Calgary/testing

From 2007.igem.org

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<p> The Primers have been designed the parts have been ordered and a plan has been put together. Now its time to start construction right? Wrong! All of these parts and ideas have to be thoroughly tested. We need to make sure that all the parts we've ordered are what their supposed to be, where their supposed to be and do what they are supposed to do. Tedious I know... </p>
 +
<p> This portion of our project provided unique challenges for a couple of reasons, which we are going to outline here so that anyone attempting to repeat our project won't have to deal with the same intense frustration and agony we did. Aren't we nice? </p>
 +
<p><em>Some of our bigger challenges in the testing phase...</em></p>
 +
<ul>
 +
  <li style="margin-bottom:20px;">Ok, first of all. When we ordered our initial batch of parts from MIT we tried to start working with them right away. This was not a great idea as full descriptions of each parts coordinates on the plates were not readily available from the registry. This lead to a brilliantly doomed attempt at reverse engineering the part locations. Things never really worked out with this batch unfortunately, did I mention it was brilliantly doomed. So The lesson is to be very careful with the parts you have ordered and take the time to work with the registry and ensure that you know 100% where everything is and how it is labled. Learn from us... seriously</li>
 +
  <li style="margin-bottom:20px;">Our second major hurdle had to do with our TOP10 cells. Those are cells that are competent to be transformed in our crazy biological experiments in case you didn't know. Our cells, for some reason had an inate resistance to chloramphenicol. This was a huge issue as most of our test plates contained chloramphenicol which we were using as a selection pressure. So while the odds of this happening are unlikely keep in mind this could be one of the problems you may face.</li>
 +
  <li style="margin-bottom:20px;">Third major issue came with one of the pivotal parts of our system, the light sensor. The registry part, M30109, is based off of the light sensor plasmids developped by the Austin iGEM '05 ad '06 teams. This part consists of the three seperate genes, which should form a working light sensor when transformed. Our current theory (as put forth by Patrick King) is that there may be a mutation at one of the biobrick restriction sites that prevented us from being able to isolate the part from its plasmid backbone. The biobrick primers we use also anneal at the biobrick restriction sites, and our attempts to PCR out the part were also unsuccesful. So we spent a few agonizing months trying to figure out why the part would not cut, and would not replicate in PCR, the way it should. Eventually we resorted to contacting the texas team and asking them for the two plasmids from their original project. It was here that we were informed of a potentially significant issue. The texas team had actually co transformed two seperate plasmids into their cells. One texas plasmid contained two of the light sensor genes, while the other contained the third. Apparently, the plasmid with two genes (pPlPCB, with genes Ho1 and PcyA) is unstable, perhaps due to the genes using too much cellular iron from transformed cells and making the cell unhealthy. The registry part had all three of them in one plasmid (M30109), and if pPLPCB is unstable then M30109 should be also. This may have also contributed to our problems.</li>
 +
</ul>
 +
<p> So with the introduction and important stuff out of the way check out our part by part testing section. Also click on any part name to link to our simplified table of parts!</p>
 +
<table width="100%" style="margin-bottom:30px;">
 +
  <tr>
 +
    <td valign="top"><ul id="sub">
 +
        <li><a href="#r0084" title="ompF promoter R0084"> ompF promoter R0084 </a></li>
 +
        <li><a href="#R0062" title="AHL Promoter R0062"> AHL Promoter R0062</a></li>
 +
        <li><a href="#R0011" title="Lac TS Promoter R0011"> Lac TS Promoter R0011 </a></li>
 +
      </ul></td>
 +
    <td valign="top"><ul id ="sub">
 +
        <li><a href="#ct" title="Construction Technique"> Construction Technique</a></li>
 +
        <li><a href="#pp" title="Plasmid Prep"> Plasmid Prep </a></li>
 +
        <li><a href="#ap" title="Antarctic Phosphatase"> Vector Dephosphorylation </a></li>
 +
      </ul></td>
 +
    <td valign="top"><ul id="sub">
 +
        <li><a href="#platePrep" title="Antarctic Phosphatase"> LB - Agar Plate </a></li>
 +
        <li><a href="#overnight" title="overnight"> Over Night Growth </a></li>
 +
        <li><a href="#gsp" title="Glycerol Stock"> Glycerol Stock Preparation </a></li>
 +
      </ul></td>
 +
    <td valign="top"><ul id="sub">
 +
        <li><a href="#age" title="Agarose Gel Electrophoresis"> Agarose Gel Electrophoresis </a></li>
 +
        <li><a href="#rd" title="Restriction Digest"> Restriction Digest </a></li>
 +
        <li><a href="#lp" title="Ligation"> Ligation </a></li>
 +
      </ul></td>
 +
  </tr>
 +
</table>
 +
<hr />
 +
<table width="100%">
 +
  <tr>
 +
    <td style="width:85%;"><p style="font-size:16px; font-weight:bold"><a style="text-decoration:none" name="r0084"> ompF promotor</a></p></td>
 +
    <td width="15%"><a style="float:right;" href="#top" title="return to top">return to top</a> </td>
 +
  </tr>
 +
</table>
 +
<table cellpadding="15px;" width="100%">
 +
  <tr>
 +
    <td><table width="100%">
 +
        <tr>
 +
          <td><b>Part</b></td>
 +
          <td>R0084</td>
 +
        </tr>
 +
        <tr>
 +
          <td><b>Plasmid</b></td>
 +
          <td>pSB1A2</td>
 +
        </tr>
 +
        <tr>
 +
          <td><b>Description</b></td>
 +
          <td>ompF promoter</td>
 +
        </tr>
 +
        <tr>
 +
          <td><b>Source</b></td>
 +
          <td>Plate</td>
 +
        </tr>
 +
        <tr>
 +
          <td><b>Transformed</b></td>
 +
          <td>YES</td>
 +
        </tr>
 +
        <tr>
 +
          <td><b>PCR Verified</b></td>
 +
          <td>June 29th 2007</td>
 +
        </tr>
 +
        <tr>
 +
          <td><b>Glycerol</b></td>
 +
          <td>YES</td>
 +
        </tr>
 +
        <tr>
 +
          <td><b>Length (base pairs)</b></td>
 +
          <td>108</td>
 +
        </tr>
 +
      </table></td>
 +
    <td><img src="June29labelled.gif" alt="Labled Gel from June 29th 2007" /> </td>
 +
  </tr>
 +
</table>
 +
 +
<table width="100%">
 +
  <tr>
 +
    <td style="width:85%;"><p style="font-size:16px; font-weight:bold"><a style="text-decoration:none" name="R0062"> AHL promotor</a></p></td>
 +
    <td width="15%"><a style="float:right;" href="#top" title="return to top">return to top</a> </td>
 +
  </tr>
 +
</table>
 +
<table cellpadding="15px;" width="100%">
 +
  <tr>
 +
    <td><table width="100%">
 +
        <tr>
 +
          <td><b>Part</b></td>
 +
          <td>R0062</td>
 +
        </tr>
 +
        <tr>
 +
          <td><b>Plasmid</b></td>
 +
          <td><table cellspacing="0" cellpadding="0">
 +
  <td height="17" width="101">pSB1A2</td>
 +
          </table>
 +
          </td>
 +
        </tr>
 +
        <tr>
 +
          <td><b>Description</b></td>
 +
          <td>AHL promoter</td>
 +
        </tr>
 +
        <tr>
 +
          <td><b>Source</b></td>
 +
          <td>Glycerol</td>
 +
        </tr>
 +
        <tr>
 +
          <td><b>Transformed</b></td>
 +
          <td>YES</td>
 +
        </tr>
 +
        <tr>
 +
          <td><b>PCR Verified</b></td>
 +
          <td>June 29th 2007</td>
 +
        </tr>
 +
        <tr>
 +
          <td><b>Glycerol</b></td>
 +
          <td>YES</td>
 +
        </tr>
 +
        <tr>
 +
          <td><b>Length (base pairs)</b></td>
 +
          <td>55</td>
 +
        </tr>
 +
      </table></td>
 +
    <td><img src="https://static.igem.org/mediawiki/2007/c/c5/June29labelled.gif" alt="Labled Gel from June 29th 2007" /> </td>
 +
  </tr>
 +
</table>
 +
 +
<table width="100%">
 +
  <tr>
 +
    <td style="width:85%;"><p style="font-size:16px; font-weight:bold"><a style="text-decoration:none" name="r0084"> lac TS promoter</a></p></td>
 +
    <td width="15%"><a style="float:right;" href="#top" title="return to top">return to top</a> </td>
 +
  </tr>
 +
</table>
 +
<table cellpadding="15px;" width="100%">
 +
  <tr>
 +
    <td><table width="100%">
 +
        <tr>
 +
          <td><b>Part</b></td>
 +
          <td><table cellspacing="0" cellpadding="0">
 +
  <td height="17" width="70">R0011</td>
 +
          </table></td>
 +
        </tr>
 +
        <tr>
 +
          <td><b>Plasmid</b></td>
 +
          <td><table cellspacing="0" cellpadding="0">
 +
  <td height="17" width="101">pSB1A2</td>
 +
          </table></td>
 +
        </tr>
 +
        <tr>
 +
          <td><b>Description</b></td>
 +
          <td><table cellspacing="0" cellpadding="0">
 +
  <td height="17" width="368">lac TS promoter</td>
 +
          </table></td>
 +
        </tr>
 +
        <tr>
 +
          <td><b>Source</b></td>
 +
          <td>Plate</td>
 +
        </tr>
 +
        <tr>
 +
          <td><b>Transformed</b></td>
 +
          <td>YES</td>
 +
        </tr>
 +
        <tr>
 +
          <td><b>PCR Verified</b></td>
 +
          <td>June 29th 2007</td>
 +
        </tr>
 +
        <tr>
 +
          <td><b>Glycerol</b></td>
 +
          <td>YES</td>
 +
        </tr>
 +
        <tr>
 +
          <td><b>Length (base pairs)</b></td>
 +
          <td>55</td>
 +
        </tr>
 +
      </table></td>
 +
    <td><img src="https://static.igem.org/mediawiki/2007/c/c5/June29labelled.gif" alt="Labled Gel from June 29th 2007" /> </td>
 +
  </tr>
 +
</table>
 +
 +
 +
<table width="100%">
 +
  <tr>
 +
    <td style="width:85%;"><p style="font-size:16px; font-weight:bold"><a style="text-decoration:none" name="R0011">lac TS promoter</a></p></td>
 +
    <td width="15%"><a style="float:right;" href="#top" title="return to top">return to top</a> </td>
 +
  </tr>
 +
</table>
 +
<table cellpadding="15px;" width="100%">
 +
  <tr>
 +
    <td><table width="100%">
 +
        <tr>
 +
          <td><b>Part</b></td>
 +
          <td><table cellspacing="0" cellpadding="0">
 +
  <td height="17" width="70">R0011</td>
 +
          </table></td>
 +
        </tr>
 +
        <tr>
 +
          <td><b>Plasmid</b></td>
 +
          <td><table cellspacing="0" cellpadding="0">
 +
  <td height="17" width="101">pSB1A2</td>
 +
          </table></td>
 +
        </tr>
 +
        <tr>
 +
          <td><b>Description</b></td>
 +
          <td><table cellspacing="0" cellpadding="0">
 +
  <td height="17" width="368">lac TS promoter</td>
 +
          </table></td>
 +
        </tr>
 +
        <tr>
 +
          <td><b>Source</b></td>
 +
          <td>Plate</td>
 +
        </tr>
 +
        <tr>
 +
          <td><b>Transformed</b></td>
 +
          <td>YES</td>
 +
        </tr>
 +
        <tr>
 +
          <td><b>PCR Verified</b></td>
 +
          <td>June 29th 2007</td>
 +
        </tr>
 +
        <tr>
 +
          <td><b>Glycerol</b></td>
 +
          <td>YES</td>
 +
        </tr>
 +
        <tr>
 +
          <td><b>Length (base pairs)</b></td>
 +
          <td>55</td>
 +
        </tr>
 +
      </table></td>
 +
    <td><img src="https://static.igem.org/mediawiki/2007/c/c5/June29labelled.gif" alt="Labled Gel from June 29th 2007" /> </td>
 +
  </tr>
 +
</table>
 +
<p> I) Colony PCR test...
<p> I) Colony PCR test...
<ul>
<ul>
-
<li> Design universal primers (valid for any biobrick out of any plasmid)</li>
+
  <li> Design universal primers (valid for any biobrick out of any plasmid)</li>
-
<li> Develop massive amounts </li>
+
  <li> Develop massive amounts </li>
-
<li> Gel to test </li>
+
  <li> Gel to test </li>
</ul>
</ul>
</p>
</p>
-
<p> II) Test Plasmids --> always grow up 5ml of overnight
+
<p> II) Test Plasmids --> always grow up 5ml of overnight
-
<ul>
+
<ul>
-
<li> Plasmid Prep --> isolating plasmid out of the cell </li>
+
  <li> Plasmid Prep --> isolating plasmid out of the cell </li>
-
<li> Sigma Plasmid Prep protocol </li>
+
  <li> Sigma Plasmid Prep protocol </li>
</ul>
</ul>
</p>
</p>
</body>
</body>
</html>
</html>

Revision as of 01:11, 23 October 2007

Choosing Our Project Designing Our Project Testing Parts and Primers Constructing our Project: The Wetlab Constructing our Project: Printer and Software Protocols Final Result of E.co Lisa

The Primers have been designed the parts have been ordered and a plan has been put together. Now its time to start construction right? Wrong! All of these parts and ideas have to be thoroughly tested. We need to make sure that all the parts we've ordered are what their supposed to be, where their supposed to be and do what they are supposed to do. Tedious I know...

This portion of our project provided unique challenges for a couple of reasons, which we are going to outline here so that anyone attempting to repeat our project won't have to deal with the same intense frustration and agony we did. Aren't we nice?

Some of our bigger challenges in the testing phase...

  • Ok, first of all. When we ordered our initial batch of parts from MIT we tried to start working with them right away. This was not a great idea as full descriptions of each parts coordinates on the plates were not readily available from the registry. This lead to a brilliantly doomed attempt at reverse engineering the part locations. Things never really worked out with this batch unfortunately, did I mention it was brilliantly doomed. So The lesson is to be very careful with the parts you have ordered and take the time to work with the registry and ensure that you know 100% where everything is and how it is labled. Learn from us... seriously
  • Our second major hurdle had to do with our TOP10 cells. Those are cells that are competent to be transformed in our crazy biological experiments in case you didn't know. Our cells, for some reason had an inate resistance to chloramphenicol. This was a huge issue as most of our test plates contained chloramphenicol which we were using as a selection pressure. So while the odds of this happening are unlikely keep in mind this could be one of the problems you may face.
  • Third major issue came with one of the pivotal parts of our system, the light sensor. The registry part, M30109, is based off of the light sensor plasmids developped by the Austin iGEM '05 ad '06 teams. This part consists of the three seperate genes, which should form a working light sensor when transformed. Our current theory (as put forth by Patrick King) is that there may be a mutation at one of the biobrick restriction sites that prevented us from being able to isolate the part from its plasmid backbone. The biobrick primers we use also anneal at the biobrick restriction sites, and our attempts to PCR out the part were also unsuccesful. So we spent a few agonizing months trying to figure out why the part would not cut, and would not replicate in PCR, the way it should. Eventually we resorted to contacting the texas team and asking them for the two plasmids from their original project. It was here that we were informed of a potentially significant issue. The texas team had actually co transformed two seperate plasmids into their cells. One texas plasmid contained two of the light sensor genes, while the other contained the third. Apparently, the plasmid with two genes (pPlPCB, with genes Ho1 and PcyA) is unstable, perhaps due to the genes using too much cellular iron from transformed cells and making the cell unhealthy. The registry part had all three of them in one plasmid (M30109), and if pPLPCB is unstable then M30109 should be also. This may have also contributed to our problems.

So with the introduction and important stuff out of the way check out our part by part testing section. Also click on any part name to link to our simplified table of parts!

ompF promotor

 return to top
Part R0084
Plasmid pSB1A2
Description ompF promoter
Source Plate
Transformed YES
PCR Verified June 29th 2007
Glycerol YES
Length (base pairs) 108
Labled Gel from June 29th 2007

AHL promotor

 return to top
Part R0062
Plasmid
pSB1A2
Description AHL promoter
Source Glycerol
Transformed YES
PCR Verified June 29th 2007
Glycerol YES
Length (base pairs) 55
Labled Gel from June 29th 2007

lac TS promoter

 return to top
Part
R0011
Plasmid
pSB1A2
Description
lac TS promoter
Source Plate
Transformed YES
PCR Verified June 29th 2007
Glycerol YES
Length (base pairs) 55
Labled Gel from June 29th 2007

lac TS promoter

 return to top
Part
R0011
Plasmid
pSB1A2
Description
lac TS promoter
Source Plate
Transformed YES
PCR Verified June 29th 2007
Glycerol YES
Length (base pairs) 55
Labled Gel from June 29th 2007

I) Colony PCR test...

  • Design universal primers (valid for any biobrick out of any plasmid)
  • Develop massive amounts
  • Gel to test

II) Test Plasmids --> always grow up 5ml of overnight

  • Plasmid Prep --> isolating plasmid out of the cell
  • Sigma Plasmid Prep protocol

Retrieved from "http://2007.igem.org/wiki/index.php/Calgary/testing"