Difference between revisions of "Team:Ecuador/Interlab"

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<title>C-lastin, Interlab</title>
 
<title>C-lastin, Interlab</title>
 
<style>
 
<style>
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</style>
 
</style>
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<div class="ec--first--section">
 
<div class="ec--first--section">
 
<div class="ec--main--title">
 
<div class="ec--main--title">
−
<a name="MOD"  style="text-decoration: none;">
+
<a name="INTER"  style="text-decoration: none;">
−
<h1>Interlab</h1>
+
<div class="ec--h1">
−
</a>
+
INTERLAB
 +
</div>
 +
</a>
 
</div>
 
</div>
 +
<center>
 +
  <p><img width="385" height="244" src="https://static.igem.org/mediawiki/2018/d/d6/T--ECUADOR--Modeling.jpeg"></p></center>
 
<div class="ec--modeling--items">
 
<div class="ec--modeling--items">
 
<ul>
 
<ul>
−
<li><a href="#MOD">Interlab</a></li>
+
<li><a href="#WWD">
−
<li><a href="#WWD">WHAT DID WE DO?</a></li>
+
WHAT DID WE DO?
−
<li><a href="#HWD">HOW DID WE DO IT?</a></li>
+
</a>
−
<li><a href="#RES">RESULTS</a></li>
+
</li>
−
<li><a href="#EXP">EXPERIENCE</a></li>
+
<li><a href="#HWD">
−
<li><a href="#ACK">ACKNOWLEDGEMENT</a></li>
+
HOW DID WE DO IT?
 +
</a>
 +
</li>
 +
<li><a href="#BIO">
 +
BioBricks
 +
</a>
 +
</li>
 +
<li><a href="#RES">
 +
RESULTS
 +
</a>
 +
</li>
 +
<li><a href="#EXP">
 +
EXPERIENCE
 +
</a>
 +
</li>
 +
<li><a href="#ACK">
 +
ACKNOWLEDGEMENT
 +
</a>
 +
</li>
 
</ul>
 
</ul>
 
</div>
 
</div>
−
<div class="ec--img--home--slider">Reference image</div>
+
 
</div>
 
</div>
 
 
 
<div class="ec--nau--content">
 
<div class="ec--nau--content">
−
<div class="ec--sect--title">
+
  <a name="WWD">
−
<a name="WWD"  style="text-decoration: none;"><h2>WHAT DID WE DO?</h2></a>
+
<div class="ec--h2">
−
  <p>Parrafo<br></p>
+
What did we do?
−
  <div class="ec--img--wwd--cont">
+
</div>
−
<p><img width="700" height="500" src="https://static.igem.org/mediawiki/2018/d/d6/T--ECUADOR--Modeling.jpeg"></p>
+
</a>
−
  </div>
+
<div class="ec--p">
−
          <h3>Converting between  absorbance of cells to absorbance of a known concentration of beads.</h3>
+
We tried to reduce the variability in the measurement of GFP expression for this reason we used a direct method to determine the mean expression level of GFP per cel
−
              <p>...</p>
+
</div>
−
              <h3>Counting  colony-forming units (CFUs) from the sample.</h3>
+
−
<p>…</p>
+
  <a name="HWD">
−
  <a name="HWD" style="text-decoration: none;"><h2>HOW DID WE DO IT?</h2></a>
+
<div class="ec--h2">
−
<p>In order to avoid distortions in the results due to differences in the experimentation processes, we follow the procedures described in the iGEM protocol (<a href="https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf">Plate reader protocol</a>, <a href="http://parts.igem.org/Help:Protocols/Competent_Cells">component_cells</a>, <a href="http://parts.igem.org/Help:Protocols/Transformation">Transformation</a>)
+
How did we do it?
−
<br>
+
</div>
−
</p>
+
</a>
−
<br>
+
<div class="ec--p">
−
              <h3>BIOBRICKS</h3>
+
In order to avoid distortions in the results due to differences in the experimentation processes, we follow the procedures described in the iGEM protocol (<a href="https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf" style="text-decoration: none">Plate reader protocol</a>, <a href="http://parts.igem.org/Help:Protocols/Competent_Cells" style="text-decoration: none">Competent cells</a>, <a href="http://parts.igem.org/Help:Protocols/Transformation" style="text-decoration: none">Transformation</a>)l
−
              <br>
+
</div>
−
+
−
  <div class="ec--biob--cont">
+
  <div class="ec--h3">
 +
Calibration
 +
</div>
 +
<div class="ec--p">Before starting the experiments, we performed the OD600 calibration measurements following the LUDOX protocol, the particle Estandar Curve with the Microsphere Protocol, and the fluorescence standard curve with the Fluorescein Protocol. This allowed us to familiarize ourselves with the plate reader required for subsequent measurements. The plate reader that we used had the following characteristics:<br />
 +
  Label: BioTek / Model: Cytation 5 / Emission: 528 / Excitation: 485 / Optic position: Top / Temperature: 25 ºC / Shake: 425 cpm for 30 sec before every measurement.
 +
</div>
 +
 +
<div class="ec--h3">Cell measurement</div>
 +
<div class="ec--p">
 +
We started by transforming E. coli DH5 alpha with the plasmid described in the protocol, and after incubation and growth in LB-medium, we diluted the culture to Abs600 0.2 and proceeded  with the samples.
 +
</div>
 +
 +
<div class="ec--h3">Colony units per 0.1 OD600 E. coli cultures</div>
 +
<div class="ec--p">
 +
In order to determine the cell concentration of the culture we followed the CFU protocol that basically consisted in diluting a culture to certain absorbance, measure the OD600 that has to be in a value of 0.1, and make serial dilutions to later count the colonies and calculate the CFU/mL.
 +
</div>
 +
 +
  <a name="BIO"><div class="ec--h2">BioBricks</div></a>
 +
 +
<div class="ec--biob--cont">
 
<div id="bigBox">
 
<div id="bigBox">
 
 
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    <table border="0" cellspacing="8px" cellpadding="0">
 
    <table border="0" cellspacing="8px" cellpadding="0">
 
          <tr>
 
          <tr>
−
            <td width="148" valign="top"><p ><strong>Device</strong><strong> </strong></p></td>
+
            <td width="148" valign="top"><center ><strong>Device</strong><strong> </strong></center></td>
−
            <td width="132" valign="top"><p ><strong>Part Number</strong><strong> </strong></p></td>
+
            <td width="132" valign="top"><center><strong>Part Number</strong><strong> </strong></center></td>
−
            <td width="104" valign="top"><p ><strong>Location</strong><strong> </strong></p></td>
+
 
              </tr>
 
              </tr>
 
          <tr>
 
          <tr>
−
            <td width="148" valign="top"><p id="label-neg-c">Negative control</p></td>
+
            <td width="148" valign="top"><p id="label-neg-c" class="ec--inter-item">Negative control</p></td>
 
            <td width="132" valign="top"><p>BBa_R0040 </p></td>
 
            <td width="132" valign="top"><p>BBa_R0040 </p></td>
−
            <td width="104" valign="top"><p>Well 2D </p></td>
 
 
              </tr>
 
              </tr>
 
          <tr>
 
          <tr>
−
            <td width="148" valign="top"><p id="type">Positive control </p></td>
+
            <td width="148" valign="top"><p id="type"class="ec--inter-item">Positive control </p></td>
 
            <td width="132" valign="top"><p>BBa_I20270 </p></td>
 
            <td width="132" valign="top"><p>BBa_I20270 </p></td>
−
            <td width="104" valign="top"><p>Well 2B</p></td>
 
 
              </tr>
 
              </tr>
 
          <tr>
 
          <tr>
−
            <td width="148" valign="top"><p id="label-td1">Test Device 1 </p></td>
+
            <td width="148" valign="top"><p id="label-td1" class="ec--inter-item">Test Device 1 </p></td>
 
            <td width="132" valign="top"><p>BBa_J364000 </p></td>
 
            <td width="132" valign="top"><p>BBa_J364000 </p></td>
−
            <td width="104" valign="top"><p>Well 2F    &nbsp; </p></td>
 
 
              </tr>
 
              </tr>
 
          <tr>
 
          <tr>
−
            <td width="148" valign="top"><p id="label-td2">Test Device 2 </p></td>
+
            <td width="148" valign="top"><p id="label-td2" class="ec--inter-item">Test Device 2 </p></td>
 
            <td width="132" valign="top"><p>BBa_J364001 </p></td>
 
            <td width="132" valign="top"><p>BBa_J364001 </p></td>
−
            <td width="104" valign="top"><p>Well 2H    &nbsp; </p></td>
 
 
              </tr>
 
              </tr>
 
          <tr>
 
          <tr>
−
            <td width="148" valign="top"><p id="label-td3">Test Device 3 </p></td>
+
            <td width="148" valign="top"><p id="label-td3" class="ec--inter-item">Test Device 3 </p></td>
 
            <td width="132" valign="top"><p>BBa_J364002 </p></td>
 
            <td width="132" valign="top"><p>BBa_J364002 </p></td>
−
            <td width="104" valign="top"><p>Well 2J </p></td>
 
 
              </tr>
 
              </tr>
 
          <tr>
 
          <tr>
−
            <td width="148" valign="top"><p id="label-td4">Test Device 4 </p></td>
+
            <td width="148" valign="top"><p id="label-td4" class="ec--inter-item">Test Device 4 </p></td>
 
            <td width="132" valign="top"><p>BBa_J364007 </p></td>
 
            <td width="132" valign="top"><p>BBa_J364007 </p></td>
−
            <td width="104" valign="top"><p>Well 2L    &nbsp; </p></td>
 
 
              </tr>
 
              </tr>
 
          <tr>
 
          <tr>
−
            <td width="148" valign="top"><p id="label-td5">Test Device 5 </p></td>
+
            <td width="148" valign="top"><p id="label-td5" class="ec--inter-item">Test Device 5 </p></td>
 
            <td width="132" valign="top"><p>BBa_J364008 </p></td>
 
            <td width="132" valign="top"><p>BBa_J364008 </p></td>
−
            <td width="104" valign="top"><p>Well 2N </p></td>
 
 
              </tr>
 
              </tr>
 
          <tr>
 
          <tr>
−
            <td width="148" valign="top"><p id="label-td6">Test Device 6 </p></td>
+
            <td width="148" valign="top"><p id="label-td6" class="ec--inter-item">Test Device 6 </p></td>
 
            <td width="132" valign="top"><p>BBa_J364009 </p></td>
 
            <td width="132" valign="top"><p>BBa_J364009 </p></td>
−
            <td width="104" valign="top"><p>Well 2P    &nbsp; </p></td>
 
 
              </tr>
 
              </tr>
 
            </table>
 
            </table>
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<div id="rightBox">
 
<div id="rightBox">
 
<div class="ec--biobr--location--container">
 
<div class="ec--biobr--location--container">
 +
<table width="338" border="0" cellpadding="0" cellspacing="8px">
 +
  <tr>
 +
<td width="330" valign="top"><p align="center"><strong>Structure<br>
 +
(Point with the cursor on the device)
 +
</strong></p></td>
 +
  </tr>
 +
</table>
 +
<div class="ec--biobr--separator"></div>
 
<div class="loc-neg-c">
 
<div class="loc-neg-c">
−
<p><strong>Location</strong></p><br>
+
 
<p><img width="359" height="75" src="https://static.igem.org/mediawiki/2018/c/c9/T--ECUADOR--L001.png"> </p>
 
<p><img width="359" height="75" src="https://static.igem.org/mediawiki/2018/c/c9/T--ECUADOR--L001.png"> </p>
 
<br>
 
<br>
 
<p>length: 54 bp </p>
 
<p>length: 54 bp </p>
−
</div>
+
  </div>
 
<div class="textdis" id="pos_c">
 
<div class="textdis" id="pos_c">
−
<p><strong>Location</strong></p><br>
+
 
<p><img width="195" height="70" src="https://static.igem.org/mediawiki/2018/b/b5/T--ECUADOR--L002.png"></p>
 
<p><img width="195" height="70" src="https://static.igem.org/mediawiki/2018/b/b5/T--ECUADOR--L002.png"></p>
 
<br>
 
<br>
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</div>
 
</div>
 
<div class="loc-td1">
 
<div class="loc-td1">
−
<p><strong>Location</strong></p><br>
+
 
<p><img width="195" height="51" src="https://static.igem.org/mediawiki/2018/8/86/T--ECUADOR--L003.png" > </p>
 
<p><img width="195" height="51" src="https://static.igem.org/mediawiki/2018/8/86/T--ECUADOR--L003.png" > </p>
 
<br>
 
<br>
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</div>
 
</div>
 
<div class="loc-td2">
 
<div class="loc-td2">
−
<p><strong>Location</strong></p><br>
+
 
<p><img width="189" height="54" src="https://static.igem.org/mediawiki/2018/c/c8/T--ECUADOR--L004.png" ></p>
 
<p><img width="189" height="54" src="https://static.igem.org/mediawiki/2018/c/c8/T--ECUADOR--L004.png" ></p>
 
<br>
 
<br>
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</div>
 
</div>
 
<div class="loc-td3">
 
<div class="loc-td3">
−
<p><strong>Location</strong></p><br>
+
 
<p><img width="197" height="63" src="https://static.igem.org/mediawiki/2018/4/43/T--ECUADOR--L005.png" > </p>
 
<p><img width="197" height="63" src="https://static.igem.org/mediawiki/2018/4/43/T--ECUADOR--L005.png" > </p>
 
<br>
 
<br>
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</div>
 
</div>
 
<div class="loc-td4">
 
<div class="loc-td4">
−
<p><strong>Location</strong></p><br>
+
 
<p><img width="196" height="52" src="https://static.igem.org/mediawiki/2018/5/53/T--ECUADOR--L006.png"></p>
 
<p><img width="196" height="52" src="https://static.igem.org/mediawiki/2018/5/53/T--ECUADOR--L006.png"></p>
 
<br>
 
<br>
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</div>
 
</div>
 
<div class="loc-td5">
 
<div class="loc-td5">
−
<p><strong>Location</strong></p><br>
+
 
<p><img width="188" height="64" src="https://static.igem.org/mediawiki/2018/9/97/T--ECUADOR--L007.png"  > </p>
 
<p><img width="188" height="64" src="https://static.igem.org/mediawiki/2018/9/97/T--ECUADOR--L007.png"  > </p>
 
<br>
 
<br>
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</div>
 
</div>
 
<div class="loc-td6">
 
<div class="loc-td6">
−
<p><strong>Location</strong></p><br>
+
 
<p><img width="194" height="55" src="https://static.igem.org/mediawiki/2018/0/02/T--ECUADOR--L008.png"></p>
 
<p><img width="194" height="55" src="https://static.igem.org/mediawiki/2018/0/02/T--ECUADOR--L008.png"></p>
 
<br>
 
<br>
 
<p>length: 918 bp</p>
 
<p>length: 918 bp</p>
 
</div>
 
</div>
−
  </div>
+
    </div>
 
</div>
 
</div>
−
  </div>
+
  </div>
−
</div>
+
  </div>
−
<a name="RES" style="text-decoration: none;"><h2>RESULTS</h2></a>
+
−
              <strong>Particle standard curves</strong>
+
<a name="RES"><div class="ec--h2">Results</div></a>
−
  <div class="ec--img--wwd--cont">
+
  <div class="ec--h3">Particle standard curves</div>
−
<p><img src="https://static.igem.org/mediawiki/2018/3/30/T--ECUADOR--A-PSC.1.png" width="775" height="445" alt=""/></p>
+
<div class="ec--img--wwd--cont">
 +
<img src="https://static.igem.org/mediawiki/2018/3/30/T--ECUADOR--A-PSC.1.png" width="537" height="307" alt=""/>
 
</div>
 
</div>
 
<div class="ec--img--wwd--cont">
 
<div class="ec--img--wwd--cont">
−
  <p><img src="https://static.igem.org/mediawiki/2018/1/1d/T--ECUADOR--A-PSC.2.png" width="779" height="445" alt=""/></p>
+
<img src="https://static.igem.org/mediawiki/2018/1/1d/T--ECUADOR--A-PSC.2.png" width="534" height="303" alt=""/>
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  </div>
+
</div>
−
<br>
+
−
              <p><strong>Fluorescein standard curves</strong> </p>
+
<div class="ec--h3">Fluorescein standard curves</div>
 +
<div class="ec--img--wwd--cont">
 +
    <p><img src="https://static.igem.org/mediawiki/2018/7/7c/T--ECUADOR--A-FSC.1.png" width="534" height="303" alt=""/><br></p>
 +
  </div>
 
               <div class="ec--img--wwd--cont">
 
               <div class="ec--img--wwd--cont">
−
    <p><img src="https://static.igem.org/mediawiki/2018/7/7c/T--ECUADOR--A-FSC.1.png" width="779" height="445" alt=""/><br></p>
+
  <img src="https://static.igem.org/mediawiki/2018/c/cf/T--ECUADOR--A-FSC.2.png" width="534" height="303" alt=""/><br>
 
  </div>
 
  </div>
−
              <div class="ec--img--wwd--cont">
+
                <div class="ec--h3">Converting between absorbance of cells to absorbance of a known concentration of beads</div>
−
  <img src="https://static.igem.org/mediawiki/2018/c/cf/T--ECUADOR--A-FSC.2.png" width="779" height="445" alt=""/><br>
+
−
  </div>
+
−
                <strong>Net  Absorbance 600 as number of particles/100 uL</strong></p>
+
 
<div class="ec--img--wwd--cont">
 
<div class="ec--img--wwd--cont">
−
               <p><img src="https://static.igem.org/mediawiki/2018/6/64/T--ECUADOR--A-NA600.png" width="752" height="452" alt=""/></p>
+
               <p><img src="https://static.igem.org/mediawiki/2018/6/64/T--ECUADOR--A-NA600.png" width="534" height="303" alt=""/></p>
 
</div>
 
</div>
 +
  <div class="ec--h3">Counting colony-forming units (CFUs) from the sample</div>
 
<div class="ec--img--wwd--cont">
 
<div class="ec--img--wwd--cont">
−
               <p><img src="https://static.igem.org/mediawiki/2018/2/2e/T--ECUADOR--A-RBT.png" width="752" height="452" alt=""/></p>
+
               <p><img src="https://static.igem.org/mediawiki/2018/2/2e/T--ECUADOR--A-RBT.png" width="534" height="303" alt=""/></p>
 
</div>
 
</div>
−
<a name="EXP" style="text-decoration: none;"><h2>EXPERIENCE</h2></a>
+
−
            <div class="ec--img--wwd--cont">
+
  <a name="EXP"><div class="ec--h2">EXPERIENCE</div></a>
−
              <p><img src="https://svgsilh.com/svg/2324013-2196f3.svg" width="752" height="452" alt=""/></p>
+
</a>
−
</div>
+
              <div class="ec--img--wwd--cont">
−
               <p><em>As a team the interlab challenge was useful&nbsp;because we learn about the protocols and the handling  of the plate reader and how it can be used in later processes of our project</em></p>
+
                <center><img src="https://svgsilh.com/svg/2324013-2196f3.svg" width="309" height="175" alt=""/></center>
−
  <br>
+
              </div>
−
<a name="ACK" style="text-decoration: none;"><h2>ACKNOWLEDGEMENT</h2></a>
+
               <div class="ec--p">The interlab protocols were easy to understand and simple. We recommend the following:</div>
−
  <br>
+
              <ul>
−
                As a team, we would like to thank the  Biomedicine team of Universidad Tecnica Equinoccial and especially Linda  Guaman, member of their research team, for allowing us to use their measurement  equipment, which were essential for the completion of the Interlab.</div>
+
                <li>
−
<div class="ec--sect--text"></div>
+
                  <div class="ec--p">Before each measurement, shake the plates in the equipment since the particles tend to precipitate in the well.</div>
 +
                </li>
 +
                <li dir="ltr">
 +
                  <div class="ec--p">If the iGEM equipment does not have the plate reader in the same place as your laboratory, they must take care of their samples in continuous refrigeration during transport and carry out the activities with time.</div>
 +
                </li>
 +
              </ul>
 +
              <div class="ec--p">We learned many things in the interlab challenge: the protocols, the management of the plate reader and how it can be used in later processes of our project. It also helped us strengthen cooperation with other universities.</div>
 +
 +
              <a name="ACK" style="text-decoration: none;">
 +
              <div class="ec--h2">ACKNOWLEDGEMENT</div>
 +
              </a>
 +
<div class="ec--p">
 +
As a team, we would like to thank the  Biomedicine team of Universidad Tecnica Equinoccial and especially Linda  Guaman, member of their research team, for allowing us to use their measurement  equipment, which were essential for the completion of the Interlab.</div>
 +
 
</div>
 
</div>
 
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Revision as of 00:17, 18 October 2018

iGEM ECUADOR
C-lastin, Interlab
INTERLAB

What did we do?
We tried to reduce the variability in the measurement of GFP expression for this reason we used a direct method to determine the mean expression level of GFP per cel
How did we do it?
In order to avoid distortions in the results due to differences in the experimentation processes, we follow the procedures described in the iGEM protocol (Plate reader protocol, Competent cells, Transformation)l
Calibration
Before starting the experiments, we performed the OD600 calibration measurements following the LUDOX protocol, the particle Estandar Curve with the Microsphere Protocol, and the fluorescence standard curve with the Fluorescein Protocol. This allowed us to familiarize ourselves with the plate reader required for subsequent measurements. The plate reader that we used had the following characteristics:
Label: BioTek / Model: Cytation 5 / Emission: 528 / Excitation: 485 / Optic position: Top / Temperature: 25 ºC / Shake: 425 cpm for 30 sec before every measurement.
Cell measurement
We started by transforming E. coli DH5 alpha with the plasmid described in the protocol, and after incubation and growth in LB-medium, we diluted the culture to Abs600 0.2 and proceeded with the samples.
Colony units per 0.1 OD600 E. coli cultures
In order to determine the cell concentration of the culture we followed the CFU protocol that basically consisted in diluting a culture to certain absorbance, measure the OD600 that has to be in a value of 0.1, and make serial dilutions to later count the colonies and calculate the CFU/mL.
<div class="ec--h2">BioBricks</div>
Device
Part Number

Negative control

BBa_R0040

Positive control

BBa_I20270

Test Device 1

BBa_J364000

Test Device 2

BBa_J364001

Test Device 3

BBa_J364002

Test Device 4

BBa_J364007

Test Device 5

BBa_J364008

Test Device 6

BBa_J364009

Structure
(Point with the cursor on the device)


length: 54 bp


length: 919 bp


length: 918 bp


length: 918 bp


length: 918 bp


length: 918 bp


length: 918 bp


length: 918 bp

<div class="ec--h2">Results</div>
Particle standard curves
Fluorescein standard curves



Converting between absorbance of cells to absorbance of a known concentration of beads

Counting colony-forming units (CFUs) from the sample

<div class="ec--h2">EXPERIENCE</div>
The interlab protocols were easy to understand and simple. We recommend the following:
  • Before each measurement, shake the plates in the equipment since the particles tend to precipitate in the well.
  • If the iGEM equipment does not have the plate reader in the same place as your laboratory, they must take care of their samples in continuous refrigeration during transport and carry out the activities with time.
We learned many things in the interlab challenge: the protocols, the management of the plate reader and how it can be used in later processes of our project. It also helped us strengthen cooperation with other universities.
<div class="ec--h2">ACKNOWLEDGEMENT</div>
As a team, we would like to thank the Biomedicine team of Universidad Tecnica Equinoccial and especially Linda Guaman, member of their research team, for allowing us to use their measurement equipment, which were essential for the completion of the Interlab.
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