Difference between revisions of "Team:WashU StLouis/parts"

Line 46: Line 46:
 
           </tr>
 
           </tr>
 
           <tr>
 
           <tr>
−
             <td>High constitutive expression cassette</td>
+
             <td>Artificial RBS for nifZ</td>
−
             <td><a href="http://parts.igem.org/Part:BBa_K314100">BBa_K314100</a></td>
+
             <td><a href="http://parts.igem.org/Part:BBa_K1605002">BBa_K1605002</a></td>
 +
          </tr>
 +
          <tr>
 +
            <td>Artificial RBS for nifE</td>
 +
            <td><a href="http://parts.igem.org/Part:BBa_K1605004">BBa_K1605004</a></td>
 +
          </tr>
 +
          <tr>
 +
            <td>Artificial RBS for nifH</td>
 +
            <td><a href="http://parts.igem.org/Part:BBa_K1605006">BBa_K1605006</a></td>
 +
          </tr>
 +
          <tr>
 +
            <td>Artificial RBS for nifS</td>
 +
            <td><a href="http://parts.igem.org/Part:BBa_K1605008">BBa_K1605008</a></td>
 +
          </tr>
 +
          <tr>
 +
            <td>Artificial RBS for nifK</td>
 +
            <td><a href="http://parts.igem.org/Part:BBa_K1605010">BBa_K1605010</a></td>
 +
          </tr>
 +
          <tr>
 +
            <td>Artificial RBS for nifB</td>
 +
            <td><a href="http://parts.igem.org/Part:BBa_K1605012">BBa_K1605012</a></td>
 +
          </tr>
 +
          <tr>
 +
            <td>Artificial RBS for nifD</td>
 +
            <td><a href="http://parts.igem.org/Part:BBa_K1605014">BBa_K1605014</a></td>
 +
          </tr>
 +
          <tr>
 +
            <td>Artificial RBS for nifN</td>
 +
            <td><a href="http://parts.igem.org/Part:BBa_K1605016">BBa_K1605016</a></td>
 +
          </tr>
 +
          <tr>
 +
            <td>Artificial RBS for hesB</td>
 +
            <td><a href="http://parts.igem.org/Part:BBa_K1605018">BBa_K1605018</a></td>
 +
          </tr>
 +
          <tr>
 +
            <td>Artificial RBS for hesA</td>
 +
            <td><a href="http://parts.igem.org/Part:BBa_K1605020">BBa_K1605020</a></td>
 +
          </tr>
 +
          <tr>
 +
            <td>Artificial RBS for cysE2</td>
 +
            <td><a href="http://parts.igem.org/Part:BBa_K1605022">BBa_K1605022</a></td>
 +
          </tr>
 +
          <tr>
 +
            <td>Artificial RBS for nifV</td>
 +
            <td><a href="http://parts.igem.org/Part:BBa_K1605024">BBa_K1605024</a></td>
 
           </tr>
 
           </tr>
 
         </tbody>
 
         </tbody>
 
       </table>
 
       </table>
 
       <h2><a href="#promoter">Promoter Characterization</a></h2>
 
       <h2><a href="#promoter">Promoter Characterization</a></h2>
−
       <table class="table table-responsive">
+
       <table class="table table-responsive table-striped">
 
         <tbody>
 
         <tbody>
 
           <tr>
 
           <tr>

Revision as of 01:57, 17 September 2015

Washington University - Penn State iGEM

Welcome To Our Website!
WashU and Penn State   iGEM 2015
Project Description Let's Talk Apply for the 2016 iGEM Team!

RBS Characterization

Name Part Number
Artificial RBS for nifZ BBa_K1605002
Artificial RBS for nifE BBa_K1605004
Artificial RBS for nifH BBa_K1605006
Artificial RBS for nifS BBa_K1605008
Artificial RBS for nifK BBa_K1605010
Artificial RBS for nifB BBa_K1605012
Artificial RBS for nifD BBa_K1605014
Artificial RBS for nifN BBa_K1605016
Artificial RBS for hesB BBa_K1605018
Artificial RBS for hesA BBa_K1605020
Artificial RBS for cysE2 BBa_K1605022
Artificial RBS for nifV BBa_K1605024

Promoter Characterization

Name Part Number
High constitutive expression cassette BBa_K314100

The 2015 WashU iGEM conducted a series of induction experiments to determine the validity of part K314100. The results obtained suggest that the part isn’t subject to inducer concentrations. The part either fluoresced at a constant, high level or didn’t fluoresce at all when tested.

The team transformed the part into DH10B twice. The colonies of the first transformation looked like this:

The cells largely appeared red. The WashU iGEM team then conducted an induction experiment to test how the cells fluoresced at different concentrations of aTc. The team pipetted cell culture into different aTc concentrations serially diluted across 8 wells. Results from this induction experiment are shown below.

While the part had a 1.3x increase between the lowest and highest inducer concentrations, the fluorescence increase was not uniform across all concentrations; fluorescence decreased at certain concentrations when it should’ve been increased.

The team proceeded with a second transformation to test the part even further. Colonies from that transformation are shown below.

Cells were largely colorless, with few red colored colonies to be found. Using the same procedure from the first experiment, the team ran a second induction experiment on these colonies to observe whether red fluorescence increased with aTc concentration. The results from that experiment are shown below.

The colonies exhibited no remarkable fluorescence regardless of the aTc concentration. At this point in time, the team hypothesized that the colonies from the first transformation were simply different than the colonies from the second transformation, perhaps as a result of homologous recombination. The team decided to run a third induction experiment, with the same protocol used in the previous two experiments, to determine the validity behind this assertion. Two colonies were picked from the first transformation; one colony was picked from the second transformation. The results from this experiment are shown below.

Values for each colony are consistent with what was obtained in their respective experiments. While the team didn’t sequence the parts in each colony, it suspects that two separate strains had been produced, in which the part had been mutated in some manner. Regardless, the team holds little confidence in the effectiveness of this part.