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| − | <h2>Overview</h2>
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| − | <p> Many variables play a role in the behavior of synthetic circuits. Most (if not all) of these variables dramatically change when transferring a circuit between organisms. However, standardization and predictability is the foundation of any engineering practice. The iGEM parts registry has provided the scientific community with a plethora of characterized BioBricks, yet, a significant portion of the registry is characterized in E. coli and only a few in B. Subtilis and Yeast. In order to overcome the dependence on the biological context we modeled and applied a commonly used control system motif, an incoherent feed-forward loop. Through the modeling of this system, we show it achieves perfect adaptation to copy number and is robust to transcriptional and translational variations. Based on modeling we designed experiments to demonstrate these properties, both within E. Coli and across different organisms. We believe our model facilitates standardization of BioBricks across bacterial species.
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| − | <img src = "https://static.igem.org/mediawiki/2019/1/11/T--TUDelft--Promoter.png" alt="promoter SOBL" style="width:15%";> To achieve a system that works predictably across bacterial species we took into account the following variables: Promoters have different strengths in different organisms. Some promoters only work in a very narrow range of bacterial species. We, therefore, explored methods to overcome this variable by utilizing orthogonal transcription through the use of T7 RNA polymerase. <br>
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| − | <img src = "https://static.igem.org/mediawiki/2019/a/a6/T--TUDelft--RBS.png" alt="RBS SOBL" style="width:15%";> A ribosome binding site contains the Shine-Dalgarno sequence where the 16s rRNA of the Ribosome binds to. However, this sequence varies across species and often ribosome binding sites are extremely inefficient when applied in phylogenetically distant species. Our solution would thus require an approach independent of the efficiency of ribosomal binding sites. <br>
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| − | <img src = "https://static.igem.org/mediawiki/2019/1/19/T--TUDelft--ORI.png" alt="Ori SOBL" style="width:16.5%";> Genetic engineering wouldn't be possible or significantly more difficult if we couldn't use plasmids. However, plasmids need an origin of replication. Although origins of replication have been heavily studied we still lack the ability to easily transfer plasmids between prokaryotes and often they behave unpredictably. We, therefore, wanted to utilize an orthogonal replication system which would function in any bacterial host. To complement this approach we developed a genetic circuit independent of copy number to minimize the unpredicitable nature when transfering between organisms. <br>
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| − | <h2>TALE - Incoherent Feed Forward Loop to control gene expression</h2>
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| − | <p> In our system we exploit a commonly applied control system known as an Incoherent feed-forward Loop (iFFL), in which an activator regulates both a gene and a repressor of the gene ... . This control system is established through the expression of a Transcription activator-like effector (TALE) protein. TALE proteins recognize DNA by a simple DNA-binding mechanism which can be altered to recognize any sequence you want … . In our system the TALE protein binds to the promoter of a gene of interest and thus represses the expression of it. .... has previously described this system and showed how it results in independence of copy number for a gene of interest. <br>
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| − | We further analyzed this system through the use of modeling, which revealed that it's insensitive to many other variables as well, which hasn't been explored yet.
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| − | Transferring genetic circuits between organisms yields large variation in most (if not all) of the parameters in the system, we exploited the robustness of this model to maintain predictability even when crossing species barriers.
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| − | Below you can find a set of questions used to establish design requirements for both our software tools and our genetic circuit.
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| − | <a class="toggle" href="javascript:void(0);" ><b>Q1 How does the TALE protein bind to DNA?</b></a>
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| − | <a class="toggle" href="javascript:void(0);" ><b>Q2 How does the iFFL behave?</b></a>
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| − | <a class="toggle" href="javascript:void(0);" ><b>Q3 How can we make our system more robust?</b></a>
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| − | <a class="toggle" href="javascript:void(0);" ><b>Q4 How does the system behave when transferring between organisms?</b></a>
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| − | <p>You can find the full model description here! </p>
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| − | <h2>Codon usage - Cross species codon harmonization</h2>
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