Difference between revisions of "Team:Birkbeck/Modeling"

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<h2> Modeling</h2>
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<h2> Modelling</h2>
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<h4>Note</h4>
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<p>In order to be considered for the <a href="https://2015.igem.org/Judging/Awards#SpecialPrizes">Best Model award</a>, you must fill out this page.</p>
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<h3>Modelling the directed evolution of bacteriophage lambda</h3>
  
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<p>Mathematical models and computer simulations provide a great way to describe the function and operation of BioBrick Parts and Devices. Synthetic Biology is an engineering discipline, and part of engineering is simulation and modeling to determine the behavior of your design before you build it. Designing and simulating can be iterated many times in a computer before moving to the lab. This award is for teams who build a model of their system and use it to inform system design or simulate expected behavior in conjunction with experiments in the wetlab.</p>
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<img src="https://static.igem.org/mediawiki/2015/7/70/Birkbeck_directed_evolution.png" height=200 width=900>  
  
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<p>To accomplish the goal of altering the host specificity of bacteriophage lambda, we took a step-by-step directed evolution approach, working according to the 'design - build - model - test - repeat' principle. This started with the design of several mutations both to alter the phage phenotype and to make segments of its genotype BioBrick-compatible. We then proceeded to 'build' individual genes through synthesis and cloning. The next steps are to recombine these genes into a recombinant bacteriophage genome, before carrying out testing and selection, and repeating this process.</p>
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Here are a few examples from previous teams:
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<br>
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</p>
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<p><u>The process is as follows:</u></p>
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<ul>
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<br><hr>
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<li><a href="https://2014.igem.org/Team:ETH_Zurich/modeling/overview">ETH Zurich 2014</a></li>
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<li><a href="https://2014.igem.org/Team:Waterloo/Math_Book">Waterloo 2014</a></li>
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</ul>
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  <th><center><img src="https://static.igem.org/mediawiki/2015/d/d4/Birkbeck_phage.png" height=150 width=100></center>
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      </th>
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  <td>Short tail fibre (stf) and reporter genes are cloned into a gt11 bacteriophage lambda vector.</td>
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  <th><img src="https://static.igem.org/mediawiki/2015/0/09/Birkbeck_circuit_development.png" height=100 width=220>
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      </th>
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  <td>Regulatory circuits are created to control the production of the tail fibre assembly (tfa) protein and the switch between lytic/lysogenic cycles of the bacteriophage. While AraC is produced, production of T7 RNA polymerase (RNAP) is inhibited, resulting in production of the cI repressor dominating and the bacteriophage remaining in lysogenic phase. On the addition of arabinose, AraC preferentially binds to to this molecule, dissociating from the promoter controlling T7 RNAP. As a result, T7 RNAP is produced, which initiates production of the Cro repressor, overriding cI production and placing the bacteriophage into the lytic phase.</td>
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</tr>
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</table>
  
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<style>
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table.lamp {
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    width:100%;
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<hr>
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  <th><img src="https://static.igem.org/mediawiki/2015/8/81/Birkbeck_phage_infection.png" height=200 width=200>
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      </th>
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  <td>The circularised gt11 vector and plasmids containing the regulatory circuits are transformed into E.coli host cells, and the host machinery is used to create a recombinant bacteriophage containing our desired mutations and additions.</td>
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</tr>
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</table>
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<style>
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table.lamp {
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    width:100%;
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    text-align:justify;
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table.lamp th {
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    width:220px;
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<hr>
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<table class="lamp">
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  <th><img src="https://static.igem.org/mediawiki/2015/a/af/Birkbeck_phages2.png" height=200 width=200>
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      </th>
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  <td>On addition of arabinose, the recombinant phage progeny goes into lytic phase and emerges from the lysed host cells.</td>
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</tr>
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</table>
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<style>
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table.lamp {
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    width:100%;
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    text-align:justify;
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}
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table.lamp th {
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    width:220px;
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}
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</style>
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<hr>
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<table class="lamp">
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<tr>
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  <th><img src="https://static.igem.org/mediawiki/2015/7/7d/Birkbeck_phage_infection2.png" height=200 width=200>
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      </th>
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  <td>Double knock-out strains of E.coli (containing neither lamB nor ompC receptor proteins) are exposed to the recombinant phages to select for baceriophage lambda mutants with widened host range.</td>
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</tr>
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</table>
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<style>
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table.lamp {
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    width:100%;
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    text-align:justify;
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}
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table.lamp th {
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    width:220px;
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}
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</style>
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<hr>
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<table class="lamp">
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  <th><img src="https://static.igem.org/mediawiki/2015/9/9c/Birkbeck_petri_dish.png" height=150 width=200>
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      </th>
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  <td>The added reporter gene will facilitate easy identification of phage progeny that has successfully infected the E.coli double knock-out strains. These bacteriophages are purified and cultured, and the process is repeated to further improve infectivity.</td>
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</tr>
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</table>
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<style>
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table.lamp {
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    width:100%;
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    text-align:justify;
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table.lamp th {
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    width:220px;
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<hr>
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<br>
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<br><hr><br>
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<!--button for Research-->
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<a href="https://2015.igem.org/Team:Birkbeck/Research"><img width="400px" height="auto" src="https://static.igem.org/mediawiki/2015/c/c7/Rachel-elliot.jpg" border="0"/></a>
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<p>Back to main Research page</p>
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<br><hr><br>
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<!--button for Results-->
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<a href="https://2015.igem.org/Team:Birkbeck/Results"><img width="400px" height="auto" src="https://static.igem.org/mediawiki/2015/1/16/Birkbeck_ORF314_construct_w_BsaI_primers_before_%26_after.png" border="0"/></a>
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<p>To find out more about our results</p>
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<!--<li><a href="https://2015.igem.org/Team:Birkbeck/Results">Continue to Results</a></li>
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<li><a href="https://2015.igem.org/Team:Birkbeck/Research">Back to main Research page</a></li>-->
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Latest revision as of 02:29, 19 September 2015

jQuery UI Accordion - Collapse content

Modelling

Modelling the directed evolution of bacteriophage lambda

To accomplish the goal of altering the host specificity of bacteriophage lambda, we took a step-by-step directed evolution approach, working according to the 'design - build - model - test - repeat' principle. This started with the design of several mutations both to alter the phage phenotype and to make segments of its genotype BioBrick-compatible. We then proceeded to 'build' individual genes through synthesis and cloning. The next steps are to recombine these genes into a recombinant bacteriophage genome, before carrying out testing and selection, and repeating this process.


The process is as follows:


 Short tail fibre (stf) and reporter genes are cloned into a gt11 bacteriophage lambda vector.
Regulatory circuits are created to control the production of the tail fibre assembly (tfa) protein and the switch between lytic/lysogenic cycles of the bacteriophage. While AraC is produced, production of T7 RNA polymerase (RNAP) is inhibited, resulting in production of the cI repressor dominating and the bacteriophage remaining in lysogenic phase. On the addition of arabinose, AraC preferentially binds to to this molecule, dissociating from the promoter controlling T7 RNAP. As a result, T7 RNAP is produced, which initiates production of the Cro repressor, overriding cI production and placing the bacteriophage into the lytic phase.
The circularised gt11 vector and plasmids containing the regulatory circuits are transformed into E.coli host cells, and the host machinery is used to create a recombinant bacteriophage containing our desired mutations and additions.
On addition of arabinose, the recombinant phage progeny goes into lytic phase and emerges from the lysed host cells.
Double knock-out strains of E.coli (containing neither lamB nor ompC receptor proteins) are exposed to the recombinant phages to select for baceriophage lambda mutants with widened host range.
The added reporter gene will facilitate easy identification of phage progeny that has successfully infected the E.coli double knock-out strains. These bacteriophages are purified and cultured, and the process is repeated to further improve infectivity.



Back to main Research page



To find out more about our results