|
|
| Line 146: |
Line 146: |
| | </div> | | </div> |
| | | | |
| − | <specialh4>Transcription Rate Sensitivity Analysis</specialh4> <br><br><br>
| |
| − | <center>
| |
| − | <figure>
| |
| − | <img src="https://static.igem.org/mediawiki/2016/8/83/T--Imperial_College--transcriptionrates1.png">
| |
| − | <figcaption><p>Figure 1: Sensitivity analysis for the transcription rates in the GP2 and GP0.4 models</p></figcaption>
| |
| − | </figure>
| |
| − | </center>
| |
| − |
| |
| − | <br><br>
| |
| − |
| |
| − | <specialh4>Translation Rate Sensitivity Analysis</specialh4>
| |
| − |
| |
| − | <br><br><br>
| |
| − | <center>
| |
| − | <figure>
| |
| − | <img src="https://static.igem.org/mediawiki/2016/f/f0/T--Imperial_College--transcriptionrategp04.png">
| |
| − | <figcaption><p>Figure 2: Sensitivity analysis for the translation rates in the GP2 and GP0.4 models</p></figcaption>
| |
| − | </figure>
| |
| − | </center>
| |
| − |
| |
| − | <br><br>
| |
| − | <specialh4>Degredation Rate Sensitivity Analysis</specialh4>
| |
| − |
| |
| − | <br><br><br>
| |
| − | <center>
| |
| − | <figure>
| |
| − | <img src="https://static.igem.org/mediawiki/2016/a/a6/T--Imperial_College--degredationsensitivity.png">
| |
| − | <figcaption><p>Figure 3: Sensitivity analysis for the degradation rates in the GP2 and GP0.4 models</p></figcaption>
| |
| − | </figure>
| |
| − | </center>
| |
| − | <br><br>
| |
| − | <specialh4>Copy Number Sensitivity Analysis</specialh4>
| |
| − |
| |
| − | <br><br><br>
| |
| − | <center>
| |
| − | <figure>
| |
| − | <img src="https://static.igem.org/mediawiki/2016/4/44/T--Imperial_College--copynumbersensitivity.png">
| |
| − | <figcaption><p>Figure 4: Sensitivity analysis for the copy numbers in the GP2 and GP0.4 models </p></figcaption>
| |
| − | </figure>
| |
| − | </center>
| |
| − |
| |
| − |
| |
| − | <br><br>
| |
| − | <p>
| |
| − | Our next process was to create a framework in which we could balance our circuit in silico. To do this we ran parameter sweeps for each of the transcription rates, translation rates and degradation rates that were indicated by the sensitivity analysis. We used known numbers from biobrick parts as we wanted our model to use the materials available to us in the lab whenever possible.</p>
| |
| − |
| |
| − |
| |
| − |
| |
| − |
| |
| − | <br><br>
| |
| − | <center>
| |
| − | <figure>
| |
| − | <img src="https://static.igem.org/mediawiki/2016/9/91/T--Imperial_College--graph1.png">
| |
| − | <figcaption><p>Figure 5: Parameter sweep for the transcription rate of C4R (k_mC4R) encompassing the anderson promoter library</p></figcaption>
| |
| − | </figure>
| |
| − | </center>
| |
| − |
| |
| − |
| |
| − | <br><br>
| |
| − |
| |
| − | <br><br>
| |
| − | <p>
| |
| − | This data was then used this data in our population level models to balance the system and predetermine the ratio of cocultures.
| |
| − | </p>
| |
| − | <br><br>
| |
| − | </p>
| |
| − |
| |
| − | </div>
| |
| − | </section>
| |
| − |
| |
| − | <section id="QuorumModel">
| |
| − | <div class="col-lg-10 col-centered">
| |
| − | <p>
| |
| − | <specialh3>Communication module</specialh3><br>
| |
| − |
| |
| − | We constructed the four quorum systems that we considered viable choices for our system (cin, rhl, lux and las) to allow us to directly compare the expected behaviour and plan our growth module experiments accordingly. We designed the overall model for the Rhl and Cin systems (Chen et al., 2015) as they have been previously shown to operate with minimal crosstalk.
| |
| − | <br>
| |
| − | <br>
| |
| − | <specialh3 class="center-text">
| |
| − | Assumptions
| |
| − | </specialh3>
| |
| − | <div class="text-center">
| |
| − | <blockquote>
| |
| − | We used numbers obtained from Chen et al for C4 and C14 production. This is a high level production term that ignores parts of the central dogma. We made this assumption due to the limited data on the enzymatic kinetics of the autoinducer synthases.
| |
| − | </blockquote>
| |
| − | </div>
| |
| − | <br>
| |
| − |
| |
| − |
| |
| − | </p>
| |
| − |
| |
| − | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true">
| |
| − | <div class="panel panel-default">
| |
| − | <div class="panel-heading" role="tab" id="RhlAcc">
| |
| − | <h4 class="panel-title">
| |
| − | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#RhlAcc-collapse" aria-expanded="false" aria-controls="RhlAcc-collapse">
| |
| − | Reactions
| |
| − | <span class="pull-right caret"></span>
| |
| − | </a>
| |
| − | </h4>
| |
| − |
| |
| − | </div>
| |
| − | <div id="RhlAcc-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="RhlAcc">
| |
| − | <div class="panel-body">
| |
| − | <center>
| |
| − | <specialh4> Communication module </specialh4></center>
| |
| − | \[\varnothing\rightarrow C14\]
| |
| − |
| |
| − | \[\varnothing\rightarrow C14\]
| |
| − |
| |
| − | \[RNA_{pol} + g_{C4R} \rightarrow RNA_{pol} + g_{C4R} + mRNA_{C4R}\]
| |
| − |
| |
| − | \[RNA_{pol} + g_{C14R} \rightarrow RNA_{pol} + g_{C14R} + mRNA_{C4R}\]
| |
| − |
| |
| − | \[mRNA_{C4R}\rightarrow C4R\]
| |
| − |
| |
| − | \[mRNA_{C14R}\rightarrow C14R\]
| |
| − |
| |
| − | \[C14+C14R \rightarrow C14_{complex}\]
| |
| − |
| |
| − | \[C4+C4R \rightarrow C4_{complex}\]
| |
| − |
| |
| − | \[C4\rightarrow\varnothing\]
| |
| − |
| |
| − | \[C4R\rightarrow\varnothing\]
| |
| − |
| |
| − | \[mRNA_{C4R}\rightarrow\varnothing\]
| |
| − |
| |
| − | \[C4_{complex}\rightarrow\varnothing\]
| |
| − |
| |
| − | \[C14\rightarrow\varnothing\]
| |
| − |
| |
| − | \[C14R\rightarrow\varnothing\]
| |
| − |
| |
| − | \[mRNA_{C14R}\rightarrow\varnothing\]
| |
| − |
| |
| − | \[C14_{complex}\rightarrow\varnothing\]
| |
| − |
| |
| − |
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="some-padding"></div>
| |
| − |
| |
| − | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true">
| |
| − | <div class="panel panel-default">
| |
| − | <div class="panel-heading" role="tab" id="CinAcc">
| |
| − | <h4 class="panel-title">
| |
| − | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#CinAcc-collapse" aria-expanded="false" aria-controls="CinAcc-collapse">
| |
| − | Simulation Results
| |
| − | <span class="pull-right caret"></span>
| |
| − | </a>
| |
| − | </h4>
| |
| − |
| |
| − | </div>
| |
| − | <div id="CinAcc-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="CinAcc">
| |
| − | <div class="panel-body">
| |
| − | <p> <center>
| |
| − |
| |
| − | <figure>
| |
| − | <img src="https://static.igem.org/mediawiki/2016/9/95/T--Imperial_College--productionofc4vs4hl.png">
| |
| − | <figcaption><p>Figure 6: Production of C4 AHL and C14 AHL against time</p> </figcaption>
| |
| − | </figure>
| |
| − |
| |
| − | <br><br>
| |
| − |
| |
| − | <figure>
| |
| − | <img src="https://static.igem.org/mediawiki/2016/e/ed/T--Imperial_College--productionc4c14.png">
| |
| − | <figcaption><p>Figure 7: Production of C4R and C14R against time</p> </figcaption>
| |
| − | </figure>
| |
| − |
| |
| − |
| |
| − | <br><br>
| |
| − |
| |
| − | <figure>
| |
| − | <img src="https://static.igem.org/mediawiki/2016/b/b5/T--Imperial_College--c4c14complexformation.png">
| |
| − | <figcaption><p>Figure 8: C4 and C14 Complex formation against time</p></figcaption>
| |
| − | </figure>
| |
| − | </center>
| |
| − |
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | </section>
| |
| − |
| |
| − |
| |
| − | <section id="StarModel">
| |
| − |
| |
| − | <div class="col-lg-10 col-centered">
| |
| − | <p>
| |
| − | <specialh3>Comparator module</specialh3><br>
| |
| − |
| |
| − | Using STAR (Short Transcription Activating RNA) technology, we were able to develop a novel method of comparing the sizes of two populations from their quorum signal concentrations.<br><br>
| |
| − | We used RNAstruct developed by <a href="http://rna.urmc.rochester.edu/RNAstructure.html">Matthews Lab</a> to help aid the development of the ANTISTAR.<br><br>
| |
| − | This software allowed us to determine the secondary structure and free energy to optimize the way in which our ANTISTAR sequence was designed. This was done so that our ANTISTAR sequence would have as high an affinity to the STAR sequence as was possible.<br><br>
| |
| − |
| |
| − | <center>
| |
| − | <figure>
| |
| − | <img src="https://static.igem.org/mediawiki/2016/3/37/T--Imperial_College--RNAstruct.png">
| |
| − | <figcaption><p>Figure 9: Secondary structure of our ANTISTAR sequence</p></figcaption>
| |
| − | </figure>
| |
| − | </center>
| |
| − |
| |
| − |
| |
| − | To calculate the kinetics of the RNA interactions that occur within this module we adapted a method developed by Eric Winfree known as DNA strand displacement kinetics (Zhang and Winfree, 2009)
| |
| − |
| |
| − | </p>
| |
| − | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true">
| |
| − | <div class="panel panel-default">
| |
| − | <div class="panel-heading" role="tab" id="StarAcc">
| |
| − | <h4 class="panel-title">
| |
| − | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#StarAcc-collapse" aria-expanded="false" aria-controls="StarAcc-collapse">
| |
| − | Star
| |
| − | <span class="pull-right caret"></span>
| |
| − | </a>
| |
| − | </h4>
| |
| − |
| |
| − | </div>
| |
| − | <div id="StarAcc-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="StarAcc">
| |
| − | <div class="panel-body">
| |
| − | <center>
| |
| − | <figure>
| |
| − | <img src="https://static.igem.org/mediawiki/2016/1/15/T--Imperial_College--STARANTISTAR.png">
| |
| − | <figcaption><p>Figure: Sensitivity analysis for the degradation rates in the GP2 and GP0.4 models</p></figcaption>
| |
| − | </figure>
| |
| − | </center>
| |
| − |
| |
| − | <center>
| |
| − | <figure>
| |
| − | <img src="https://static.igem.org/mediawiki/2016/a/a6/T--Imperial_College--degredationsensitivity.png">
| |
| − | <figcaption><p>Figure 3: Sensitivity analysis for the degradation rates in the GP2 and GP0.4 models</p></figcaption>
| |
| − | </figure>
| |
| − | </center>
| |
| − |
| |
| − | <center>
| |
| − | <figure>
| |
| − | <img src="https://static.igem.org/mediawiki/2016/a/a6/T--Imperial_College--degredationsensitivity.png">
| |
| − | <figcaption><p>Figure 3: Sensitivity analysis for the degradation rates in the GP2 and GP0.4 models</p></figcaption>
| |
| − | </figure>
| |
| − | </center>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | </section>
| |
| − |
| |
| − | <section id="GrowthModel">
| |
| − |
| |
| − | <div class="col-lg-10 col-centered">
| |
| − | <p>
| |
| − | <specialh3>Growth regulator module</specialh3><br>
| |
| − | We modelled 4 different growth regulator systems in silico in order to assess the speed and effectiveness of each case.
| |
| − |
| |
| − | <br><br>
| |
| − | Auxotrophy (LeuB)
| |
| − |
| |
| − | <br><br>
| |
| − | Antibiotic resistance (Chloramphenicol resistance)
| |
| − |
| |
| − | <br><br>
| |
| − | Gene product 2
| |
| − |
| |
| − | <br><br>
| |
| − | Gene Product 0.4
| |
| − |
| |
| − |
| |
| − |
| |
| − | </p>
| |
| − |
| |
| − | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true">
| |
| − | <div class="panel panel-default">
| |
| − | <div class="panel-heading" role="tab" id="GrowthAcc">
| |
| − | <h4 class="panel-title">
| |
| − | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#GrowthAcc-collapse" aria-expanded="false" aria-controls="GrowthAcc-collapse">
| |
| − | Growth Regulator Results
| |
| − | <span class="pull-right caret"></span>
| |
| − | </a>
| |
| − | </h4>
| |
| − |
| |
| − | </div>
| |
| − | <div id="GrowthAcc-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="GrowthAcc">
| |
| − | <div class="panel-body">
| |
| − |
| |
| − |
| |
| − |
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| | </section> | | </section> |
| | | | |
