates, because all the ligations were done in pSB1K3 (kanamycine resistence) backbone. Pink and with colonies appeared after incubation by 37 degrees Celcius for about 12h. The white colonies will be used for further experiments.</p>
+
<div class="description">
−
</p></div>
+
<p><p>Transformations of the biobrick&nbsp;combinations in NEB10b�ta. The transformations are plated on kanamycine agar plates, because all the ligations were done in pSB1K3 (kanamycine resistence) backbone. Pink and with colonies appeared after incubation by 37 degrees Celcius for about 12h. The white colonies will be used for further experiments.</p>
 +
</p>
 +
<p class="experiment"><p><strong>Experiment 11: transformation of the biobrickombinations in NEB10b�ta.</strong><br>
 +
03-09-18<br>
 +
<br>
 +
<strong>Materials</strong><br>
 +
See materials&nbsp;experiment 5.<br>
 +
-Kanamycin&nbsp;(40 mg/mL)<br>
 +
-Ampicillin (50 mg/mL)<br>
 +
<br>
 +
<br>
 +
<strong>Methods</strong><br>
 +
See methods experiment 5<br>
 +
<br>
 +
Make 19 agar plates with kanamycine (Kana) (17,5 ul/20mL) and 1 agar plate with ampicillin (Amp) (40 ul/mL). Plate all the transformations on agar plates with Kana , because all the ligations are done in the pSB1K3 (Kana resistence) backbone.&nbsp;<br>
 +
Devide one Amp plate and one Kana plate in about 30 parts. Because the biobrick combinations with biobrick K133116 have originally Amp and Kana resistence, the right backbone plasmid (with hopefully the right ligation) is only growing on the agar plate with Kana.<br>
 +
<br>
 +
<br>
 +
<strong>Results</strong><br>
 +
There is grown n every transformation plates, so the transformations are done well.<br>
 +
There are pink and white colonies present. Only the white colonies will be used for further experiments.<br>
 +
&nbsp;</p>
 +
</p>
 +
</div>
 
</div>
 
</div>
  
Line 3,752: Line 5,574:
 
</div>
 
</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
−
<div class="description"><p><p>Plasmid DNA was isolated of 20 different colonies. The first time something went wrong. The second time we had good concentrations of isolated plasmid DNA. After this we will digest the DNA to see if the plasmids all have the required biobricks. The tube have a code from now on, see table 1.<br>
+
<div class="description">
 +
<p><p>Plasmid DNA was isolated of 20 different colonies. The first time something went wrong. The second time we had good concentrations of isolated plasmid DNA. After this we will digest the DNA to see if the plasmids all have the required biobricks. The tube have a code from now on, see table 1.<br>
 
&nbsp;</p>
 
&nbsp;</p>
  
Line 3,813: Line 5,636:
 
<tr>
 
<tr>
 
<td style="width: 122px;">J</td>
 
<td style="width: 122px;">J</td>
−
<td style="width: 133px;">II13453</td>
+
<td style="width: 133px;">I13453</td>
 
<td style="width: 116px;">K133071</td>
 
<td style="width: 116px;">K133071</td>
 
<td style="width: 106px;">pSB1K3</td>
 
<td style="width: 106px;">pSB1K3</td>
Line 3,821: Line 5,644:
  
 
<p><em>Table 1: list of biobricks of abbreviations</em></p>
 
<p><em>Table 1: list of biobricks of abbreviations</em></p>
−
</p></div>
+
</p>
 +
<p class="experiment"><p><strong>Experiment&nbsp;12: Overnight cultures and minipreps</strong><br>
 +
05-09-18/06-09-18<br>
 +
<br>
 +
<strong>Materials</strong><br>
 +
See materials experiment 3<br>
 +
<br>
 +
<strong>Methods</strong><br>
 +
See methods experiment 3</p>
 +
 
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 500px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 122px;"><strong>Number</strong></td>
 +
<td style="width: 131px;"><strong>Promotor</strong></td>
 +
<td style="width: 118px;"><strong>Gene</strong></td>
 +
<td style="width: 106px;"><strong>Backbone</strong></td>
 +
</tr>
 +
<tr>
 +
<td style="width: 122px;">A</td>
 +
<td style="width: 131px;">K352002 (CooF)</td>
 +
<td style="width: 118px;">K173013</td>
 +
<td style="width: 106px;">pSB1K3</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 122px;">B</td>
 +
<td style="width: 131px;">K352002 (CooF)</td>
 +
<td style="width: 118px;">K173003</td>
 +
<td style="width: 106px;">pSB1K3</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 122px;">C</td>
 +
<td style="width: 131px;">K352002 (CooF)</td>
 +
<td style="width: 118px;">K133071</td>
 +
<td style="width: 106px;">pSB1K3</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 122px;">D</td>
 +
<td style="width: 131px;">K352003 (CooM)</td>
 +
<td style="width: 118px;">K133071</td>
 +
<td style="width: 106px;">pSB1K3</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 122px;">E</td>
 +
<td style="width: 131px;">K352003 (CooM)</td>
 +
<td style="width: 118px;">K173003</td>
 +
<td style="width: 106px;">pSB1K3</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 122px;">F</td>
 +
<td style="width: 131px;">K352003 (CooM)</td>
 +
<td style="width: 118px;">K173013</td>
 +
<td style="width: 106px;">pSB1K3</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 122px;">G</td>
 +
<td style="width: 131px;">B0032</td>
 +
<td style="width: 118px;">J23105</td>
 +
<td style="width: 106px;">pSB1A3</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 122px;">H</td>
 +
<td style="width: 131px;">I13453</td>
 +
<td style="width: 118px;">K173013</td>
 +
<td style="width: 106px;">pSB1K3</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 122px;">I</td>
 +
<td style="width: 131px;">I13453</td>
 +
<td style="width: 118px;">K173003</td>
 +
<td style="width: 106px;">pSB1K3</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 122px;">J</td>
 +
<td style="width: 131px;">I13453</td>
 +
<td style="width: 118px;">K133071</td>
 +
<td style="width: 106px;">pSB1K3</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
Table 1: list of biobricks of abbreviations<br>
 +
&nbsp;</p>
 +
</div>
 
</div>
 
</div>
  
Line 3,862: Line 5,767:
 
</div>
 
</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
−
<div class="description"><p><p>The minipreps of experiment 12 are digested with the restriction enzymes: SmaI and ScaI. Only Sca and Sma are both incubated at 37 degrees Celcius. Sma has to be incubated at 25 degrees Celcius. This is done in experiment 15.</p>
+
<div class="description">
−
</p></div>
+
<p><p>The minipreps of experiment 12 are digested with the restriction enzymes: SmaI and ScaI. Only Sca and Sma are both incubated at 37 degrees Celcius. Sma has to be incubated at 25 degrees Celcius. This is done in experiment 15.</p>
 +
</p>
 +
<p class="experiment"><p><strong>Experiment 12: Digestions of the minipreps</strong><br>
 +
10-09-18<br>
 +
<br>
 +
<strong>Materials</strong><br>
 +
-SmaI (restriction-enzyme)<br>
 +
-ScaI (restriction-enzyme)<br>
 +
See materials experiment 10<br>
 +
<br>
 +
<strong>Methods</strong><br>
 +
See methods experiment 10<br>
 +
&nbsp;</p>
 +
 
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 346px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 221px;">DNA (A1 396.7 ng/&mu;l)&nbsp;~ 2 &mu;g</td>
 +
<td style="width: 114px;">5.0&nbsp;&mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Cutsmart (10x)</td>
 +
<td style="width: 114px;">5&nbsp;&mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sca1-HF (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sma1 (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Nuclease free water</td>
 +
<td style="width: 114px;">Fill to 50 &mu;L</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<em>Table 1: Digestion scheme 1</em><br>
 +
&nbsp;
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 346px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 221px;">DNA (B1 15.65 ng/&mu;l)&nbsp;~ 2 &mu;g</td>
 +
<td style="width: 114px;">127.8</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Cutsmart (10x)</td>
 +
<td style="width: 114px;">5 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sca1-HF (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sma1&nbsp; (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Nuclease free water</td>
 +
<td style="width: 114px;">Fill to 50 &mu;L</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<em>Table 2: Digestion scheme 2</em><br>
 +
&nbsp;
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 346px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 221px;">DNA (C1 186.6 ng/&mu;l)&nbsp;~ 2 &mu;g</td>
 +
<td style="width: 114px;">10.7&nbsp;&mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Cutsmart (10x)</td>
 +
<td style="width: 114px;">5 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sca1-HF 1 (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sma1&nbsp; (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Nuclease free water</td>
 +
<td style="width: 114px;">Fill to 50 &mu;L</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
&nbsp;<em>Table 3: Digestion scheme 3</em><br>
 +
&nbsp;
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 346px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 221px;">DNA (D1 79.0&nbsp;&nbsp;&nbsp;&nbsp;ng/&mu;l)&nbsp;~ 2 &mu;g</td>
 +
<td style="width: 114px;">25.3 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Cutsmart (10x)</td>
 +
<td style="width: 114px;">5 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sca1-HF 1 (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sma1&nbsp; (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Nuclease free water</td>
 +
<td style="width: 114px;">Fill to 50 &mu;L</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
&nbsp;<em>Table 4: Digestion scheme 4</em><br>
 +
&nbsp;
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 346px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 221px;">DNA (E1 97.7 ng/&mu;l)&nbsp;~ 2 &mu;g</td>
 +
<td style="width: 114px;">20.7 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Cutsmart (10x)</td>
 +
<td style="width: 114px;">5 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sca1-HF 1 (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sma1&nbsp; (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Nuclease free water</td>
 +
<td style="width: 114px;">Fill to 50 &mu;L</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
&nbsp;<em>Table 5: Digestion scheme 5</em><br>
 +
&nbsp;
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 346px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 221px;">DNA (F1 106.7 ng/&mu;l)&nbsp;~ 2 &mu;g</td>
 +
<td style="width: 114px;">18.7 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Cutsmart (10x)</td>
 +
<td style="width: 114px;">5 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sca1-HF 1 (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sma1&nbsp; (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Nuclease free water</td>
 +
<td style="width: 114px;">Fill to 50 &mu;L</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
&nbsp;<em>Table 6: Digestion scheme 6</em><br>
 +
&nbsp;
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 346px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 221px;">DNA (G1 328.9 ng/&mu;l)&nbsp;~ 2 &mu;g</td>
 +
<td style="width: 114px;">6.1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Cutsmart (10x)</td>
 +
<td style="width: 114px;">5 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sca1-HF 1 (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sma1&nbsp; (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Nuclease free water</td>
 +
<td style="width: 114px;">Fill to 50 &mu;L</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
&nbsp;<em>Table 7: Digestion scheme 7</em><br>
 +
&nbsp;
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 346px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 221px;">DNA (H1 121.0 ng/&mu;l)&nbsp;~ 2 &mu;g</td>
 +
<td style="width: 114px;">16.5 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Cutsmart (10x)</td>
 +
<td style="width: 114px;">5 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sca1-HF 1 (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sma1&nbsp; (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Nuclease free water</td>
 +
<td style="width: 114px;">Fill to 50 &mu;L</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
&nbsp;<em>Table 8: Digestion scheme 8</em><br>
 +
&nbsp;
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 346px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 221px;">DNA (I1 1129.5 ng/&mu;l)&nbsp;~ 2 &mu;g</td>
 +
<td style="width: 114px;">&nbsp;1.7 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Cutsmart (10x)</td>
 +
<td style="width: 114px;">5 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sca1-HF 1 (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sma1&nbsp; (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Nuclease free water</td>
 +
<td style="width: 114px;">Fill to 50 &mu;L</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
&nbsp;<em>Table 9: Digestion scheme 9</em><br>
 +
&nbsp;
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 346px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 221px;">DNA (J1 1465.7 ng/&mu;l)&nbsp;~ 2 &mu;g</td>
 +
<td style="width: 114px;">&nbsp;1.4 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Cutsmart (10x)</td>
 +
<td style="width: 114px;">5 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sca1-HF 1 (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sma1&nbsp; (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Nuclease free water</td>
 +
<td style="width: 114px;">Fill to 50 &mu;L</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
&nbsp;<em>Table 10: Digestion scheme 10</em><br>
 +
&nbsp;
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 346px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 221px;">DNA (A2 310.4 ng/&mu;l)&nbsp;~ 2 &mu;g</td>
 +
<td style="width: 114px;">6.4 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Cutsmart (10x)</td>
 +
<td style="width: 114px;">5 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sca1-HF 1 (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sma1&nbsp; (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Nuclease free water</td>
 +
<td style="width: 114px;">Fill to 50 &mu;L</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
&nbsp;<em>Table 11: Digestion scheme 11</em><br>
 +
&nbsp;
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 346px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 221px;">DNA (B2 364.9l)&nbsp;~ 2 &mu;g</td>
 +
<td style="width: 114px;">5.5 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Cutsmart (10x)</td>
 +
<td style="width: 114px;">5 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sca1-HF 1 (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sma1&nbsp; (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Nuclease free water</td>
 +
<td style="width: 114px;">Fill to 50 &mu;L</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
&nbsp;<em>Table 12: Digestion scheme 12</em><br>
 +
&nbsp;
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 346px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 221px;">DNA (C2 228.3 ng/&mu;l)&nbsp;~ 2 &mu;g</td>
 +
<td style="width: 114px;">8.8 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Cutsmart (10x)</td>
 +
<td style="width: 114px;">5 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sca1-HF 1 (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sma1&nbsp; (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Nuclease free water</td>
 +
<td style="width: 114px;">Fill to 50 &mu;L</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
&nbsp;<em>Table 13: Digestion scheme 13</em><br>
 +
&nbsp;
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 346px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 221px;">DNA (D2 701.7 ng/&mu;l)&nbsp;~ 2 &mu;g</td>
 +
<td style="width: 114px;">2.9 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Cutsmart (10x)</td>
 +
<td style="width: 114px;">5 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sca1-HF 1 (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sma1&nbsp; (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Nuclease free water</td>
 +
<td style="width: 114px;">Fill to 50 &mu;L</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
&nbsp;<em>Table 14: Digestion scheme 14</em><br>
 +
&nbsp;
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 346px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 221px;">DNA (E2 119.8 ng/&mu;l)&nbsp;~ 2 &mu;g</td>
 +
<td style="width: 114px;">16.7 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Cutsmart (10x)</td>
 +
<td style="width: 114px;">5 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sca1-HF 1 (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sma1&nbsp; (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Nuclease free water</td>
 +
<td style="width: 114px;">Fill to 50 &mu;L</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
&nbsp;<em>Table 15: Digestion scheme 15</em><br>
 +
&nbsp;
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 346px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 221px;">DNA (F2 493.3 ng/&mu;l)&nbsp;~ 2 &mu;g</td>
 +
<td style="width: 114px;">4.1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Cutsmart (10x)</td>
 +
<td style="width: 114px;">5 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sca1-HF 1 (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sma1&nbsp; (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Nuclease free water</td>
 +
<td style="width: 114px;">Fill to 50 &mu;L</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
&nbsp;<em>Table 16: Digestion scheme 16</em><br>
 +
&nbsp;
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 346px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 221px;">DNA (G2 211.7 ng/&mu;l)&nbsp;~ 2 &mu;g</td>
 +
<td style="width: 114px;">9.5 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Cutsmart (10x)</td>
 +
<td style="width: 114px;">5 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sca1-HF 1 (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sma1&nbsp; (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Nuclease free water</td>
 +
<td style="width: 114px;">Fill to 50 &mu;L</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
&nbsp;<em>Table 17: Digestion scheme 17</em>
 +
 
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 346px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 221px;">DNA (H2 482.2 ng/&mu;l)&nbsp;~ 2 &mu;g</td>
 +
<td style="width: 114px;">4.2 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Cutsmart (10x)</td>
 +
<td style="width: 114px;">5 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sca1-HF 1 (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sma1&nbsp; (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Nuclease free water</td>
 +
<td style="width: 114px;">Fill to 50 &mu;L</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
&nbsp;<em>Table 18: Digestion scheme 18</em><br>
 +
&nbsp;
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 346px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 221px;">DNA (I2 1364.7 ng/&mu;l)&nbsp;~ 2 &mu;g</td>
 +
<td style="width: 114px;">1.5 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Cutsmart (10x)</td>
 +
<td style="width: 114px;">5 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sca1-HF 1 (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sma1&nbsp; (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Nuclease free water</td>
 +
<td style="width: 114px;">Fill to 50 &mu;L</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
&nbsp;<em>Table 19: Digestion scheme 19</em><br>
 +
&nbsp;
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 346px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 221px;">DNA (J2 1665.5 ng/&mu;l)&nbsp;~ 2 &mu;g1.2</td>
 +
<td style="width: 114px;">&nbsp;&mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Cutsmart (10x)</td>
 +
<td style="width: 114px;">5 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sca1-HF 1 (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sma1&nbsp; (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Nuclease free water</td>
 +
<td style="width: 114px;">Fill to 50 &mu;L</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<em>Table 20: Digestion scheme 20</em><br>
 +
<br>
 +
<br>
 +
<br>
 +
<strong>Results</strong><br>
 +
The results of the digestions can be found in figure 1 and 2 (Images experiment 13)<br>
 +
<br>
 +
Ladder, A1, B1, C1, D1, E1, F1, G1, H1, I1, (J1), Ladder, A2, B2, C2, D2, E2, F2, G2, Ladder<br>
 +
Ladder, H2, I2, J2, Ladder, K3, J1, ladder<br>
 +
Something went wrong with the first (J1), so look at the second one.<br>
 +
<br>
 +
<strong>Conclusion/discussion</strong><br>
 +
Restriction-enzyme Sma was incubated at 37 degrees Celcius (same as Sca). This must be 25 degrees Celcius.<br>
 +
Therefore, Sma was added again and was incubated at 25 degrees Celcius. See results experiment 16.</p>
 +
</div>
 
</div>
 
</div>
  
Line 3,904: Line 6,355:
 
</div>
 
</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
−
<div class="description"><p><p>Digestions have been done of the biobrickcombinations&nbsp;K352002 +K133116 + pSB1K3 and K352003 + K133116 + pSB1K3 with restriction-enzymes ScaI and SmaI</p>
+
<div class="description">
−
</p></div>
+
<p><p>Digestions have been done of the biobrickcombinations&nbsp;K352002 +K133116 + pSB1K3 and K352003 + K133116 + pSB1K3 with restriction-enzymes ScaI and SmaI</p>
 +
</p>
 +
<p class="experiment"><p><strong>Experiment 14: Digestions</strong><br>
 +
13-09-18<br>
 +
<br>
 +
Digestions of the biobrick combinations:&nbsp;K352002+K133116+pSB1K3&nbsp;and&nbsp;K352003+K133116+pSB1K3<br>
 +
<br>
 +
<strong>Materials</strong><br>
 +
-SmaI (restriction-enzyme)<br>
 +
-ScaI (restricion-enzyme)<br>
 +
See experiment..<br>
 +
<br>
 +
<br>
 +
<strong>Methods</strong><br>
 +
1&nbsp;&mu;L SmaI restriction-enzyme has been added to the digetions from experiment 13, except for pSB1K3.<br>
 +
After an our at room temperature the digestions were put in -20 degrees Celcius.</p>
 +
&nbsp;
 +
 
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 346px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 221px;">DNA (K352002+K133116+pSB1K3 189,3 ng/&mu;l)&nbsp;~ 2 &mu;g</td>
 +
<td style="width: 114px;">32,4 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Cutsmart (10x)</td>
 +
<td style="width: 114px;">5&nbsp;&mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sca1-HF (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sma1 (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Nuclease free water</td>
 +
<td style="width: 114px;">Fill to 50 &mu;L</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
 
 +
<p><em>Table 1: Digestion scheme 1</em></p>
 +
 
 +
 
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 346px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 221px;">DNA (K352003+K133116_pSB1K3 355,3 ng/&mu;l)&nbsp;~ 2 &mu;g</td>
 +
<td style="width: 114px;">5,6 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Cutsmart (10x)</td>
 +
<td style="width: 114px;">5&nbsp;&mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sca1-HF (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Sma1 (2 U/&mu;L)</td>
 +
<td style="width: 114px;">1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 221px;">Nuclease free water</td>
 +
<td style="width: 114px;">Fill to 50 &mu;L</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
 
 +
<p><em>Table 2: Digestion scheme 2</em></p>
 +
&nbsp;
 +
 
 +
<p>&nbsp;</p>
 +
</p>
 +
</div>
 
</div>
 
</div>
  
Line 3,946: Line 6,473:
 
</div>
 
</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
−
<div class="description"><p><p>All the digestions of experiment 13 have digested again with SmaI for an our at 25 degrees Celcius. Because earlier the digestions have been put immediately bt 37 degrees Celcius. It looks like H1, J1 and D2 include the right biobricks.&nbsp;</p>
+
<div class="description">
−
</p></div>
+
<p><p>All the digestions of experiment 13 have digested again with SmaI for an our at 25 degrees Celcius. Because earlier the digestions have been put immediately bt 37 degrees Celcius. It looks like H1, J1 and D2 include the right biobricks.&nbsp;</p>
 +
</p>
 +
<p class="experiment"><p><span style="font-size:14px;"><strong>Experiment 16: Gelelectroforesis</strong></span><br>
 +
14-09-18<br>
 +
<br>
 +
<strong>Materials</strong><br>
 +
See experment ...<br>
 +
<br>
 +
<strong>Methods</strong><br>
 +
See experiment ...<br>
 +
<br>
 +
<strong>Gel 1</strong><br>
 +
L, A1, B1, C1, D1, E1, F1, G1, G1 with only smaI, H1, I1, J1, A2, B2, C2, D2, E2, F2, G2, L<br>
 +
<br>
 +
<strong>Gel 2</strong><br>
 +
L,&nbsp;</p>
 +
</p>
 +
</div>
 
</div>
 
</div>
  
Line 3,988: Line 6,532:
 
</div>
 
</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
−
<div class="description"><p><p>Minipreps of the biobrick combinations:&nbsp;K352002+K133116+pSB1K3 (1) and&nbsp;K352003+K133116+pSB1K3 (2) are done. The DNA concentration is for 1:189,3 ng/&mu;l and for 2: 355,3&nbsp;ng/&mu;l.</p>
+
<div class="description">
−
</p></div>
+
<p><p>Minipreps of the biobrick combinations:&nbsp;K352002+K133116+pSB1K3 (1) and&nbsp;K352003+K133116+pSB1K3 (2) are done. The DNA concentration is for 1:189,3 ng/&mu;l and for 2: 355,3&nbsp;ng/&mu;l.</p>
 +
</p>
 +
<p class="experiment"><p><span style="font-size:14px;"><strong>Experiment 14: Minipreps</strong></span><br>
 +
13-09-18<br>
 +
<br>
 +
Minipreps of the&nbsp;biobrick combinations:&nbsp;K352002+K133116+pSB1K3&nbsp;and&nbsp;K352003+K133116+pSB1K3<br>
 +
<br>
 +
<strong>Materials</strong><br>
 +
See experiment 3<br>
 +
<br>
 +
<strong>Methods</strong><br>
 +
See experiment 3<br>
 +
<br>
 +
<strong>Results</strong><br>
 +
K352002+K133116+pSB1K3: 189,3&nbsp;ng/&mu;l<br>
 +
K352003+K133116+pSB1K3: 355,3&nbsp;ng/&mu;l</p>
 +
</p>
 +
</div>
 
</div>
 
</div>
  
Line 4,030: Line 6,591:
 
</div>
 
</div>
 
<div class="attendees">Elise Grootscholten | Paul Reusink</div>
 
<div class="attendees">Elise Grootscholten | Paul Reusink</div>
−
<div class="description"><p><p>Different&nbsp;strains&nbsp;of E.coli are tested as negative control. Those strains were:&nbsp;NEB10B�ta, BL21 (DE3), BL21, HB101, DH5alpha and&nbsp;JM109. NEB10B�ta produced the most gas. BL21, HB101 and JM101 produced none/almost none gas. Because BL21 isn't a K12 strain we can not use that one. That is why we will test further gasproduction in HB101 and JM109. And maybe those will be our final E.coli strains.</p>
+
<div class="description">
−
</p></div>
+
<p><p>Different&nbsp;strains&nbsp;of E.coli are tested as negative control. Those strains were:&nbsp;NEB10B�ta, BL21 (DE3), BL21, HB101, DH5alpha and&nbsp;JM109. NEB10B�ta produced the most gas. BL21, HB101 and JM101 produced none/almost none gas. Because BL21 isn't a K12 strain we can not use that one. That is why we will test further gasproduction in HB101 and JM109. And maybe those will be our final E.coli strains.</p>
 +
</p>
 +
<p class="experiment"><p><span style="font-size:14px;"><strong>Experiment 5: Testing different strains&nbsp;of E.coli</strong></span><br>
 +
<br>
 +
<strong>Materials</strong><br>
 +
All the following types of bacteria are E.coli:<br>
 +
-glycerolstock BL21<br>
 +
-glycerolstock HB101<br>
 +
-glycerolstock DH5alpha<br>
 +
-glycerolstock JM109<br>
 +
-agarplate with NEB10beta<br>
 +
-agarplat with BL21 (DE3)&nbsp;<br>
 +
<br>
 +
<strong>Methods</strong><br>
 +
See experiment 1<br>
 +
Make overnight cultures of the following E.coli tribes: NEB10B�ta, BL21 (DE3), BL21, HB101, DH5alpha, JM109.<br>
 +
All the bacteria are de deluted in normal LB agar, LB agar with urea and LB agar with pyruvate.&nbsp;<br>
 +
<br>
 +
<br>
 +
<strong>Results</strong><br>
 +
&nbsp;</p>
 +
 
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 500px;">
 +
<tbody>
 +
<tr>
 +
<td>Bacteria</td>
 +
<td>LB</td>
 +
<td>LB</td>
 +
<td>Urea</td>
 +
<td>Urea</td>
 +
<td>Pyruvate</td>
 +
<td>Pyruvate</td>
 +
</tr>
 +
<tr>
 +
<td>NEB10beta</td>
 +
<td>+</td>
 +
<td>+++</td>
 +
<td>+++</td>
 +
<td>+++</td>
 +
<td>+</td>
 +
<td>++/+++</td>
 +
</tr>
 +
<tr>
 +
<td>BL21 (DE3)&nbsp;</td>
 +
<td>-</td>
 +
<td>++</td>
 +
<td>-</td>
 +
<td>-</td>
 +
<td>-</td>
 +
<td>-</td>
 +
</tr>
 +
<tr>
 +
<td>BL21</td>
 +
<td>-</td>
 +
<td>-</td>
 +
<td>-</td>
 +
<td>-</td>
 +
<td>-</td>
 +
<td>-</td>
 +
</tr>
 +
<tr>
 +
<td>DH5a</td>
 +
<td>+++</td>
 +
<td>+/-</td>
 +
<td>-</td>
 +
<td>-</td>
 +
<td>+/-</td>
 +
<td>+</td>
 +
</tr>
 +
<tr>
 +
<td>HB101</td>
 +
<td>-</td>
 +
<td>-</td>
 +
<td>-</td>
 +
<td>-</td>
 +
<td>+</td>
 +
<td>-</td>
 +
</tr>
 +
<tr>
 +
<td>JM109</td>
 +
<td>-</td>
 +
<td>-</td>
 +
<td>-</td>
 +
<td>-</td>
 +
<td>-</td>
 +
<td>-</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
</p>
 +
</div>
 
</div>
 
</div>
  
Line 4,072: Line 6,723:
 
</div>
 
</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
−
<div class="description"><p>32 minipreps have been performed. A lot of them have a low concentration nucleic acid and/or have a high 260/280 and 260/230 rate. Therefore those will be done again.</p></div>
+
<div class="description">
 +
<p>32 minipreps have been performed. A lot of them have a low concentration nucleic acid and/or have a high 260/280 and 260/230 rate. Therefore those will be done again.</p>
 +
<p class="experiment"><span style="font-size:14px;"><strong>Experiment 17: Minipreps</strong></span><br>
 +
20-09-18<br>
 +
Overnight cultures (35)<br>
 +
21-09-18<br>
 +
Minipreps (32)<br>
 +
<br>
 +
<strong>Materials&nbsp;</strong><br>
 +
See experiment 3<br>
 +
-Kanamycin (40 mg/mL)<br>
 +
<br>
 +
<strong>Methods</strong><br>
 +
See experiment 3<br>
 +
Make 32 minipreps:<br>
 +
A: 6<br>
 +
B: 6<br>
 +
C:6<br>
 +
D:1<br>
 +
E:6<br>
 +
F:6<br>
 +
H:1<br>
 +
I: 1<br>
 +
J:2<br>
 +
<br>
 +
<strong>Results/conclusion</strong><br>
 +
There was no grow in B6, C2 and C5.<br>
 +
A lot of them have a low concentration nucleic acid and/or have a high 260/280 and 260/230 rate. Therefore those will be done again.<br>
 +
Thise are: A1, A4, A5, A6, B1, B5, B6 C1, C2, C3, C4, C5, C6, E1, E2, E5, E6, F4, F5</p>
 +
</div>
 
</div>
 
</div>
  
Line 4,113: Line 6,793:
 
</div>
 
</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
−
<div class="description"><p><p>Making overnight cultures of the numbers: A1, A4, A5, A6, B1, B5, B6 C1, C2, C3, C4, C5, C6, E1, E2, E5, E6, F4, F5 with Kanamycin. These biobrick combinations can be found in experiment 12. Also&nbsp;the first digestions with XbaI, Eco0109I, HindIII and SspI-HF have been done.</p>
+
<div class="description">
−
</p></div>
+
<p><p>Making overnight cultures of the numbers: A1, A4, A5, A6, B1, B5, B6 C1, C2, C3, C4, C5, C6, E1, E2, E5, E6, F4, F5 with Kanamycin. These biobrick combinations can be found in experiment 12. Also&nbsp;the first digestions with XbaI, Eco0109I, HindIII and SspI-HF have been done.</p>
 +
</p>
 +
<p class="experiment"><span style="font-size:14px;"><strong>Experiment 19: Making overnight cultures + first digestions</strong></span><br>
 +
27-09-18<br>
 +
<br>
 +
<strong>Materials</strong><br>
 +
-Kanamycine&nbsp;<br>
 +
-XbaI<br>
 +
-SspI-HF<br>
 +
-Eco019I<br>
 +
-HindIII<br>
 +
See experiment ...<br>
 +
<br>
 +
<strong>Methods</strong><br>
 +
See experiment ...<br>
 +
Make new overnight cultures of the numbers:&nbsp;A1, A4, A5, A6, B1, B5, B6 C1, C2, C3, C4, C5, C6, E1, E2, E5, E6, F4, F5 with Kanamycin.&nbsp;<br>
 +
<br>
 +
&nbsp;</p>
 +
</div>
 
</div>
 
</div>
  
Line 4,155: Line 6,853:
 
</div>
 
</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
−
<div class="description"><p><p>Minipreps have been made of the numbers:&nbsp;A1, A4, A5, A6, B1, B5, B6 C1, C2, C3, C4, C5, C6, E1, E2, E5, E6, F4, F5.</p>
+
<div class="description">
−
</p></div>
+
<p><p>Minipreps have been made of the numbers:&nbsp;A1, A4, A5, A6, B1, B5, B6 C1, C2, C3, C4, C5, C6, E1, E2, E5, E6, F4, F5.</p>
 +
</p>
 +
<p class="experiment"><p><span style="font-size:14px;"><strong>Experiment 20: Minipreps of gasproduction biobricks</strong></span><br>
 +
27-09-18<br>
 +
<br>
 +
Make minipreps of A1, A4, A5, A6, B1, B5, B6 C1, C2, C3, C4, C5, C6, E1, E2, E5, E6, F4, F5.<br>
 +
See experiment 3.<br>
 +
<br>
 +
Also make master mixes for the control digestions and code&nbsp;the tubes.<br>
 +
&nbsp;</p>
 +
</p>
 +
</div>
 
</div>
 
</div>
  
Line 4,197: Line 6,906:
 
</div>
 
</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
−
<div class="description"><p><p>From 27-09 until 04-10 more than 200 digestions and 17 gelelectrophoresis have been performed. This was done as a control for our composite parts. We digested with XbaI, Eco01019 and SspI-HF/HindIII. Every restriction-enzyme has an unique place to cut in our constructs. With this we could test if the promoter, backbone and gas production gene was present. However, the digestions didn't show us very good results. After this we started with&nbsp;PCR.</p>
+
<div class="description">
−
</p></div>
+
<p><p>From 27-09 until 04-10 more than 200 digestions and 17 gelelectrophoresis have been performed. This was done as a control for our composite parts. We digested with XbaI, Eco01019 and SspI-HF/HindIII. Every restriction-enzyme has an unique place to cut in our constructs. With this we could test if the promoter, backbone and gas production gene was present. However, the digestions didn't show us very good results. Only I1 and J1 looked good.&nbsp;After this we started with&nbsp;PCR.</p>
 +
</p>
 +
<p class="experiment"><p><span style="font-size:14px;"><strong>Experiment 21: Digestions and gelelectrophoresis</strong></span><br>
 +
01-10-18<br>
 +
<br>
 +
Digestions of the gasproduction biobrick combinations (D, H, I and J) and a start with the gelelectrophoresis.<br>
 +
<br>
 +
<strong>Methods</strong><br>
 +
Digestions of D2, H1, I2 and J2 of experiment 12.<br>
 +
J2 will from now on be J3.<br>
 +
&nbsp;</p>
 +
 
 +
<table border="1" cellpadding="1" cellspacing="1">
 +
<tbody>
 +
<tr>
 +
<td>NEB 2.1 (10x)</td>
 +
<td>4 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>Eco0109I (20 U/&mu;L)</td>
 +
<td>1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>Nuclease free water</td>
 +
<td>35 &mu;L</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
 
 +
<p><em>Table 1: Master mix digestion 1</em><br>
 +
&nbsp;</p>
 +
 
 +
<table border="1" cellpadding="1" cellspacing="1">
 +
<tbody>
 +
<tr>
 +
<td>NEB 2.1 (10x)</td>
 +
<td>4 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>Eco0109I (20 U/&mu;L)</td>
 +
<td>1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>XbaI (20 U/&mu;L)</td>
 +
<td>1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>Nuclease free water</td>
 +
<td>34 &mu;L<br>
 +
&nbsp;</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<em>Table 2: Master mix digestion 2</em><br>
 +
&nbsp;
 +
<table border="1" cellpadding="1" cellspacing="1">
 +
<tbody>
 +
<tr>
 +
<td>NEB 2.1 (10x)</td>
 +
<td>2 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>Eco0109I (20 U/&mu;L)</td>
 +
<td>1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>XbaI (20 U/&mu;L)</td>
 +
<td>1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>HindIII&nbsp;(20 U/&mu;L)</td>
 +
<td>1&nbsp;&mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>Nuclease free water</td>
 +
<td>15 &mu;L</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<em>Table 3: Master mix digestion 3</em><br>
 +
&nbsp;
 +
<table border="1" cellpadding="1" cellspacing="1">
 +
<tbody>
 +
<tr>
 +
<td>NEB 2.1 (10x)</td>
 +
<td>2 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>Eco0109I (20 U/&mu;L)</td>
 +
<td>1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>XbaI (20 U/&mu;L)</td>
 +
<td>1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>SspI-HF</td>
 +
<td>1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>Nuclease free water</td>
 +
<td>15&nbsp; &mu;L</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<em>Table 4: Master mix digestion 4</em><br>
 +
&nbsp;
 +
<table border="1" cellpadding="1" cellspacing="1">
 +
<tbody>
 +
<tr>
 +
<td>NEB 2.1 (10x)</td>
 +
<td>2 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>HindIII (20 U/&mu;L)</td>
 +
<td>1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>XbaI (20 U/&mu;L)</td>
 +
<td>1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>Nuclease free water</td>
 +
<td>16 &mu;L<br>
 +
&nbsp;</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<em>Table 5: Master mix digestion 5</em><br>
 +
&nbsp;
 +
<table border="1" cellpadding="1" cellspacing="1">
 +
<tbody>
 +
<tr>
 +
<td>NEB 2.1 (10x)</td>
 +
<td>2 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>SspI-HF (20 U/&mu;L)</td>
 +
<td>1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>XbaI (20 U/&mu;L)</td>
 +
<td>1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>Nuclease free water</td>
 +
<td>16 &mu;L<br>
 +
&nbsp;</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<em>Table 6: Master mix digestion 6</em><br>
 +
&nbsp;
 +
<table border="1" cellpadding="1" cellspacing="1">
 +
<tbody>
 +
<tr>
 +
<td>NEB 2.1 (10x)</td>
 +
<td>4 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>XbaI (20 U/&mu;L)</td>
 +
<td>1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>Nuclease free water</td>
 +
<td>35 &mu;L<br>
 +
&nbsp;</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<em>Table 7: Master mix digestion 7</em><br>
 +
&nbsp;
 +
<table border="1" cellpadding="1" cellspacing="1">
 +
<tbody>
 +
<tr>
 +
<td>NEB 2.1 (10x)</td>
 +
<td>2 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>HindIII (20 U/&mu;L)</td>
 +
<td>1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>Nuclease free water</td>
 +
<td>17 &mu;L<br>
 +
&nbsp;</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<em>Table 8: Master mix digestion 8</em><br>
 +
&nbsp;
 +
<table border="1" cellpadding="1" cellspacing="1">
 +
<tbody>
 +
<tr>
 +
<td>NEB 2.1 (10x)</td>
 +
<td>2 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>SspI-HF (20 U/&mu;L)</td>
 +
<td>1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>Nuclease free water</td>
 +
<td>17 &mu;L<br>
 +
&nbsp;</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<em>Table 9: Master mix digestion 9</em><br>
 +
&nbsp;
 +
<table border="1" cellpadding="1" cellspacing="1">
 +
<tbody>
 +
<tr>
 +
<td>NEB 2.1 (10x)</td>
 +
<td>2 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>Eco0109I (20 U/&mu;L)</td>
 +
<td>1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>HindIII (20 U/&mu;L)</td>
 +
<td>1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>Nuclease free water</td>
 +
<td>16 &mu;L<br>
 +
&nbsp;</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<em>Table 10: Master mix digestion 10</em><br>
 +
&nbsp;
 +
<table border="1" cellpadding="1" cellspacing="1">
 +
<tbody>
 +
<tr>
 +
<td>NEB 2.1 (10x)</td>
 +
<td>2 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>Eco0109I (20 U/&mu;L)</td>
 +
<td>1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>SspI-HF (20 U/&mu;L)</td>
 +
<td>1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>Nuclease free water</td>
 +
<td>16 &mu;L<br>
 +
&nbsp;</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<em>Table 11: Master mix digestion 11</em><br>
 +
<br>
 +
Pipette 9&nbsp;&mu;L&nbsp;of the mastermix with 2&nbsp;&mu;L&nbsp;of H1/ 1&nbsp;&mu;L&nbsp;of D2/ 1 &mu;L&nbsp;of I2/ 1 &mu;L&nbsp;of J23<br>
 +
<br>
 +
D2 and J3 are digested with mastermix: 1, 2, 3, 5, 7, 8 and 10.<br>
 +
H1 and I2 are digested with mastermix: 1, 2, 4, 6, 7, 9, and 11.<br>
 +
&nbsp;
 +
<p><strong>Gelelectrophoresis</strong><br>
 +
01-10-18<br>
 +
<br>
 +
<br>
 +
02-10-18<br>
 +
<strong>Gelelectrophoresis</strong><br>
 +
<br>
 +
<br>
 +
03-10-18<br>
 +
<strong>Gelelectrophoresis</strong><br>
 +
<br>
 +
<strong>Gel 1</strong><br>
 +
L, D2.1, D2.2, D2.3, D2.5, D2.7, D2.8, D2.10, D not digested, L, J3.1, J3.2, J3.3, J3.5, J3.7, J3.8, J3.10, J not digested, L.<br>
 +
<br>
 +
<strong>Gel 2</strong><br>
 +
L, H2.1, H2.2 H2.4, H2.6, H2.7, H2.9, H2.11, H2 not digested, L, I2.1, I2.2, I2.4, I2.6, H2.7, H2.9, H2.11, H2 not digested, L<br>
 +
<br>
 +
<strong>Gel 3</strong><br>
 +
L, C1.1, C1.2, C1.4, C1.6, C1.7, C1.8, C1.10, C1 not digested, L, C2.1, C2.2, C2.4, C2.6, C2.7, C2.8, C2.10, C2 not dgested, L<br>
 +
<br>
 +
<strong>Gel 4</strong><br>
 +
L, C3.1, C3.2, C3.4, C3.6, C3.7, C3.8, C3.10, L, C4.1, C4.2, C4.4, C4.6, C4.7, C4.8, C4.10, C4 not digested, L<br>
 +
<br>
 +
<strong>Gel 5</strong><br>
 +
L, C6.1, C6.2, C6.4, C6.6, C6.7, C6.8, C6.10, C6 not digested, L, B5.1, B5.3, B5.4, B5.4 (other tube), B5.6, B5..., B5.9, B5.11, L<br>
 +
<br>
 +
04-10-18<br>
 +
<strong>Gelelectrophoresis</strong><br>
 +
<br>
 +
Gel 1<br>
 +
L, J2 Eco0109, J2 HindIII, J2 XbaI, J2 Eco0109 + XbaI, J2 Eco0109 + HindIII, J2 HindIII + XbaI, J2 Eco0109 + HindIII + XbaI, J2 not digested, L<br>
 +
Gel 2<br>
 +
<br>
 +
Gel 3<br>
 +
<br>
 +
Gel 4</p>
 +
</p>
 +
</div>
 
</div>
 
</div>
  
Line 4,239: Line 7,246:
 
</div>
 
</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
 
<div class="attendees">Elise Grootscholten | Randall de Waard</div>
−
<div class="description"><p><p>Digestions with EcoRI-HF and PstI are done for the biobrick numbers A2, B1, B5, B6, D1, E1, E3, I1, J1, J1.2, CooA (8) and pSB1C3. This is done because now the Kanamycin backbone can be replaced for the Chloramphenicol backbone. This is done by dephosphorylation and ligation.</p>
+
<div class="description">
−
</p></div>
+
<p><p>Digestions with EcoRI-HF and PstI are done for the biobrick numbers A2, B1, B5, B6, D1, E1, E3, I1, J1, J1.2, CooA (8) and pSB1C3. This is done because now the Kanamycin backbone can be replaced for the Chloramphenicol backbone. This is done by dephosphorylation and ligation.</p>
 +
</p>
 +
<p class="experiment"><p><span style="font-size:14px;"><strong>Experiment 24: Digestions, gelelectrophoresis and dephosphorylation and ligation.</strong></span><br>
 +
08-10-18 &lt;---- klopt dit?<br>
 +
<br>
 +
<strong>Materals</strong><br>
 +
See experiment ...<br>
 +
-Restriction enzymes EcoRI-HF and PstI<br>
 +
<br>
 +
<strong>Methods</strong><br>
 +
Digestion<br>
 +
<br>
 +
Mastermix</p>
 +
 
 +
<table border="1" cellpadding="1" cellspacing="1">
 +
<tbody>
 +
<tr>
 +
<td>Cutsmart (10x)</td>
 +
<td>55 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>EcoRI-HF (20 U/&mu;L)</td>
 +
<td>1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>PstI (20 U/&mu;L)</td>
 +
<td>1 &mu;L</td>
 +
</tr>
 +
<tr>
 +
<td>Nuclease free water</td>
 +
<td>382,7 &mu;L<br>
 +
&nbsp;</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
 
 +
<p><em>Table 1: Master mix</em><br>
 +
<br>
 +
50&nbsp;&mu;L mastermix&nbsp;- ...&nbsp;&mu;L DNA (see below)<br>
 +
A2: 12,6&nbsp;&mu;L<br>
 +
B1: 15,7&nbsp;&mu;L<br>
 +
B5: 9,4&nbsp;&mu;L<br>
 +
B6: 9,6&nbsp;&mu;L<br>
 +
D1: 10,2&nbsp;&mu;L<br>
 +
E1: 15,7&nbsp;&mu;L (1&nbsp;&mu;g)<br>
 +
I1: 14,3&nbsp;&mu;L (1 &mu;g)<br>
 +
J1.2: 17,4&nbsp;&mu;L<br>
 +
CooA (8): 5,4&nbsp;&mu;L<br>
 +
<br>
 +
Dephosphorylation + ligation scheme<br>
 +
.........<br>
 +
<br>
 +
<strong>Results</strong><br>
 +
See figure 1.</p>
 +
</p>
 +
</div>
 
</div>
 
</div>
  
Line 4,281: Line 7,343:
 
</div>
 
</div>
 
<div class="attendees">Elise Grootscholten | Paul Reusink</div>
 
<div class="attendees">Elise Grootscholten | Paul Reusink</div>
−
<div class="description"><p><p>Overnight cultures made of the following strains with the right plasmid:</p>
+
<div class="description">
 +
<p><p>Overnight cultures made of the following strains with the right plasmid:</p>
  
 
<table border="1" cellpadding="1" cellspacing="1" style="width: 322px;">
 
<table border="1" cellpadding="1" cellspacing="1" style="width: 322px;">
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<p><em>Table 1: Overnight culture scheme.</em></p>
 
<p><em>Table 1: Overnight culture scheme.</em></p>
−
</p></div>
+
</p>
 +
<p class="experiment"><p><span style="font-size:14px;"><strong>Experiment 6: Testing gasproduction with our new biobricks.</strong></span><br>
 +
<br>
 +
Testing our new biobricks K260401 in JM109 and HB101. As a negative control we used the other new biobricks ...<br>
 +
<br>
 +
<strong>Materials</strong><br>
 +
-LB culture medium<br>
 +
-Kanamycin<br>
 +
-Bacteria on agar plates with the right plasmid<br>
 +
<br>
 +
<strong>Methods</strong><br>
 +
Make overnight cultures of the following strains with the right plasmid:</p>
 +
 
 +
<table border="1" cellpadding="1" cellspacing="1" style="width: 322px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 88px;"><strong>Number</strong></td>
 +
<td style="width: 105px;"><strong>HB101</strong></td>
 +
<td style="width: 113px;"><strong>JM109</strong></td>
 +
</tr>
 +
<tr>
 +
<td style="width: 88px;">J1</td>
 +
<td style="width: 105px;">1x</td>
 +
<td style="width: 113px;">1x</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 88px;">I1</td>
 +
<td style="width: 105px;">3x</td>
 +
<td style="width: 113px;">3x</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 88px;">D1</td>
 +
<td style="width: 105px;">2x</td>
 +
<td style="width: 113px;">2x</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 88px;">B1</td>
 +
<td style="width: 105px;">1x</td>
 +
<td style="width: 113px;">1x</td>
 +
</tr>
 +
<tr>
 +
<td style="width: 88px;">B6</td>
 +
<td style="width: 105px;">1x</td>
 +
<td style="width: 113px;">1x</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<em>Table 1: Overnight culture scheme.</em><br>
 +
<br>
 +
Only the numbers J1, I1 and D1 are tested. The rest is made for making glycerolstocks.<br>
 +
J1 and D1 are the negative controls in this experiment.<br>
 +
<br>
 +
LB: 582 mL +18 mL 100 mM sodiumpyruvate and 525 &mu;L kanamycin (40 mg/mL).<br>
 +
After this the LB is devided in 2. Number 1: 0,5 &mu;L arabinose, number 2: 1,0 &mu;L arabinose.<br>
 +
The rest is done just like earlier experiments with a total of 20 tubes because everthing is done twice.<br>
 +
<br>
 +
Results<br>
 +
<br>
 +
&nbsp;</p>
 +
</div>
 
</div>
 
</div>
  
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</div></div>
 
</div></div>
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<div id="footer"></div>
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<script src="https://2018.igem.org/Template:Rotterdam_HR/js/main?action=raw&ctype=text/javascript"></script>
 +
 +
</div>
 +
</body>
 +
</html>

Revision as of 13:41, 15 October 2018

Notebook

Notebook

show wetlab entries
 show hardware entries
 show software entries

Team page

May 10

Rens Boeser

Created the first prototype for the team page

01

Team page

May 10

Footer

May 20

Rens Boeser

Social media created

01

Footer

May 20

Footer

May 21

Rens Boeser

Social media buttons same color and fixed iGEM style issues

02

Footer

May 21

Notebook

May 24

Rens Boeser

Multiple week summary styles created for the notes

01

Notebook

May 24

Header

May 27

Rens Boeser

Added the header with mobile compatibility als resized logos and readded them to the header

01

Header

May 27

Main template

May 28

Rens Boeser

Added template header and footer with 'under construction' content

01

Main template

May 28

Header

May 28

Rens Boeser

Small fixes and menu adjustments

02

Header

May 28

Team

May 29

Rens Boeser

Added a PI list with the secondary PI

02

Team

May 29

Main page

June 7

Rens Boeser

Designed and added content holders to the index Page

01

Main page

June 7

Team

June 9

Rens Boeser

Added myself to the teammember Page and did some bugfixing

03

Team

June 9

Header

June 9

Rens Boeser

Started working on a better looking menubar

03

Header

June 9

Header

June 10

Rens Boeser

Added the ability to add sub-menus to items in the menu bar. Integration into the website and added indication for which Page you are on

04

Header

June 10

Construction pages

June 13

Rens Boeser

Updated all Pages to the new style with an under construction sign

01

Construction pages

June 13

Header

June 14

Rens Boeser

Added a way to recognise Pages under construction using the menubar

05

Header

June 14

Main page

June 14

Rens Boeser

Updated the content containers so that they are responsive

02

Main page

June 14

Architecture design [TC]

June 18

Tom van Dijk | Mike Vrieswijk

The first draft of the architecture design for the temperature controller was made.

01

Architecture design [TC]

June 18

Human Practices page

June 22

Rens Boeser

Content added to the human practices page

01

Human Practices page

June 22

Main page

June 23

Rens Boeser

Content created and added to the main page

03

Main page

June 23

Architecture design [TC]

June 24

Tom van Dijk | Mike Vrieswijk

Today a few changes have been made to the architecture design of the temperature controller. The choice was made to use a premade power supply as opposed to a self-made one, because this was considering the time left to finish this device the best choice. A few alterations were made to make the architecture design better readable.

02

Architecture design [TC]

June 24

First prototype [TC]

June 25

Tom van Dijk | Mike Vrieswijk

A 3d drawing was made for the casing that will hold the temperature controller. The first prototype was printed, but was determent to be inadequate due to the lack of enough space for the cooler block.

01

First prototype [TC]

June 25

Team

June 26

Rens Boeser

Added the remaining instructors to the team

04

Team

June 26

Second prototype [TC]

June 26

Tom van Dijk | Mike Vrieswijk

In the drawing space was added to fit the cooler block for the temperature controller. Slits were added to the drawing for the nuts to slide into. The altered drawing was printed. The newly printed parts, cooler block, fan and Peltier element were assembled. Everything fit together.

02

Second prototype [TC]

June 26

Assessing CooA Production

June 26

Dustin van der Meulen

To start testing the production of CooA, we firstly resuspended and transformed the BioBricks from the kit witch we wanted to use (Table 1). This has been done using the iGEM Kit plate instructions (Protocol 1, step 1 - 3) and the NEB 10 béta transformation protocol adapted New England Biolabs (Protocol 2), using bought cells with a reported transformation efficienty of 1-3 10^9 cfu/μg pUC19. Inucbation was at 37°C.

Table 1 : Resuspended BioBricks from the iGEM 2018 Distribution kit and their uses.

BBa_K592009 Blue Chromoprotein AmilCP
BBa_B0031 Ribosomal binding site (strong), derived from BBa_B0030
BBa_B0032 Ribosomal binding site (medium), derived from BBa_B0030
BBa_B0030 Ribosomal binding site (weak)
BBa_B0015 Double terminator (BBa_B0010 & BBa_B0012)
BBa_J23100 Strong Constitutive Anderson Promotor
BBa_J23105 Medium Constitutive Anderson Promotor
BBa_J23113 Weak Constitutive Anderson Promotor
BBa_J45199 Banana odor enzyme (ATF1) generator
BBa_K1184000 Killer Red
BBa_K352001 CooA
BBa_K352011 CooA responsive system

After checking the plates on the 28th of June, we recieved the following results (Table 2):

Table 2 : Kolonies found after overnight growth.
BBa_K592009 20 colonies
BBa_B0031 4 colonies
BBa_B0032 5 colonies
BBa_B0030 1 colonies
BBa_B0015 6 colonies
BBa_J23100 1 colonies
BBa_J23105 98 colonies
BBa_J23113 19 colonies
BBa_J45199 0 colonies
BBa_K1184000 4 colonies
BBa_K352001 2 colonies
BBa_K352011 4 colonies

01

Assessing CooA Production

June 26

Testrun [TC]

June 27

Tom van Dijk | Mike Vrieswijk

A short test run was done with the newly assembled cooler. The test concluded that the temperature controller could cool a water droplet to the point of freezing and heat it back up to its boiling point.

03

Testrun [TC]

June 27

Assessing CooA Production

June 29

Dustin van der Meulen

After making a 20 mL overnight culture of our NEB 10 béta cells containing our BioBricks, we performed a mini prep (Protocol 3). Using our nanodrop spectrophotometer  which gave us the following DNA concentrations:
 

BBa_K592009 72,5 ng/μL
BBa_B0031 118,9 ng/μL
BBa_B0032 52,5 ng/μL
BBa_B0030 488,6 ng/μL
BBa_B0015 53,5 ng/μL
BBa_J23100 148,3 ng/μL
BBa_J23105 96,3 ng/μL
BBa_J23113 151,8 ng/μL
BBa_K118400 140,4 ng/μL
BBa_K352001 77,7 ng/μL
BBa_K352011 76,8 ng/μL

After assessign our results, in the future we will perform the ethanol carry-over steps.

Also from the previously mentioned overnight culture 1 mL has been used to make Glycerol Stock (Protocol 4)

02

Assessing CooA Production

June 29

Draft decision

July 2

Tom van Dijk | Mike Vrieswijk

The choice to use a computer PSU as a power supply was made. The choice was made on the grounds, that computer PSU have a standard 12, 5 and 3.3V output. This is beneficial because these are the voltages necessary for the selected components.

01

Draft decision

July 2

Assessing CooA Production

July 2

Dustin van der Meulen

Today we performed a digestion, dephosphorilation and ligation (following protocols 4 and 5) of a few promotor and RBS BioBricks. Because of administrative compliations only the variants with J23113 and a RBS could be made.

03

Assessing CooA Production

July 2

Architecture design [TC]

July 5

Tom van Dijk | Mike Vrieswijk

Changes were made to the architecture design of the temperature controller. Different voltage regulators were selected to fit the newly chosen power supply. To better readability of the architecture design a legend was added.

03

Architecture design [TC]

July 5

Electrical circuit design [TC]

July 8

Tom van Dijk | Mike Vrieswijk

Custom footprints were made for multiple components and added to the parts library in the PCB design software.

01

Electrical circuit design [TC]

July 8

Electrical circuit design [TC]

July 9

Tom van Dijk | Mike Vrieswijk

The provisional electronic circuit design for the temperature controller was made.

02

Electrical circuit design [TC]

July 9

Electrical circuit design [TC]

July 11

Tom van Dijk | Mike Vrieswijk

The electronic circuit design for the temperature controller was finished. Measuring point were added to the electronic circuit design.

03

Electrical circuit design [TC]

July 11

Stock up

July 11

Elise Grootscholten | Loraine Nelson | Paul Reusink

To have some basic biobricks ready, we stocked them by doing a plasmid isolation and freeze the DNA.

01

Stock up

July 11

Basic parts

July 11

Elise Grootscholten | Loraine Nelson | Paul Reusink

To start on a basic construct we did a digestion and ligation with the parts we isolated earlier. To prevent original constructs we did a defosphorylation after the digestion. We controlled the digestion by doing a gelelktroforeses.

01

Basic parts

July 11

Board layout [TC]

July 12

Tom van Dijk | Mike Vrieswijk

Started working on the board layout for the PCB of the temperature controller.

01

Board layout [TC]

July 12

Basic parts

July 12

Tom van Dijk | Randall de Waard

We continued with the ligated DNA by transformating it to Neb10Beta. This was plated onto agarplates and incubated for 1 day.

02

Basic parts

July 12

Board layout [TC]

July 13

Tom van Dijk | Mike Vrieswijk

Further work was done to the board layout for the temperature controller.

02

Board layout [TC]

July 13

Basic parts

July 13

Elise Grootscholten | Randall de Waard

The results of 7/12/2018 were collected, in action of this we decided to plate some ligated and transferred DNA from 7/12/2018 again. This we did because there wasn't a clear grow of colonies on the plates.

03

Basic parts

July 13

Blue White screening on paper

July 13

Elise Grootscholten | Randall de Waard

We tried to let bacteria grow on paper, therefore we used our own buisnesscards. We let the buisnesscards absorb some LB-medium and put them inside petridishes.

01

Blue White screening on paper

July 13

Board layout [TC]

July 15

Tom van Dijk | Mike Vrieswijk

Work has been done on the board layout for the temperature controller. The board layout is now finished.

03

Board layout [TC]

July 15

Basic parts

July 16

Mei Ju Goemans | Elise Grootscholten | Dustin van der Meulen | Randall de Waard

To know if the ligation was succesfull we will controll the DNA, therefor we ented the bacteria's with DNA in LB-medium to grow overnight. We also plated the used bacteria's onto new plates to use for further experiments.

04

Basic parts

July 16

Blue White screening on paper

July 16

Mei Ju Goemans | Elise Grootscholten | Dustin van der Meulen | Randall de Waard

We did a blue white screening on our buisnesscards to see if bacteria had grew on them. We transferred the buisnesscards to new petrydishes and added ITPG and X-gal to the petrydishes.

02

Blue White screening on paper

July 16

Notebook

July 17

Rens Boeser

Frontend design for the notebook

02

Notebook

July 17

Basic parts

July 17

Rens Boeser | Dustin van der Meulen | Loraine Nelson | Suzanne Romeijn | Randall de Waard

The resulst of 7/16/2018 showed that the DNA didnt ligate as planned. To be able to digest enough DNA we did a plasmid isolation again. We digested this DNA and froze it for further use.

05

Basic parts

July 17

Stock up

July 18

Dustin van der Meulen | Loraine Nelson | Suzanne Romeijn | Mike Vrieswijk | Randall de Waard

We did another round of plasmid isolation for stocking.

02

Stock up

July 18

Notebook

July 19

Rens Boeser

Designed a way to follow experiments that take up multiple days and eventually designed a notebook entry page and made it a responsive page

03

Notebook

July 19

Basic parts

July 19

Dustin van der Meulen | Mei Ju Goemans| Randall de Waard

We digested all DNA we have in stock to test for our basic construct. We tested the DNA by doing a gelelktroforeses.

06

Basic parts

July 19

Notebook

July 21

Rens Boeser

Created a one page notebook. Made the notebook entry page more suited for the entry length

04

Notebook

July 21

Assessing Gas Production BioBricks in E.Coli

July 23

Elise Grootscholten | Randall de Waard

Transformations of the biobricks K133071, K173003 and I13453 are performed. K133071 will produce CO2 if there's pyruvate present and K173003 will produce CO2 if urea is present. I13453 is a promotor which will work if there's arabinose present.                                                                                                        
The biobricks were transformed first into electrocometent cells and later into chemical competent cells from the strain NEb10Beta. After transformations the culture was plated on agar plates with antibiotics. The first transformations didn't work out, but the second did, because then the right competent cells were used. 

Experiment 1: Transformation biobricks

Transformation of the biobricks K133071, K173003 and I13453 in NEB10B�ta.

Materials 

- Biobricks (Bba_K133071, Bba_K173003 and Bba_I3453) 
- Nuclease free water
- LB- agar
- Variable Volume Pipettes 
- Sterile pipetpoints
- Chemically competent cells ( NEB10Beta, 1x10^9) 
- Heater (42�C)  
- Incubator + shaker (37�C and 250 rpm) 
- Eppendorf tubes (1,5 and 2 ml) 
- Stable outgrow medium for NEB10Beta or SOC- outgrow medium 
-Chloramphenicol (40 mg/mL)

Methods
-K173003 --> pSB1C3 backbone, plate 6, well 15N
-K133071 --> pSB1C3 backbone, plate 6, wel 21A
-I13453 --> pSB1C3 backbone, plate 3, well 19O

To be able to use the DNA from the biobricks, they were first diluted in Nuclease free water. This was done by adding 10ul of nuclease free water to the DNA and incubated for 5 minutes at room temperature (18-24�C). After incubation the DNA is transferred to Eppendorf tubes and put on ice. 

For the transformation 1 ul of the DNA was added to 50 ul chemically competent cells strain NEB10Beta. The mixture was incubated for 30 minutes on ice. The mixture got a heat shock by putting the tube in a heater (42�C) for 30 seconds. After the heat shock the tube was immediately put on ice for 5 minutes. 
For good grow of the cells, 950 ul of outgrow medium was added to the mixture. After mixing the cells with outgrow medium, the culture was incubated for 1 hour at 37�C and 250 rpm.  

After incubation the culture was plated onto LB-agar plates. For specific growth of bacteria there was antibiotics added to the plates. The antibiotics that were used, depended on the plasmid. The biobricks used in this experiment were resistent to chloramphenicol (Can). We worked with a work concentration of 35 ug/ml for chloramphenicol. For good results we plated 100 ul undiluted culture onto a plate and centrifuged the rest of the culture. The supernatant was discarded and the pellet was resuspended and plated onto a LB-agar plate. The plates were then incubated by 37�C. 

Results
The first transformation was done in electrocompetent cells instead of chemocompetent cells so the first transformation did not succeed.

 

Conclusion

The second transformation from the biobricks was successful. The bacteria with the biobricks plasmids will be stored by 4�C and can be used for further experiments. 
The CFU from the negative controle will be plated on a plate with antibiotics to make sure there isn't a plasmid in the bacteria. The outgrow medium will be sterilized by filter again. 
 

01

Assessing Gas Production BioBricks in E.Coli

July 23

ATP sensor

July 24

Rens Boeser | Mei Ju Goemans | Elise Grootscholten | Dustin van der Meulen | Loraine Nelson | Suzanne Romeijn | Randall de Waard

New biobricks, used to make an ATP sensor, were transformed into NEB10Beta. The culture was than plated onto agar and incubated for 1 day.

01

ATP sensor

July 24

Gas output

July 24

Rens Boeser | Mei Ju Goemans | Elise Grootscholten | Dustin van der Meulen | Loraine Nelson | Suzanne Romeijn | Randall de Waard

New biobricks, which produces gasses, were transformed into NEB10Beta. The culture was than plated onto agar and incubated for 1 day.

01

Gas output

July 24

Notebook generator

July 24

Rens Boeser

Started working on an automatic notebook creator

01

Notebook generator

July 24

Transformation

July 24

Loraine Nelson | Elise Grootscholten | Paul Reusink

Today we did a transformation with new biobricks. The biobricks were built into chemically competent cells from the strain NEb10Beta. After transformation the culture was plated on agar plates with antibiotics. The results were checked the next day. 

Materials: 

- Biobricks (Bba-K1499004 and Bba- K1614019) 
- Nuclease free water
- LB- agar
- Variable Volume Pipettes 
- Sterile pipetpoints
- Chemically competent cells ( NEB10Beta, 1x10^9) 
- Heater (42�C)  
- Incubator + shaker (37�C and 250 rpm) 
- Eppendorf tubes (1,5 and 2 ml) 
- Stable outgrow medium for NEB10Beta or SOC- outgrow medium 

Method: 

To be able to use the DNA from the biobricks, they were first diluted in Nuclease free water. This was done by adding 10ul of nuclease free water to the DNA and incubated for 5 minutes at room temperature (18-24�C). After incubation the DNA is transferred to Eppendorf tubes and put on ice. 

For the transformation 1 ul of the DNA was added to 50 ul chemically competent cells strain NEB10Beta. The mixture was incubated for 30 minutes on ice. The mixture got a heat shock by putting the tube in a heater (42�C) for 30 seconds. After the heat shock the tube was immediately put on ice for 5 minutes. 
For good grow of the cells, 950 ul of outgrow medium was added to the mixture. After mixing the cells with outgrow medium, the culture was incubated for 1 hour at 37�C and 250 rpm.  

After incubation the culture was plated onto LB-agar plates. For specific growth of bacteria there was antibiotics added to the plates. The antibiotics that were used, depended on the plasmid. The biobricks used in this experiment were resistent to chloramphenicol (Can). We worked with a work concentration of 35 ug/ml for chloramphenicol. For good results we plated 100 ul undiluted culture onto a plate and centrifuged the rest of the culture. The supernatant was discarded and the pellet was resuspended and plated onto a LB-agar plate. The plates were then incubated by 37�C. 

Results:
Tabel 1 shows the results of the plates which were incubated. 

Biobricks:  Antibiotics:  Dilution:  Colonies: CFU: 
Bba-K1614019  Can  0x  109 1*10^3 /ml
Bba-K1614019 Can Centrifuged >300    -
Bba-K1499004     Can 0x 33 3*10^2 /ml
Bba-K1499004         Can Centrifuged 228    -         
Positive controle: Bba- K1499004 None 0x > 300    -
Negative controle: outgrow medium none 0x 18    - 

Tabel 1: results from the transformation with the biobricks Bba-K1614019 and Bba-K1499004. 

The plates show CFU which can be used for further experiments.  The negative controle also has CFU, this shows that the outgrow medium was infected and not sterile. 
The plates are stored by 4�C. 

Conclusion: 

The transformation from the biobricks was successful. The bacteria with the biobricks plasmids will be stored by 4�C and can be used for further experiments. 
The CFU from the negative controle will be plated on a plate with antibiotics to make sure there isn't a plasmid in the bacteria. The outgrow medium will be sterilized by filter again. 

 

01

Transformation

July 24

Assessing Gas Production BioBricks in E.Coli

July 24

Elise Grootscholten | Randall de Waard

To be able to always get the necessary biobricks, there have been made glycerolstocks of the transformed biobricks K133071, K173003 and I13453. The glycerolstocks are stored at -80 degrees Celsius. If needed, they can be retrieved from this storage to use for experiments. 

Experiment 2: Glycerolstocks

Making glycerol stocks of the biobricks K133071, K173003, I13453. 

Materials: 
- 87% glycerol 
- culture with biobrick plasmids
- LB-medium
- Variable Volume Pipette
- Sterile pipetpoints

Methods: 
The colonies from the transformation were used to make an overnight culture. The culture was made by putting a colony in LB-medium and then the culture was incubated for a night at 37�C.  

The next day the cultures had grown close and were ready for a glycerol stock. For making glycerol stocks there was added 250 ul 87% glycerol to 1 ml culture. This was frozen in -80�C.  

The stocks were stored at -80�C in drawer 3, tower 1, drawer 

02

Assessing Gas Production BioBricks in E.Coli

July 24

Notebook generator

July 25

Rens Boeser

Automatic generation of entries completed

02

Notebook generator

July 25

Google Drive API

July 25

Rens Boeser

Used the Google drive REST API to download the entries from drive

01

Google Drive API

July 25

Automatic uploader

July 25

Rens Boeser

Used the igemwiki API to update pages using software

01

Automatic uploader

July 25

Transformation

July 27

Loraine Nelson | Elise Grootscholten | Paul Reusink

To be able to always get the necessary biobricks, we made an glycerol stock after every transformation. The glycerol stocks are stored at -80 degrees Celsius. If needed, they can retrieved from this storage to use for experiments. 

Making glycerol stocks of the biobricks. 

Materials: 
- 87% glycerol 
- culture with biobrick plasmids
- LB-medium
- Variable Volume Pipette
- Sterile pipetpoints


Method: 

The colonies from the transformation were used to make an overnight culture. The culture was made by putting a colony in LB-medium and then the culture was incubated for a night at 37�C.  

The next day the cultures had grown close and were ready for a glycerol stock. For making glycerol stocks there was added 250 ul 87% glycerol to 1 ml culture. This was frozen in -80�C.  

The stocks were stored at -80�C in drawer 3, tower 1, drawer 

02

Transformation

July 27

Assessing Gas Production BioBricks in E.Coli

July 27

Elise Grootscholten | Randall de Waard

For further experiments there is isolated DNA needed of the biobricks J23100, K133071, K173003 and I134353. The DNA is isolated out of the bacteria with the help of a plasmid purifaction kit. After isolation this DNA can be used for digestion and ligation or other experiments. J23100 (from the glycerolstock): 325,59 ng/ul, K173003: 217,06 ng/ul, K133071: 186,79 ng/ul, I13453: 88,18 ng/ul

Experiment 3: Minipreps

Minipreps of the following biobricks in NEB10B�ta:
-J23100: Constitutive promoter
-K133071: Urea --> ammonia + CO2
-K173003: Pyruvate --> acetaldehyde + CO2
-I134353: Promoter (AraC protein binds with arabinose)

Materials: 
- Mini prep, Plasmid Purification Kit, Machery Michels 
- Eppendorf cups
- Variable Volume Pipets
- sterile pipet points
-Chloramphenicol (40 mg/mL)

Method: 
Miniprep protocol...??
After the transformation we made an overnight culture from the colonies. The colonies were anted into 20 ml LB and incubated overnight at 37�C and 150 rpm. The culture was mini prepped the next day. For isolation, 5,4 ml was added step wise in a 2ml Eppendorf cup. The culture was then centrifuged at >12000x g for 30 seconds. The supernatant was deposed, and the pellet was resupended with A1 buffer and vortexed. To the resuspended culture was then.....???

Results

J23100: 325,59 ng/ul
K173003: 217,06 ng/ul
K133071: 186,79 ng/ul
I13453: 88,18 ng/ul

03

Assessing Gas Production BioBricks in E.Coli

July 27

Automation program

July 29

Rens Boeser

Connected the page uploader to the notebook generator

01

Automation program

July 29

Automatic uploader

July 29

Rens Boeser

Created a program that uploads all updated pages

02

Automatic uploader

July 29

Page generator

July 31

Rens Boeser

Built a page builder that automatically adds the header and footer before uploading

01

Page generator

July 31

Google Drive API

July 31

Rens Boeser

Redone the google API handler for reusability and readability and made the google API handler generic

02

Google Drive API

July 31

Automation program

July 31

Rens Boeser

revamped the main program

02

Automation program

July 31

Automation program

August 1

Rens Boeser

Finished redoing the application for generating and uploading pages

03

Automation program

August 1

Notebook

August 2

Rens Boeser

Added month categorization to the page

05

Notebook

August 2

Transformation

August 9

Loraine Nelson | Elise Grootscholten | Paul Reusink

For further experiments we needed isolated DNA. The DNA is isolated out of the bacteria with the help of a plasmid purifaction kit. After isolation this DNA can be used for digestion and ligation or other experiments.

Materials: 
- Mini prep, Plasmid Purification Kit, Machery Michels 
- Eppendorf cups
- Variable Volume Pipets
- sterile pipet points


Method: 
After the transformation we made an overnight culture from the colonies. The colonies were anted into 20 ml LB and incubated overnight at 37�C and 150 rpm. The culture was mini prepped the next day. For isolation, 5,4 ml was added step wise in a 2ml Eppendorf cup. The culture was then centrifuged at >12000x g for 30 seconds. The supernatant was deposed, and the pellet was resupended with A1 buffer and vortexed. To the resuspended culture was then. 

03

Transformation

August 9

Assessing Gas Production BioBricks in E.Coli

August 10

Elise Grootscholten | Randall de Waard

The biobricks J23100, K133071, K173003 and I134353 were sucessfully digested after the second time. After the digestions the biobricks K133071 and K173003 were dephosphorylated and ligated with the inserts J23100 and I13453. This was done in the original backbone of K133071 and K173003, and not another control backbone. To know if the biobricks were right ligated this was done by testing practically. See the experiments: Testing gas production. 

Experiment 4: Digestion, Defosforylation and ligation
Digestions
30-07-18 and 08-08-18

Biobricks: J23100, K133071, K173003 and I13453

Used pipette scheme (Table 1) to prepare the DNA to be cut with multiple combinations of EcoRI-HF, SpeI-HF, PstI and XbaI.

DNA ~ 2 μg
NEBbuffer 2.1 5 μL
Restriction enzyme 1 (1 U/μL) 2 μL
Restriction enzyme 2 (1 U/μL) 2 μL
Nuclease free water Fill to 100 μL
(Table 1 ; Double Digest Pipette Scheme using : https://nebcloner.neb.com/#!/redigest, and doubling the amount)


Biobricks:
J23100: Constitutive promotor
K133071: Urea --> ammonia + CO2
K173003: Pyruvate --> acetaldehyde + CO2
I134353: Promotor (AraC protein binds with arabinose)

With this we started a digest of the following BioBricks using the following Restriction enzyme couples (Table 2)
 
Number BioBrick BBa_ code Restriction enzyme 1 Restriction enzyme 2
9 J23100 (35 bp) EcoRI SpeI
11 K133071 (1707 bp) EcoRI XbaI
12 K173003 (3052 bp) EcoRI XbaI
10 I13453 (130 bp) EcoRI SpeI

Table 2: Biobricks and restriction enzymes

Digestion was started on the 30th of July, ~16:00, 37°C and 185 RPM.
Digestion was stopped on the 31st of July, ~10:00.

This has been done in tandem with the corresponding part from Cloning Mulitple BioBricks to Assess CooA Production Part X 



Dephosphorylation
10-08-18

K133071 and K173003 were dephosphoralized:
 

Volume Compound
25 μL Vector DNA (0,5 μg)
3 μL phosphatase buffer (10x)
1 μL milli Q
1 μL phosphatase (1 U/μL)
30 μL Total
Table 3: Pipetting scheme

The new DNA concentration of the reaction mix is 0,5 μg/30 μL
The reaction mix was incubated for 10 minutes at 37 °C
The phosphatase was inactivated for 2 minutes at 75 °C
 
For the ligation mix 100 ng of vector DNA was used from the dephosphorylation reaction mix.
The following vector-insert combinations were ligated:
Vector Insert 1 Insert 2
K133071 J23100 I13453
K173003 J23100 I13453
Table 4: Biobrick combination

The original plasmids of the vector and inserts that were digested are about the same size in bp and the ratio vector DNA-insert DNA is determined to be 1:3. Therefore, the amount of insert DNA that was used for the ligation was 140 ng (rounded up to 7 μL of the digestion reaction mix). Because there was only 5 μL left of the K352001 insert DNA, the total 5 μL were used and an extra 2 μL of milli Q water were added to the pre-ligation mix.

Pre-Ligationmix:
Volume Compound
6 μL Vector DNA (0,5 μg/30 μL)
7 μL Insert DNA (2 μg/100 μL)
4 μL Delutionbuffer (5x)
3 μL Milli Q
20 μL Total
Table 5: Pre-ligationmix pipette scheme

Ligationmix:
incubation at room-temperature for 20 minutes.
Volume Compound
10 μL Pre-ligationmix
2 μL 10x T4 ligationbuffer
1 μL T4 DNA ligase (1U/μL)
8 μL Milli Q
20 μL Total
Table 6: Ligationmix pipette scheme
 
All the vectors contain the CamR gene. 30 plates were poured with Cam (14 for the transformed cells (100μL), 14 for the transformed cells (900 μL centrifuged and resuspended in 100 μL and 1 positive and 1 negative control)

 

04

Assessing Gas Production BioBricks in E.Coli

August 10

Urea and sodium pyruvate test for resistance E.coli

August 10

Randall de Waard | Elise Grootscholten

Testing different amounts of urea and sodiumpyruvate to know which concentrations the bacteria survive.

Experiment 1: Testing different concentrations of Urea and Sodium pyruvate
08-08-18

Testing different amountsof urea and sodiumpyruvate to know which concentrations the bacteria survive.

-First let the LB agarose heat up (95 degrees Celcius)
-Second, weigh urea and sodium pyruvate in beakers (see table 1)

What How much Solution
Urea 1 g 1 mL purified water
Urea 2,4 g 2 mL purified water
Urea 4,8 g 4 mL purified water
Sodium pyruvate 5,7 mL None

Table 1: weighing urea and sodium pyruvate for LB agarose plates.
-Purify these substances, so it is free of bacteria. Work beneath the flame and pour it in to plastic 50 mL tubes.
-Pour the LB agarose in the plastic tubes when it's about 45 degrees Celcius and mix gently.
-Pour the LB agarose mix in a petridish and wait until it's dry. Do this for all the plastic tubes.
-Also fill 2 petridishes with LB agarose without other substances (positive and negative control).
-prick a BL21(DE3) bacteria and put it in a 100 mL erlenmeyer with 20 mL culture medium.

09-08-18

Pipette 100 ul overnight culture on each plate, except on the negative control, and divide it. (24h 37 degrees Celcius)

 

Results
 

What How much Solution Result
Urea 1 g 1 mL purified water Grow
Urea 2,4 g 2 mL purified water No grow
Urea 4,8 g 4 mL purified water No grow
Sodium pyruvate 5,7 mL - Grow
Positive control - - Grow
Negative control - - No grow

Table 1: Results experiment 1

Conclusion

More tests will be done with urea and sodiumpyruvate because with 1 g urea the bacteria live and with 2,4 g urea the bacteria die.
The bacteria survive 5,7 mL sodiumpyruvate so we will test with higher concentrations.
 

01

Urea and sodium pyruvate test for resistance E.coli

August 10

Assessing Gas Production BioBricks in E.Coli

August 13

Elise Grootscholten | Randall de Waard

Transformations of the biobricks (K133071 + J23100), (K13071 + I1345), (K173003 + J23100) and (K173003 + I13453) in NEB10B�ta. There was no grow except for the biobrick combination K133071 + J23100.

Experiment 5: Transformation

13-08-2018
Transformations of the ligations of experiment 4

Materials

-Chloramphenicol (40 mg/mL)
-LB-agar
-Ligations
-Heat block,42 degrees Celcius
-Shaker, 37 degrees Celsius 
-Chemo competent cells NEB10b�ta (New England Biolabs)

Methods

10 ul ligationmix is used for every transformation

Transformation Protocol
Overview

Quick Ligation products may be transformed by many different methods. The following protocol is recommended by New England Biolabs.

Protocol

  1. Thaw competent cells on ice.
  2. Chill approximately 5 ng (2 μl) of the ligation mixture in a 1.5 ml microcentrifuge tube.
  3. Add 50 μl of competent cells to the DNA. Mix gently by pipetting up and down or flicking the tube 4�5 times to mix the cells and DNA. Do not vortex.
  4. Place the mixture on ice for 30 minutes. Do not mix.
  5. Heat shock at 42°C for 30 seconds*. Do not mix.
  6. Add 950 μl of room temperature media* to the tube.
  7. Place tube at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
  8. Warm selection plates to 37°C.
  9. Spread 50�100 μl of the cells and ligation mixture onto the plates.
  10. Incubate overnight at 37°C.

    * Please note: For the duration and temperature of the heat shock step as well as for the media to be used during the recovery period, please follow the recommendations provided by the competent cells� manufacturer.


Instead of 2 ul of the ligation mix, use 10 ul ligation mix.

05

Assessing Gas Production BioBricks in E.Coli

August 13

Assessing Gas Production BioBricks in E.Coli

August 13

Elise Grootscholten | Randall de Waard

A colony PCR is done for the NEB10B�ta E.coli cells with expected the biobrick combination of K133071 with J23100. Nevertheless, on a gel the difference with and without promotor couldn't be seen. So there must be another way of proving the right biobricks are there.

Expermiment 6: Colony PCR K133071 + J23100

Mastermix

What 1 reaction 30 reactions
Taq buffer(10x) 5 ul 150 ul
10 mM dntp 1 ul 30 ul
10 uM Forward primer 1 ul 30 ul
10 uM Reverse primer 1 ul 30 ul
Taq polymerase 0,25 ul 7,5 ul
Nucease free hydrogen 41,75 ul 1252,5 ul
Total 50 ul 1500 ul

 

06

Assessing Gas Production BioBricks in E.Coli

August 13

Assessing Gas Production BioBricks in E.Coli

August 13

Elise Grootscholten | Randall de Waard

The ligations and transformations of the biobricks K173003 + J23100, K173003 + I13453, and K133071 + I13453 have been performed again. There was a lot of grow on the agar plates. To know if the biobricks were right ligated this was done by testing practically. See the experiments: Testing gas production. 

Experiment 7: Ligation of biobricks
K173003 + J23100
K173003 + I13453
K133071 + I13453
 

Volume Compound
6 μL vector DNA (0,5 μg/30 μL) (100 ng)
14 μL insert DNA (2 μg/100 μL) (280 ng)
4 μL dilutionbuffer (5x)
1 μL milli Q water
25 μL Total


Use 10 mL

Ligationmix:

Volume Compound
10 μL pre-ligationmix 
2 μL 10x T4 ligationbuffer
1 μL T4 DNA ligase (1U/μL)
8 μL milli Q water
20 μL Total

incubation at room-temperature for 20 minutes.
Store at -20 degrees Celcius

07

Assessing Gas Production BioBricks in E.Coli

August 13

Testing gas production

August 16

Elise Grootscholten | Paul Reusink

Making a set up for the gas production testing and testing it with NEB10B�ta with the expected biobricks in it (K133071 + J23100) and a negative control.

Experiment 1: Gasproduction testing

15-08-18
Making a set up for the gas production testing and testing it with NEB10B�ta with the expected biobricks in it (K133071 + J23100) and a negative control.

Materials
-15 mL tubes
-Little glass tubes
-Cocktail prickers
-Culture medium
-Urea
-Chloramphenicol (40 mg/mL)


Methods
Stick tape on the little tube and cover the little tubes with tin foil. This can now enter the autoclave.
Make overnight cultures (20 mL)of the desired colonies in culture medium with 17,5 ul/ 20mL Chloramphenicol.

After a day, prepare the culture medium for the tests.
Put 1 g urea and 17,5 ul Chloramphenicol in 20 mL culture medium.

Pipette 10 mL of the overnight culture in the 15 mL tube en centrifuge 5000 rpm fot 5 minutes. Throw the supernatant away.
Fill the 15 mL tube and the little tube to the edge with culture medium. Work sterile.
Put a paper on the little tube and turn it around.
Then hold it above the 15 mL tube and pull the paper away and let the little tube fall into the 15 mL tube.
Try to do this so there's no gas in the little tube.
Now wait until gas is produced.

16-08-18
Making a set up for the gas production testing and testing it with NEB10B�ta with the expected biobricks in it (K133071 + J23100).
20 different colonies are tested.

Materials
Use the same materials as above.

Methods
Use the protocol above.
Make 700 mL culture medium with 35 g urea and 612,5 ul Chloramphenicol (40 mg/mL). This cannot be done sterile.
The culture medium can be used for the 20 different colonies with expected biobricks: K133071 + J23100.

 


Results

15-08-18
Gas was let into the little tubes so it was difficult to see whether there was gasproduction or not. Mainly in the negative control this was 

 

01

Testing gas production

August 16

Testing gas production

August 23

Elise Grootscholten | Paul Reusink

Making a set up for the gas production testing and testing it with NEB10B�ta with the expected biobricks in it (K133071 + I13453), (K173003 + J23100), (K173003 + I13453) and a negative control.

Experiment 2: Gasproduction testing
20-08-18

Making a set up for the gas production testing and testing it with NEB10B�ta with the expected biobricks in it (K133071 + I13453), (K173003 + J23100), (K173003 + I13453) and a negative control

Materials
See materials Experiment 1

Methods
See methods Experiment 1
850 mL culture medium is made with 21,5 g LB for 42 erlenmeyers (also for CooA production).
Overnight cultures are made of 10 colonies each of K173003 + I13453 and K173003 + J23100.

21-08-18

K173003 + J23100 are tested (see methods experiment 1)
Overnight cultures are made of  K133071 + I13453 and K173003 + I13453 are tested later.

Results
Colonies 9 and 10 are chosen use for further tests.

02

Testing gas production

August 23

Testing gas production

August 23

Elise Grootscholten | Paul Reusink

Gasproduction testing for the biobricks (K173003 + I13453), (K133071 + I13453) and negative controls (B0015 and K133071 without urea and arabinose).
K173003 + I13453 is tested with sodiumpyruvate and arabinose for gasproduction and K133071 + I13453 is tested with urea and arabinose for gasproduction.
The negative control also produces a little bit gas.

Experiment 3: Gasproduction testing

Gasproduction testing for the biobricks K173003 + I13453, K133071 + I13453 and negative controls.
K173003 + I13453 is tested with sodiumpyruvate and arabinose for gasproduction and K133071 + I13453 is tested with urea and arabinose for gasproduction.

Materials
See experiment 1 and 2.

Methods
See experiment 1 and 2.
375 mL culture medium with CAM (already in it) + 18,75 g urea + 0,5 ul arabinose.
340 mL culture medium with CAM (already in it) + 51 mL natriumpyruvate (20 mM) + 0,5 ul arabinose.


Results and conclusion
Colonies 5 and 7 of the biobricks K173003 + I13453 (pyruvate)  and number 4 of the biobricks K133071 + I13453 (urea).
In both the negative controls there's been gas produced. Futher gasproduction test will be done.

03

Testing gas production

August 23

Testing gas production

August 23

Elise Grootscholten | Paul Reusink

Testing the gasproduction of the colonies 9 and 10 of biobricks (K173003 + J23100), colonies 5 and 7 of biobricks (K173003 + I13453) and colony 4 of biobricks (K133071 + I13453) with and without centrifuging the bacteria. There is also a negative control (J04450 pSB1K3) with Kanamycine. The negative contol started with a lot of gas inside the tube. We can not see wether there is produced more after a day or not. This have to be tested later.

Experiment 4: Gasproduction testing 
24-08-18

Testing the gasproduction with and without centrifuging the bacteria.

methods
See methods experiment 1
2 negative controls
colonies 9 and 10 of biobricks K173003 + J23100.
Colonies 5 and 7 of biobricks K173003 + I13453
Colony 4 of biobricks K133071 + I13453

Culture medium:
150 mL: Sodiumpyruvate (20 mM) 22,5 mL + Chloramphenicol (40 mg/mL) 131 ul
150 mL: Sodiumpyruvate (20mM) 22,5 mL +  Chloramphenicol (40 mg/mL) 131 ul + Arabinose (300 mg/mL) 0,5 ul
80 mL: Urea 4 g + Chloramphenicol (40 mg/mL) 70 ul + Arabinose (300 mg/mL) 0,5 ul
80 mL: Kanamycine (40 mg/mL) 70 ul

 

04

Testing gas production

August 23

Assessing Gas Production BioBricks in E.Coli

August 27

Elise Grootscholten | Randall de Waard

Minipreps are made of colonies 11 and 19 of biobrick combination K133071 + J23100. Results: 11. 270,57 ng/ul 19. 253,59 ng/ul.

Minipreps
21-08-18

Minipreps of colonies 11 and 19 (see Testing gasproduction experiment 1)

Materials
See experiment 3

Methods 
See experiment 3

Results

11. K133071 + J23100: 270,57 ng/ul
19. K133071 + J23100: 253,59 ng/ul

08

Assessing Gas Production BioBricks in E.Coli

August 27

Assessing Gas Production BioBricks in E.Coli

August 27

Elise Grootscholten | Randall de Waard

A miniprep of the biobrick K352002 is made. The concentration is 69,77 ng/ul.

Experiment 9: Minipreps
28-08-18

Materials
See experiment 3

Methods
See experiment 3
Make minipreps of the biobricks K352002, K352003

Results
See figure 1

29-08-18
Materials
See experiment 3

Methods
See experiment 3
Make minipreps of the biobrick K352002

Results
See figure 2

30-08-18
Materials
See experiment 3

Methods
See experiment 3
Make minipreps of the biobricks K133116 and K173013

Results
See image experiment 9

09

Assessing Gas Production BioBricks in E.Coli

August 27

Urea and sodium pyruvate test for resistance E.coli

August 28

Randall de Waard | Elise Grootscholten

Testing different amounts of urea and sodiumpyruvate to know which concentrations the bacteria survive.

Experiment 2: Testing different concentrations of Urea and Sodium pyruvate
13-08-18


Same testing like experiment 1 but with different concentrations.

What How much Solution
Urea 1,2 g 1 mL purified water
Urea 1,6 g 2 mL purified water
Sodium pyruvate (10 mM) 10 mL None


Results
 
What How much Solution Result
Urea 1,2 g 1 mL purified water Little grow
Urea 1,6 g 2 mL purified water No grow
Sodium pyruvate (10 mM) 10 mL - Grow
Positive control - - Grow
Negative control - - No Grow
Table 2: Results experiment 2

Conclusion

For urea is 1 g used/20 mL and for sodiumpyruvate is 6 mL (10mM) or 3 mL (20 mM) used.
 

02

Urea and sodium pyruvate test for resistance E.coli

August 28

Assessing Gas Production BioBricks in E.Coli

September 7

Elise Grootscholten | Randall de Waard

Digestions, gelelectrophoresis, dephosphorylations and ligations of different biobrick combinations. These biobrick combinations are 4 different promoters (J23100, I13453, K352002, K352003)  with 4 different gasproduction biobricks (k173003, K173013, K133071, K133116). J23100 is about 1 kb to long. The rest seems likely to be right digested. The ligations will be transformed in NEB10b�ta and digested again as control.

Experiment 10: Digestion, defosforylation and ligation

Materials
See experiment...

Methods
See experiment ..

 

DNA (J23100 325,59 ng/μl) ~ 2 μg 6,1
Cutsmart (10x) 5 μL
Restriction enzyme 1 (1 U/μL) 1 μL
Restriction enzyme 2 (1 U/μL) 1 μL
Nuclease free water Fill to 50 μL

Table 1: Digestion scheme 1
 

DNA (J23100 325,59 ng/μl) ~ 2 μg 6,1
Cutsmart (10x) 5 μL
Restriction enzyme 1 (1 U/μL) 1 μL
Nuclease free water Fill to 50 μL
Table 2: Digestion scheme 2
 
DNA (J23100 325,59 ng/μl) ~ 2 μg 6,1
Cutsmart (10x) 5 μL
Restriction enzyme 1 (1 U/μL) 1 μL
Nuclease free water Fill to 50 μL
Table 3: Digestion scheme 3
 
DNA (K352003 180,65 ng/μl) ~ 2 μg 11,1 μL
Cutsmart (10x) 5 μL
Restriction enzyme 1 (1 U/μL) 2 μL
Restriction enzyme 2 (1 U/μL) 2 μL
Nuclease free water Fill to 50 μL
Table 4: Digestion scheme 4
 
DNA (K352002 69,77 ng/μl) ~ 2 μg 28,7 μL
Cutsmart (10x) 5 μL
Restriction enzyme 1 (1 U/μL) 2 μL
Restriction enzyme 2 (1 U/μL) 2 μL
Nuclease free water Fill to 50 μL
Table 5: Digestion scheme 5
 
DNA (K173003 217,06 ng/μL) ~ 2 μg 9,2 μL
Cutsmart (10x) 5 μL
Restriction enzyme 1 (1 U/μL) 2 μL
Restriction enzyme 2 (1 U/μL) 2 μL
Nuclease free water Fill to 50 μL
Table 6: Digestion scheme 6
 
DNA (I13453 88,18 ng/μL) ~ 2 μg 22,7 μL
Cutsmart (10x) 5 μL
Restriction enzyme 1 (1 U/μL) 1 μL
Restriction enzyme 2 (1 U/μL) 1 μL
Nuclease free water Fill to 50 μL
 Table 7: Digestion scheme 7
 
DNA (K133071 186,79 ng/μL) ~ 2 μg 10,7 μL
Cutsmart (10x) 5 μL
Restriction enzyme 1 (1 U/μL) 1 μL
Restriction enzyme 2 (1 U/μL) 1 μL
Nuclease free water Fill to 50 μL
 Table 8: Digestion scheme 8
 
DNA (pSB1K3 163,93 ng/μL) ~ 2 μg  12,2 μL
Cutsmart (10x) 5 μL
Restriction enzyme 1 (1 U/μL) 2 μL
Restriction enzyme 2 (1 U/μL) 2 μL
Nuclease free water Fill to 50 μL
Table 9: Digestion scheme 9
 
DNA (K173013 345,1 ng/μl) ~ 2 μg  5,8 μL
Cutsmart (10x) 5 μL
Restriction enzyme 1 (1 U/μL) 1 μL
Restriction enzyme 2 (1 U/μL) 1 μL
Nuclease free water Fill to 50 μL
 Table 10: Digestion scheme 10
DNA (K133116 194,1 ng/μl) ~ 2 μg 10,3 μL
Cutsmart (10x) 5 μL
Restriction enzyme 1 (1 U/μL) 1 μL
Restriction enzyme 2 (1 U/μL) 1 μL
Nuclease free water Fill to 50 μL
Table 11: Digestion scheme 11
 
Biobrick Restriction-enzyme 1 Restriction enzyme 2
K173003 XbaI PstI-HF
K173013 XbaI PstI-HF
K133071 XbaI PstI-HF
K133116 XbaI PstI-HF
J23100 EcoRI-HF SpeI-HF
I13453 EcoRI-HF SpeI-HF
K352002 EcoRI-HF SpeI-HF
K352003 EcoRI-HF SpeI-HF
pSB1K3 EcoRI-HF PstI-HF
Table 12: Digestion scheme used enzymes

Gelelectrophoresis
Number What Amount
1. Ladder 5 μL
2. J23100 EcoRI-HF+ SPeI-HF 5 μL
3. J23100 EcoRI-HF 5 μL
4. J23100 SpeI-HF 5 μL
5. pSB1K3 EcoRI-HF + PstI-HF 5 μL
6 K173013 XbaI + PstI-HF 5 μL
7. K173003 XbaI + PstI-HF 5 μL
8. K133071 XbaI + PstI-HF 5 μL
9. K133116 XbaI + PstI-HF 5 μL
10. K352002 EcoRI-HF + SpeI-HF 5 μL
11. K352003 EcoRI-HF + SpeI-HF 5 μL
12. I13453 EcoRI-HF + SpeI-HF 5 μL
13. Ladder  5 μL
14. Ladder 10 μL
Table 13: Gelelectrophoresis order of digestions

dephosphorylation

 

Volume Compound
12,5 μL Vector DNA (0,5 μg)
2 μL phosphatase buffer (10x)
4,5 μL milli Q
1 μL phosphatase (1 U/μL)
20 μL Total
Table 14: Pipetting scheme dephosphorylation
 
Volume Compound
37,5 μL Vector DNA (0,5 μg)
6 μL phosphatase buffer (10x)
13,5 μL milli Q
3μL phosphatase (1 U/μL)
60 μL Total
Table 15: Pipetting scheme dephosphorylation

Pre-Ligationmix:
Volume Compound
7,5 μL Insert DNA (2 μg/50 μL) 300 ng
7,5 μL Insert DNA (2 μg/50 μL) 300 ng
4 μL Backbone (0,5 μg/ 20 μL) 100 ng
5 μL Delutionbuffer (5x)
1 μL Milli Q
25 μL Total
Table 16: Pre-ligationmix pipette scheme

Ligationmix:
Volume Compound
10 μL Pre-ligationmix
2 μL 10x T4 ligationbuffer
1 μL T4 DNA ligase (1U/μL)
8 μL Milli Q
20 μL Total
Table 17: Ligationmix pipette scheme

Ligation started at 15.45 (31-08-18) at room temperature.
end ligation around 11.00 (01-09-18)
Store at -20 degrees Celcius.
 

10

Assessing Gas Production BioBricks in E.Coli

September 7

Assessing Gas Production BioBricks in E.Coli

September 7

Elise Grootscholten | Randall de Waard

Transformations of the biobrick combinations in NEB10b�ta. The transformations are plated on kanamycine agar plates, because all the ligations were done in pSB1K3 (kanamycine resistence) backbone. Pink and with colonies appeared after incubation by 37 degrees Celcius for about 12h. The white colonies will be used for further experiments.

Experiment 11: transformation of the biobrickombinations in NEB10b�ta.
03-09-18

Materials
See materials experiment 5.
-Kanamycin (40 mg/mL)
-Ampicillin (50 mg/mL)


Methods
See methods experiment 5

Make 19 agar plates with kanamycine (Kana) (17,5 ul/20mL) and 1 agar plate with ampicillin (Amp) (40 ul/mL). Plate all the transformations on agar plates with Kana , because all the ligations are done in the pSB1K3 (Kana resistence) backbone. 
Devide one Amp plate and one Kana plate in about 30 parts. Because the biobrick combinations with biobrick K133116 have originally Amp and Kana resistence, the right backbone plasmid (with hopefully the right ligation) is only growing on the agar plate with Kana.


Results
There is grown n every transformation plates, so the transformations are done well.
There are pink and white colonies present. Only the white colonies will be used for further experiments.
 

11

Assessing Gas Production BioBricks in E.Coli

September 7

Assessing Gas Production BioBricks in E.Coli

September 11

Elise Grootscholten | Randall de Waard

Plasmid DNA was isolated of 20 different colonies. The first time something went wrong. The second time we had good concentrations of isolated plasmid DNA. After this we will digest the DNA to see if the plasmids all have the required biobricks. The tube have a code from now on, see table 1.
 

Number Promotor Gene Backbone
A K352002 (CooF) K173013 pSB1K3
B K352002 (CooF) K173003 pSB1K3
C K352002 (CooF) K133071 pSB1K3
D K352003 (CooM) K133071 pSB1K3
E K352003 (CooM) K173003 pSB1K3
F K352003 (CooM) K173013 pSB1K3
H I13453 K173013 pSB1K3
I I13453 K173003 pSB1K3
J I13453 K133071 pSB1K3

Table 1: list of biobricks of abbreviations

Experiment 12: Overnight cultures and minipreps
05-09-18/06-09-18

Materials
See materials experiment 3

Methods
See methods experiment 3

Number Promotor Gene Backbone
A K352002 (CooF) K173013 pSB1K3
B K352002 (CooF) K173003 pSB1K3
C K352002 (CooF) K133071 pSB1K3
D K352003 (CooM) K133071 pSB1K3
E K352003 (CooM) K173003 pSB1K3
F K352003 (CooM) K173013 pSB1K3
G B0032 J23105 pSB1A3
H I13453 K173013 pSB1K3
I I13453 K173003 pSB1K3
J I13453 K133071 pSB1K3
Table 1: list of biobricks of abbreviations
 

12

Assessing Gas Production BioBricks in E.Coli

September 11

Assessing Gas Production BioBricks in E.Coli

September 11

Elise Grootscholten | Randall de Waard

The minipreps of experiment 12 are digested with the restriction enzymes: SmaI and ScaI. Only Sca and Sma are both incubated at 37 degrees Celcius. Sma has to be incubated at 25 degrees Celcius. This is done in experiment 15.

Experiment 12: Digestions of the minipreps
10-09-18

Materials
-SmaI (restriction-enzyme)
-ScaI (restriction-enzyme)
See materials experiment 10

Methods
See methods experiment 10
 

DNA (A1 396.7 ng/μl) ~ 2 μg 5.0 μL
Cutsmart (10x) 5 μL
Sca1-HF (2 U/μL) 1 μL
Sma1 (2 U/μL) 1 μL
Nuclease free water Fill to 50 μL
Table 1: Digestion scheme 1
 
DNA (B1 15.65 ng/μl) ~ 2 μg 127.8
Cutsmart (10x) 5 μL
Sca1-HF (2 U/μL) 1 μL
Sma1  (2 U/μL) 1 μL
Nuclease free water Fill to 50 μL
Table 2: Digestion scheme 2
 
DNA (C1 186.6 ng/μl) ~ 2 μg 10.7 μL
Cutsmart (10x) 5 μL
Sca1-HF 1 (2 U/μL) 1 μL
Sma1  (2 U/μL) 1 μL
Nuclease free water Fill to 50 μL
 Table 3: Digestion scheme 3
 
DNA (D1 79.0    ng/μl) ~ 2 μg 25.3 μL
Cutsmart (10x) 5 μL
Sca1-HF 1 (2 U/μL) 1 μL
Sma1  (2 U/μL) 1 μL
Nuclease free water Fill to 50 μL
 Table 4: Digestion scheme 4
 
DNA (E1 97.7 ng/μl) ~ 2 μg 20.7 μL
Cutsmart (10x) 5 μL
Sca1-HF 1 (2 U/μL) 1 μL
Sma1  (2 U/μL) 1 μL
Nuclease free water Fill to 50 μL
 Table 5: Digestion scheme 5
 
DNA (F1 106.7 ng/μl) ~ 2 μg 18.7 μL
Cutsmart (10x) 5 μL
Sca1-HF 1 (2 U/μL) 1 μL
Sma1  (2 U/μL) 1 μL
Nuclease free water Fill to 50 μL
 Table 6: Digestion scheme 6
 
DNA (G1 328.9 ng/μl) ~ 2 μg 6.1 μL
Cutsmart (10x) 5 μL
Sca1-HF 1 (2 U/μL) 1 μL
Sma1  (2 U/μL) 1 μL
Nuclease free water Fill to 50 μL
 Table 7: Digestion scheme 7
 
DNA (H1 121.0 ng/μl) ~ 2 μg 16.5 μL
Cutsmart (10x) 5 μL
Sca1-HF 1 (2 U/μL) 1 μL
Sma1  (2 U/μL) 1 μL
Nuclease free water Fill to 50 μL
 Table 8: Digestion scheme 8
 
DNA (I1 1129.5 ng/μl) ~ 2 μg  1.7 μL
Cutsmart (10x) 5 μL
Sca1-HF 1 (2 U/μL) 1 μL
Sma1  (2 U/μL) 1 μL
Nuclease free water Fill to 50 μL
 Table 9: Digestion scheme 9
 
DNA (J1 1465.7 ng/μl) ~ 2 μg  1.4 μL
Cutsmart (10x) 5 μL
Sca1-HF 1 (2 U/μL) 1 μL
Sma1  (2 U/μL) 1 μL
Nuclease free water Fill to 50 μL
 Table 10: Digestion scheme 10
 
DNA (A2 310.4 ng/μl) ~ 2 μg 6.4 μL
Cutsmart (10x) 5 μL
Sca1-HF 1 (2 U/μL) 1 μL
Sma1  (2 U/μL) 1 μL
Nuclease free water Fill to 50 μL
 Table 11: Digestion scheme 11
 
DNA (B2 364.9l) ~ 2 μg 5.5 μL
Cutsmart (10x) 5 μL
Sca1-HF 1 (2 U/μL) 1 μL
Sma1  (2 U/μL) 1 μL
Nuclease free water Fill to 50 μL
 Table 12: Digestion scheme 12
 
DNA (C2 228.3 ng/μl) ~ 2 μg 8.8 μL
Cutsmart (10x) 5 μL
Sca1-HF 1 (2 U/μL) 1 μL
Sma1  (2 U/μL) 1 μL
Nuclease free water Fill to 50 μL
 Table 13: Digestion scheme 13
 
DNA (D2 701.7 ng/μl) ~ 2 μg 2.9 μL
Cutsmart (10x) 5 μL
Sca1-HF 1 (2 U/μL) 1 μL
Sma1  (2 U/μL) 1 μL
Nuclease free water Fill to 50 μL
 Table 14: Digestion scheme 14
 
DNA (E2 119.8 ng/μl) ~ 2 μg 16.7 μL
Cutsmart (10x) 5 μL
Sca1-HF 1 (2 U/μL) 1 μL
Sma1  (2 U/μL) 1 μL
Nuclease free water Fill to 50 μL
 Table 15: Digestion scheme 15
 
DNA (F2 493.3 ng/μl) ~ 2 μg 4.1 μL
Cutsmart (10x) 5 μL
Sca1-HF 1 (2 U/μL) 1 μL
Sma1  (2 U/μL) 1 μL
Nuclease free water Fill to 50 μL
 Table 16: Digestion scheme 16
 
DNA (G2 211.7 ng/μl) ~ 2 μg 9.5 μL
Cutsmart (10x) 5 μL
Sca1-HF 1 (2 U/μL) 1 μL
Sma1  (2 U/μL) 1 μL
Nuclease free water Fill to 50 μL
 Table 17: Digestion scheme 17
DNA (H2 482.2 ng/μl) ~ 2 μg 4.2 μL
Cutsmart (10x) 5 μL
Sca1-HF 1 (2 U/μL) 1 μL
Sma1  (2 U/μL) 1 μL
Nuclease free water Fill to 50 μL
 Table 18: Digestion scheme 18
 
DNA (I2 1364.7 ng/μl) ~ 2 μg 1.5 μL
Cutsmart (10x) 5 μL
Sca1-HF 1 (2 U/μL) 1 μL
Sma1  (2 U/μL) 1 μL
Nuclease free water Fill to 50 μL
 Table 19: Digestion scheme 19
 
DNA (J2 1665.5 ng/μl) ~ 2 μg1.2  μL
Cutsmart (10x) 5 μL
Sca1-HF 1 (2 U/μL) 1 μL
Sma1  (2 U/μL) 1 μL
Nuclease free water Fill to 50 μL
Table 20: Digestion scheme 20



Results
The results of the digestions can be found in figure 1 and 2 (Images experiment 13)

Ladder, A1, B1, C1, D1, E1, F1, G1, H1, I1, (J1), Ladder, A2, B2, C2, D2, E2, F2, G2, Ladder
Ladder, H2, I2, J2, Ladder, K3, J1, ladder
Something went wrong with the first (J1), so look at the second one.

Conclusion/discussion
Restriction-enzyme Sma was incubated at 37 degrees Celcius (same as Sca). This must be 25 degrees Celcius.
Therefore, Sma was added again and was incubated at 25 degrees Celcius. See results experiment 16.

13

Assessing Gas Production BioBricks in E.Coli

September 11

Assessing Gas Production BioBricks in E.Coli

September 13

Elise Grootscholten | Randall de Waard

Digestions have been done of the biobrickcombinations K352002 +K133116 + pSB1K3 and K352003 + K133116 + pSB1K3 with restriction-enzymes ScaI and SmaI

Experiment 14: Digestions
13-09-18

Digestions of the biobrick combinations: K352002+K133116+pSB1K3 and K352003+K133116+pSB1K3

Materials
-SmaI (restriction-enzyme)
-ScaI (restricion-enzyme)
See experiment..


Methods
1 μL SmaI restriction-enzyme has been added to the digetions from experiment 13, except for pSB1K3.
After an our at room temperature the digestions were put in -20 degrees Celcius.

 
DNA (K352002+K133116+pSB1K3 189,3 ng/μl) ~ 2 μg 32,4 μL
Cutsmart (10x) 5 μL
Sca1-HF (2 U/μL) 1 μL
Sma1 (2 U/μL) 1 μL
Nuclease free water Fill to 50 μL

Table 1: Digestion scheme 1

DNA (K352003+K133116_pSB1K3 355,3 ng/μl) ~ 2 μg 5,6 μL
Cutsmart (10x) 5 μL
Sca1-HF (2 U/μL) 1 μL
Sma1 (2 U/μL) 1 μL
Nuclease free water Fill to 50 μL

Table 2: Digestion scheme 2

 

 

15

Assessing Gas Production BioBricks in E.Coli

September 13

Assessing Gas Production BioBricks in E.Coli

September 14

Elise Grootscholten | Randall de Waard

All the digestions of experiment 13 have digested again with SmaI for an our at 25 degrees Celcius. Because earlier the digestions have been put immediately bt 37 degrees Celcius. It looks like H1, J1 and D2 include the right biobricks. 

Experiment 16: Gelelectroforesis
14-09-18

Materials
See experment ...

Methods
See experiment ...

Gel 1
L, A1, B1, C1, D1, E1, F1, G1, G1 with only smaI, H1, I1, J1, A2, B2, C2, D2, E2, F2, G2, L

Gel 2
L, 

16

Assessing Gas Production BioBricks in E.Coli

September 14

Assessing Gas Production BioBricks in E.Coli

September 17

Elise Grootscholten | Randall de Waard

Minipreps of the biobrick combinations: K352002+K133116+pSB1K3 (1) and K352003+K133116+pSB1K3 (2) are done. The DNA concentration is for 1:189,3 ng/μl and for 2: 355,3 ng/μl.

Experiment 14: Minipreps
13-09-18

Minipreps of the biobrick combinations: K352002+K133116+pSB1K3 and K352003+K133116+pSB1K3

Materials
See experiment 3

Methods
See experiment 3

Results
K352002+K133116+pSB1K3: 189,3 ng/μl
K352003+K133116+pSB1K3: 355,3 ng/μl

14

Assessing Gas Production BioBricks in E.Coli

September 17

Testing gas production

September 17

Elise Grootscholten | Paul Reusink

Different strains of E.coli are tested as negative control. Those strains were: NEB10B�ta, BL21 (DE3), BL21, HB101, DH5alpha and JM109. NEB10B�ta produced the most gas. BL21, HB101 and JM101 produced none/almost none gas. Because BL21 isn't a K12 strain we can not use that one. That is why we will test further gasproduction in HB101 and JM109. And maybe those will be our final E.coli strains.

Experiment 5: Testing different strains of E.coli

Materials
All the following types of bacteria are E.coli:
-glycerolstock BL21
-glycerolstock HB101
-glycerolstock DH5alpha
-glycerolstock JM109
-agarplate with NEB10beta
-agarplat with BL21 (DE3) 

Methods
See experiment 1
Make overnight cultures of the following E.coli tribes: NEB10B�ta, BL21 (DE3), BL21, HB101, DH5alpha, JM109.
All the bacteria are de deluted in normal LB agar, LB agar with urea and LB agar with pyruvate. 


Results
 

Bacteria LB LB Urea Urea Pyruvate Pyruvate
NEB10beta + +++ +++ +++ + ++/+++
BL21 (DE3)  - ++ - - - -
BL21 - - - - - -
DH5a +++ +/- - - +/- +
HB101 - - - - + -
JM109 - - - - - -

05

Testing gas production

September 17

Assessing Gas Production BioBricks in E.Coli

September 20

Elise Grootscholten | Randall de Waard

32 minipreps have been performed. A lot of them have a low concentration nucleic acid and/or have a high 260/280 and 260/230 rate. Therefore those will be done again.

Experiment 17: Minipreps
20-09-18
Overnight cultures (35)
21-09-18
Minipreps (32)

Materials 
See experiment 3
-Kanamycin (40 mg/mL)

Methods
See experiment 3
Make 32 minipreps:
A: 6
B: 6
C:6
D:1
E:6
F:6
H:1
I: 1
J:2

Results/conclusion
There was no grow in B6, C2 and C5.
A lot of them have a low concentration nucleic acid and/or have a high 260/280 and 260/230 rate. Therefore those will be done again.
Thise are: A1, A4, A5, A6, B1, B5, B6 C1, C2, C3, C4, C5, C6, E1, E2, E5, E6, F4, F5

17

Assessing Gas Production BioBricks in E.Coli

September 20

Assessing Gas Production BioBricks in E.Coli

September 26

Elise Grootscholten | Randall de Waard

Making overnight cultures of the numbers: A1, A4, A5, A6, B1, B5, B6 C1, C2, C3, C4, C5, C6, E1, E2, E5, E6, F4, F5 with Kanamycin. These biobrick combinations can be found in experiment 12. Also the first digestions with XbaI, Eco0109I, HindIII and SspI-HF have been done.

Experiment 19: Making overnight cultures + first digestions
27-09-18

Materials
-Kanamycine 
-XbaI
-SspI-HF
-Eco019I
-HindIII
See experiment ...

Methods
See experiment ...
Make new overnight cultures of the numbers: A1, A4, A5, A6, B1, B5, B6 C1, C2, C3, C4, C5, C6, E1, E2, E5, E6, F4, F5 with Kanamycin. 

 

19

Assessing Gas Production BioBricks in E.Coli

September 26

Assessing Gas Production BioBricks in E.Coli

September 27

Elise Grootscholten | Randall de Waard

Minipreps have been made of the numbers: A1, A4, A5, A6, B1, B5, B6 C1, C2, C3, C4, C5, C6, E1, E2, E5, E6, F4, F5.

Experiment 20: Minipreps of gasproduction biobricks
27-09-18

Make minipreps of A1, A4, A5, A6, B1, B5, B6 C1, C2, C3, C4, C5, C6, E1, E2, E5, E6, F4, F5.
See experiment 3.

Also make master mixes for the control digestions and code the tubes.
 

20

Assessing Gas Production BioBricks in E.Coli

September 27

Assessing Gas Production BioBricks in E.Coli

September 27

Elise Grootscholten | Randall de Waard

From 27-09 until 04-10 more than 200 digestions and 17 gelelectrophoresis have been performed. This was done as a control for our composite parts. We digested with XbaI, Eco01019 and SspI-HF/HindIII. Every restriction-enzyme has an unique place to cut in our constructs. With this we could test if the promoter, backbone and gas production gene was present. However, the digestions didn't show us very good results. Only I1 and J1 looked good. After this we started with PCR.

Experiment 21: Digestions and gelelectrophoresis
01-10-18

Digestions of the gasproduction biobrick combinations (D, H, I and J) and a start with the gelelectrophoresis.

Methods
Digestions of D2, H1, I2 and J2 of experiment 12.
J2 will from now on be J3.
 

NEB 2.1 (10x) 4 μL
Eco0109I (20 U/μL) 1 μL
Nuclease free water 35 μL

Table 1: Master mix digestion 1
 

NEB 2.1 (10x) 4 μL
Eco0109I (20 U/μL) 1 μL
XbaI (20 U/μL) 1 μL
Nuclease free water 34 μL
 
Table 2: Master mix digestion 2
 
NEB 2.1 (10x) 2 μL
Eco0109I (20 U/μL) 1 μL
XbaI (20 U/μL) 1 μL
HindIII (20 U/μL) 1 μL
Nuclease free water 15 μL
Table 3: Master mix digestion 3
 
NEB 2.1 (10x) 2 μL
Eco0109I (20 U/μL) 1 μL
XbaI (20 U/μL) 1 μL
SspI-HF 1 μL
Nuclease free water 15  μL
Table 4: Master mix digestion 4
 
NEB 2.1 (10x) 2 μL
HindIII (20 U/μL) 1 μL
XbaI (20 U/μL) 1 μL
Nuclease free water 16 μL
 
Table 5: Master mix digestion 5
 
NEB 2.1 (10x) 2 μL
SspI-HF (20 U/μL) 1 μL
XbaI (20 U/μL) 1 μL
Nuclease free water 16 μL
 
Table 6: Master mix digestion 6
 
NEB 2.1 (10x) 4 μL
XbaI (20 U/μL) 1 μL
Nuclease free water 35 μL
 
Table 7: Master mix digestion 7
 
NEB 2.1 (10x) 2 μL
HindIII (20 U/μL) 1 μL
Nuclease free water 17 μL
 
Table 8: Master mix digestion 8
 
NEB 2.1 (10x) 2 μL
SspI-HF (20 U/μL) 1 μL
Nuclease free water 17 μL
 
Table 9: Master mix digestion 9
 
NEB 2.1 (10x) 2 μL
Eco0109I (20 U/μL) 1 μL
HindIII (20 U/μL) 1 μL
Nuclease free water 16 μL
 
Table 10: Master mix digestion 10
 
NEB 2.1 (10x) 2 μL
Eco0109I (20 U/μL) 1 μL
SspI-HF (20 U/μL) 1 μL
Nuclease free water 16 μL
 
Table 11: Master mix digestion 11

Pipette 9 μL of the mastermix with 2 μL of H1/ 1 μL of D2/ 1 μL of I2/ 1 μL of J23

D2 and J3 are digested with mastermix: 1, 2, 3, 5, 7, 8 and 10.
H1 and I2 are digested with mastermix: 1, 2, 4, 6, 7, 9, and 11.
 

Gelelectrophoresis
01-10-18


02-10-18
Gelelectrophoresis


03-10-18
Gelelectrophoresis

Gel 1
L, D2.1, D2.2, D2.3, D2.5, D2.7, D2.8, D2.10, D not digested, L, J3.1, J3.2, J3.3, J3.5, J3.7, J3.8, J3.10, J not digested, L.

Gel 2
L, H2.1, H2.2 H2.4, H2.6, H2.7, H2.9, H2.11, H2 not digested, L, I2.1, I2.2, I2.4, I2.6, H2.7, H2.9, H2.11, H2 not digested, L

Gel 3
L, C1.1, C1.2, C1.4, C1.6, C1.7, C1.8, C1.10, C1 not digested, L, C2.1, C2.2, C2.4, C2.6, C2.7, C2.8, C2.10, C2 not dgested, L

Gel 4
L, C3.1, C3.2, C3.4, C3.6, C3.7, C3.8, C3.10, L, C4.1, C4.2, C4.4, C4.6, C4.7, C4.8, C4.10, C4 not digested, L

Gel 5
L, C6.1, C6.2, C6.4, C6.6, C6.7, C6.8, C6.10, C6 not digested, L, B5.1, B5.3, B5.4, B5.4 (other tube), B5.6, B5..., B5.9, B5.11, L

04-10-18
Gelelectrophoresis

Gel 1
L, J2 Eco0109, J2 HindIII, J2 XbaI, J2 Eco0109 + XbaI, J2 Eco0109 + HindIII, J2 HindIII + XbaI, J2 Eco0109 + HindIII + XbaI, J2 not digested, L
Gel 2

Gel 3

Gel 4

21

Assessing Gas Production BioBricks in E.Coli

September 27

Assessing Gas Production BioBricks in E.Coli

October 8

Elise Grootscholten | Randall de Waard

Digestions with EcoRI-HF and PstI are done for the biobrick numbers A2, B1, B5, B6, D1, E1, E3, I1, J1, J1.2, CooA (8) and pSB1C3. This is done because now the Kanamycin backbone can be replaced for the Chloramphenicol backbone. This is done by dephosphorylation and ligation.

Experiment 24: Digestions, gelelectrophoresis and dephosphorylation and ligation.
08-10-18 <---- klopt dit?

Materals
See experiment ...
-Restriction enzymes EcoRI-HF and PstI

Methods
Digestion

Mastermix

Cutsmart (10x) 55 μL
EcoRI-HF (20 U/μL) 1 μL
PstI (20 U/μL) 1 μL
Nuclease free water 382,7 μL
 

Table 1: Master mix

50 μL mastermix - ... μL DNA (see below)
A2: 12,6 μL
B1: 15,7 μL
B5: 9,4 μL
B6: 9,6 μL
D1: 10,2 μL
E1: 15,7 μL (1 μg)
I1: 14,3 μL (1 μg)
J1.2: 17,4 μL
CooA (8): 5,4 μL

Dephosphorylation + ligation scheme
.........

Results
See figure 1.

25

Assessing Gas Production BioBricks in E.Coli

October 8

Testing gas production

October 11

Elise Grootscholten | Paul Reusink

Overnight cultures made of the following strains with the right plasmid:

Number HB101 JM109
J1 1x 1x
I1 3x 3x
D1 2x 2x
B1 1x 1x
B6 1x 1x

Table 1: Overnight culture scheme.

Experiment 6: Testing gasproduction with our new biobricks.

Testing our new biobricks K260401 in JM109 and HB101. As a negative control we used the other new biobricks ...

Materials
-LB culture medium
-Kanamycin
-Bacteria on agar plates with the right plasmid

Methods
Make overnight cultures of the following strains with the right plasmid:

Number HB101 JM109
J1 1x 1x
I1 3x 3x
D1 2x 2x
B1 1x 1x
B6 1x 1x
Table 1: Overnight culture scheme.

Only the numbers J1, I1 and D1 are tested. The rest is made for making glycerolstocks.
J1 and D1 are the negative controls in this experiment.

LB: 582 mL +18 mL 100 mM sodiumpyruvate and 525 μL kanamycin (40 mg/mL).
After this the LB is devided in 2. Number 1: 0,5 μL arabinose, number 2: 1,0 μL arabinose.
The rest is done just like earlier experiments with a total of 20 tubes because everthing is done twice.

Results

 

06

Testing gas production

October 11