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

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<div class="attendees">UNKNOWN</div>
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<div class="attendees">Dustin van der Meulen</div>
 
<div class="description">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 b�ta transformation protocol adapted New England Biolabs (Protocol 2), using bought cells with a reported transformation efficienty of 1-3 10^9 cfu/�g pUC19. Inucbation was at 37�C.<br>
 
<div class="description">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 b�ta transformation protocol adapted New England Biolabs (Protocol 2), using bought cells with a reported transformation efficienty of 1-3 10^9 cfu/�g pUC19. Inucbation was at 37�C.<br>
 
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<div class="attendees">UNKNOWN</div>
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<div class="attendees">Dustin van der Meulen</div>
 
<div class="description"><p>After making a 20 mL overnight culture of our NEB 10 b�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>After making a 20 mL overnight culture of our NEB 10 b�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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<div class="attendees">UNKNOWN</div>
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<div class="attendees">Dustin van der Meulen</div>
 
<div class="description"><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>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>
 
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</div>
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<div class="attendees">UNKNOWN</div>
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<div class="attendees">None</div>
 
<div class="description"><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>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>
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<div class="attendees">UNKNOWN</div>
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<div class="attendees">Loraine Nelson | Elise Grootscholten | Paul Reusink</div>
 
<div class="description"><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>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>
 
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<div class="attendees">UNKNOWN</div>
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<div class="attendees">None</div>
 
<div class="description"><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>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>
 
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<div class="attendees">UNKNOWN</div>
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<div class="attendees">Loraine Nelson | Elise Grootscholten | Paul Reusink</div>
 
<div class="description"><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>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>
 
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<div class="attendees">UNKNOWN</div>
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<div class="attendees">None</div>
 
<div class="description"><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>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>
 
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</div>
<div class="attendees">UNKNOWN</div>
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<div class="attendees">Loraine Nelson | Elise Grootscholten | Paul Reusink</div>
 
<div class="description"><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>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>
 
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<div class="attendees">UNKNOWN</div>
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<div class="attendees">None</div>
 
<div class="description"><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>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>
 
</div>
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<path d="M9.46,6.28L11.05,9C8.47,9.26 6.5,11.41 6.5,14A5,5 0 0,0 11.5,19C13.55,19 15.31,17.77 16.08,16H13.5V14H21.5V16H19.25C18.84,17.57 17.97,18.96 16.79,20H19.5V22H3.5V20H6.21C4.55,18.53 3.5,16.39 3.5,14C3.5,10.37 5.96,7.2 9.46,6.28M12.74,2.07L13.5,3.37L14.36,2.87L17.86,8.93L14.39,10.93L10.89,4.87L11.76,4.37L11,3.07L12.74,2.07Z"></path>
 
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</svg>
<h1 class="title">Urea and sodium pyruvate test for resistence E.coli</h1>
+
<h1 class="title">Urea and sodium pyruvate test for resistance E.coli</h1>
 
<h3 class="date">August 10</h3>
 
<h3 class="date">August 10</h3>
 
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</div>
<div class="attendees">UNKNOWN</div>
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<div class="attendees">Randall de Waard | Elise Grootscholten</div>
 
<div class="description"><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><span style="font-size:14px;">Testing different amounts of urea and sodiumpyruvate to know which concentrations the bacteria survive.</span></p>
 
</div>
 
</div>
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<div class="number"><b>1</b></div>
 
<div class="number"><b>1</b></div>
<h3 class="title" style="color: white; padding: 10px 5px 5px 5px;">Urea and sodium pyruvate test for resistence E.coli</h3>
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<h3 class="title" style="color: white; padding: 10px 5px 5px 5px;">Urea and sodium pyruvate test for resistance E.coli</h3>
 
<svg viewBox="0 0 24 24">
 
<svg viewBox="0 0 24 24">
 
<path d="M9.46,6.28L11.05,9C8.47,9.26 6.5,11.41 6.5,14A5,5 0 0,0 11.5,19C13.55,19 15.31,17.77 16.08,16H13.5V14H21.5V16H19.25C18.84,17.57 17.97,18.96 16.79,20H19.5V22H3.5V20H6.21C4.55,18.53 3.5,16.39 3.5,14C3.5,10.37 5.96,7.2 9.46,6.28M12.74,2.07L13.5,3.37L14.36,2.87L17.86,8.93L14.39,10.93L10.89,4.87L11.76,4.37L11,3.07L12.74,2.07Z"></path>
 
<path d="M9.46,6.28L11.05,9C8.47,9.26 6.5,11.41 6.5,14A5,5 0 0,0 11.5,19C13.55,19 15.31,17.77 16.08,16H13.5V14H21.5V16H19.25C18.84,17.57 17.97,18.96 16.79,20H19.5V22H3.5V20H6.21C4.55,18.53 3.5,16.39 3.5,14C3.5,10.37 5.96,7.2 9.46,6.28M12.74,2.07L13.5,3.37L14.36,2.87L17.86,8.93L14.39,10.93L10.89,4.87L11.76,4.37L11,3.07L12.74,2.07Z"></path>
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<div class="attendees">UNKNOWN</div>
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<div class="attendees">None</div>
 
<div class="description"><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>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>
 
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<div class="attendees">UNKNOWN</div>
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<div class="attendees">None</div>
 
<div class="description"><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>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>
 
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<div class="attendees">UNKNOWN</div>
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<div class="attendees">None</div>
 
<div class="description">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;</div>
 
<div class="description">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;</div>
 
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<div class="attendees">UNKNOWN</div>
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<div class="attendees">Elise Grootscholten | Paul Reusink</div>
 
<div class="description"><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>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>
 
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<div class="attendees">UNKNOWN</div>
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<div class="attendees">Elise Grootscholten | Paul Reusink</div>
 
<div class="description"><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>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>
 
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<div class="attendees">UNKNOWN</div>
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<div class="attendees">Elise Grootscholten | Paul Reusink</div>
 
<div class="description"><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>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>
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<div class="attendees">UNKNOWN</div>
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<div class="attendees">Elise Grootscholten | Paul Reusink</div>
 
<div class="description"><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>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>
 
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<div class="attendees">UNKNOWN</div>
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<div class="attendees">None</div>
 
<div class="description"><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>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>
 
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<div class="attendees">UNKNOWN</div>
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<div class="attendees">None</div>
 
<div class="description"><p>A miniprep of the biobrick K352002 is made. The concentration is 69,77 ng/ul.</p>
 
<div class="description"><p>A miniprep of the biobrick K352002 is made. The concentration is 69,77 ng/ul.</p>
 
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<path d="M9.46,6.28L11.05,9C8.47,9.26 6.5,11.41 6.5,14A5,5 0 0,0 11.5,19C13.55,19 15.31,17.77 16.08,16H13.5V14H21.5V16H19.25C18.84,17.57 17.97,18.96 16.79,20H19.5V22H3.5V20H6.21C4.55,18.53 3.5,16.39 3.5,14C3.5,10.37 5.96,7.2 9.46,6.28M12.74,2.07L13.5,3.37L14.36,2.87L17.86,8.93L14.39,10.93L10.89,4.87L11.76,4.37L11,3.07L12.74,2.07Z"></path>
 
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<h1 class="title">Urea and sodium pyruvate test for resistence E.coli</h1>
+
<h1 class="title">Urea and sodium pyruvate test for resistance E.coli</h1>
 
<h3 class="date">August 28</h3>
 
<h3 class="date">August 28</h3>
 
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</div>
 
</div>
<div class="attendees">UNKNOWN</div>
+
<div class="attendees">Randall de Waard | Elise Grootscholten</div>
 
<div class="description"><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><span style="font-size:14px;">Testing different amounts of urea and sodiumpyruvate to know which concentrations the bacteria survive.</span></p>
 
</div>
 
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<div class="cardform">
 
<div class="number"><b>2</b></div>
 
<div class="number"><b>2</b></div>
<h3 class="title" style="color: white; padding: 10px 5px 5px 5px;">Urea and sodium pyruvate test for resistence E.coli</h3>
+
<h3 class="title" style="color: white; padding: 10px 5px 5px 5px;">Urea and sodium pyruvate test for resistance E.coli</h3>
 
<svg viewBox="0 0 24 24">
 
<svg viewBox="0 0 24 24">
 
<path d="M9.46,6.28L11.05,9C8.47,9.26 6.5,11.41 6.5,14A5,5 0 0,0 11.5,19C13.55,19 15.31,17.77 16.08,16H13.5V14H21.5V16H19.25C18.84,17.57 17.97,18.96 16.79,20H19.5V22H3.5V20H6.21C4.55,18.53 3.5,16.39 3.5,14C3.5,10.37 5.96,7.2 9.46,6.28M12.74,2.07L13.5,3.37L14.36,2.87L17.86,8.93L14.39,10.93L10.89,4.87L11.76,4.37L11,3.07L12.74,2.07Z"></path>
 
<path d="M9.46,6.28L11.05,9C8.47,9.26 6.5,11.41 6.5,14A5,5 0 0,0 11.5,19C13.55,19 15.31,17.77 16.08,16H13.5V14H21.5V16H19.25C18.84,17.57 17.97,18.96 16.79,20H19.5V22H3.5V20H6.21C4.55,18.53 3.5,16.39 3.5,14C3.5,10.37 5.96,7.2 9.46,6.28M12.74,2.07L13.5,3.37L14.36,2.87L17.86,8.93L14.39,10.93L10.89,4.87L11.76,4.37L11,3.07L12.74,2.07Z"></path>
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</div>
 
</div>
<div class="attendees">UNKNOWN</div>
+
<div class="attendees">None</div>
 
<div class="description"><p></p></div>
 
<div class="description"><p></p></div>
 
</div>
 
</div>
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</div>
 
</div>
<div class="attendees">UNKNOWN</div>
+
<div class="attendees">None</div>
 
<div class="description"><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>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>
 
</div>
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</div>
 
</div>
<div class="attendees">UNKNOWN</div>
+
<div class="attendees">None</div>
 
<div class="description"><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>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>
 
</div>
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</div>
 
</div>
<div class="attendees">UNKNOWN</div>
+
<div class="attendees">None</div>
 
<div class="description"><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>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>
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</div>
 
</div>
<div class="attendees">UNKNOWN</div>
+
<div class="attendees">None</div>
 
<div class="description"><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>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>
 
</div>
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</div>
<div class="attendees">UNKNOWN</div>
+
<div class="attendees">None</div>
 
<div class="description"><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>Digestions have been done of the biobrickcombinations&nbsp;K352002 +K133116 + pSB1K3 and K352003 + K133116 + pSB1K3 with restriction-enzymes ScaI and SmaI</p>
 
</div>
 
</div>
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</div>
<div class="attendees">UNKNOWN</div>
+
<div class="attendees">None</div>
 
<div class="description"><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>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>
 
</div>
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</div>
 
</div>
<div class="attendees">UNKNOWN</div>
+
<div class="attendees">None</div>
 
<div class="description"><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/�l and for 2: 355,3&nbsp;ng/�l.</p>
 
<div class="description"><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/�l and for 2: 355,3&nbsp;ng/�l.</p>
 
</div>
 
</div>
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</div>
 
</div>
<div class="attendees">UNKNOWN</div>
+
<div class="attendees">Elise Grootscholten | Paul Reusink</div>
 
<div class="description"><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>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>
 
</div>
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</div>
 
</div>
<div class="attendees">UNKNOWN</div>
+
<div class="attendees">None</div>
 
<div class="description">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.</div>
 
<div class="description">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.</div>
 
</div>
 
</div>
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<label class="back" for="switch-89">&times;</label>
 
</div>
 
</div>
<div class="attendees">UNKNOWN</div>
+
<div class="attendees">None</div>
 
<div class="description"><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>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>
 
</div>
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</div>
 
</div>
<div class="attendees">UNKNOWN</div>
+
<div class="attendees">None</div>
 
<div class="description"><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>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>
 
</div>
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</div>
 
</div>
<div class="attendees">UNKNOWN</div>
+
<div class="attendees">None</div>
 
<div class="description"><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>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>
 
</div>
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</div>
 
</div>
<div class="attendees">UNKNOWN</div>
+
<div class="attendees">None</div>
 
<div class="description"><p></p></div>
 
<div class="description"><p></p></div>
 
</div>
 
</div>
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<label class="back" for="switch-93">&times;</label>
 
</div>
 
</div>
<div class="attendees">UNKNOWN</div>
+
<div class="attendees">Randall de Waard | Elise Grootscholten</div>
 
<div class="description"><p></p></div>
 
<div class="description"><p></p></div>
 
</div>
 
</div>
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</div>
 
</div>
<div class="attendees">UNKNOWN</div>
+
<div class="attendees">None</div>
 
<div class="description"><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>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>
 
</div>
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</div>
 
</div>
<div class="attendees">UNKNOWN</div>
+
<div class="attendees">None</div>
 
<div class="description"><p></p></div>
 
<div class="description"><p></p></div>
 
</div>
 
</div>
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</div>
 
</div>
<div class="attendees">UNKNOWN</div>
+
<div class="attendees">None</div>
 
<div class="description"><p></p></div>
 
<div class="description"><p></p></div>
 
</div>
 
</div>
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</div>
<div class="attendees">UNKNOWN</div>
+
<div class="attendees">None</div>
 
<div class="description"><p></p></div>
 
<div class="description"><p></p></div>
 
</div>
 
</div>
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</div>
<div class="attendees">UNKNOWN</div>
+
<div class="attendees">Elise Grootscholten | Paul Reusink</div>
 
<div class="description"><p>Overnight cultures made of the following strains with the right plasmid:</p>
 
<div class="description"><p>Overnight cultures made of the following strains with the right plasmid:</p>
  

Revision as of 10:31, 12 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 b�ta transformation protocol adapted New England Biolabs (Protocol 2), using bought cells with a reported transformation efficienty of 1-3 10^9 cfu/�g 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
1

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

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)
2

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.

3

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

None

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. 

1

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. 

1

Transformation

July 24

Assessing Gas Production BioBricks in E.Coli

July 24

None

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. 

2

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. 

2

Transformation

July 27

Assessing Gas Production BioBricks in E.Coli

July 27

None

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

3

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.

3

Transformation

August 9

Assessing Gas Production BioBricks in E.Coli

August 10

None

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. 

4

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.

1

Urea and sodium pyruvate test for resistance E.coli

August 10

Assessing Gas Production BioBricks in E.Coli

August 13

None

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.

5

Assessing Gas Production BioBricks in E.Coli

August 13

Assessing Gas Production BioBricks in E.Coli

August 13

None

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.

6

Assessing Gas Production BioBricks in E.Coli

August 13

Assessing Gas Production BioBricks in E.Coli

August 13

None
The ligations and transformations of the biobricks K173003 + J23100, K173003 + I13453, and K133071 + I13453 has 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. 
7

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.

1

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.

2

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.

3

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.

4

Testing gas production

August 23

Assessing Gas Production BioBricks in E.Coli

August 27

None

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

8

Assessing Gas Production BioBricks in E.Coli

August 27

Assessing Gas Production BioBricks in E.Coli

August 27

None

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

9

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.

2

Urea and sodium pyruvate test for resistance E.coli

August 28

Chemo competent cells

August 30

None

1

Chemo competent cells

August 30

Assessing Gas Production BioBricks in E.Coli

September 7

None

Digestions, gelelectrophoresis, dephosphorylations and ligations of different biobrick combinations. These biobrick combinations are 4 different promotors (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.

10

Assessing Gas Production BioBricks in E.Coli

September 7

Assessing Gas Production BioBricks in E.Coli

September 7

None

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.

11

Assessing Gas Production BioBricks in E.Coli

September 7

Assessing Gas Production BioBricks in E.Coli

September 11

None

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) K133071 pSB1K3
C K352002 (CooF) K173003 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 II13453 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

None

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.

13

Assessing Gas Production BioBricks in E.Coli

September 11

Assessing Gas Production BioBricks in E.Coli

September 13

None

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

15

Assessing Gas Production BioBricks in E.Coli

September 13

Assessing Gas Production BioBricks in E.Coli

September 14

None

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. 

16

Assessing Gas Production BioBricks in E.Coli

September 14

Assessing Gas Production BioBricks in E.Coli

September 17

None

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/�l and for 2: 355,3 ng/�l.

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.

5

Testing gas production

September 17

Assessing Gas Production BioBricks in E.Coli

September 20

None
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.
17

Assessing Gas Production BioBricks in E.Coli

September 20

Assessing Gas Production BioBricks in E.Coli

September 26

None

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.

19

Assessing Gas Production BioBricks in E.Coli

September 26

Assessing Gas Production BioBricks in E.Coli

September 27

None

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.

20

Assessing Gas Production BioBricks in E.Coli

September 27

Assessing Gas Production BioBricks in E.Coli

September 27

None

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 PCR.

21

Assessing Gas Production BioBricks in E.Coli

September 27

Assessing Gas Production BioBricks in E.Coli

October 5

None

22

Assessing Gas Production BioBricks in E.Coli

October 5

PCR

October 7

Randall de Waard | Elise Grootscholten

1

PCR

October 7

Assessing Gas Production BioBricks in E.Coli

October 8

None

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.

25

Assessing Gas Production BioBricks in E.Coli

October 8

Assessing Gas Production BioBricks in E.Coli

October 11

None

23

Assessing Gas Production BioBricks in E.Coli

October 11

Assessing Gas Production BioBricks in E.Coli

October 11

None

24

Assessing Gas Production BioBricks in E.Coli

October 11

Assessing Gas Production BioBricks in E.Coli

October 11

None

26

Assessing Gas Production BioBricks in E.Coli

October 11

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.

6

Testing gas production

October 11