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| − | <div id="Overview">
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| − | <img class="banneroh" src = "https://static.igem.org/mediawiki/2019/7/74/T--TUDelft--Results_logo.png" alt="Results">
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| − | <ul class=" nav nav-pills nav-stacked accordion" data-spy= "affix" data-offset-top="180">
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| − | <li><a class="jump" href="#Overview"><b>Overview</b></a></li>
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| − | <li><a class="jump" href="#PartConstruction"><b>Parts Construction</b></a></li>
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| − | <li><a class="jump" href="#PartCharacterization"><b>Parts Characterization</b></a></li>
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| − | <li><a class="jump" href="#Orthogonality"><b>Orthogonality</b></a></li>
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| − | <li style="padding-left:5%;"><a class="jump" href="#Replication">Replication</a></li>
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| − | <li style="padding-left:5%;"><a class="jump" href="#ToxicityAssay">Toxicity Assay</a></li>
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| − | <li><a class="jump" href="#Controllability"><b>Controllability</b></a></li>
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| − | <li style="padding-left:5%;"><a class="jump" href="#CopyNumber">Plasmid Copy Number</a></li>
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| − | <li style="padding-left:5%;"><a class="jump" href="#Transcription">Transcriptional Variations</a></li>
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| − | <li style="padding-left:5%;"><a class="jump" href="#Translation">Translational Variations</a></li>
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| − | <li style="padding-left:5%;"><a class="jump" href="#Harmonization">Harmonization</a></li>
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| − | <li><a class="jump" href="#Future"><b>Future Plans</b></a></li>
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| − | Overview
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| − | <div id="PartConstruction">
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| − | <h1>Parts Construction</h1>
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| − | <p>text</p>
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| − | <h1>Part Characterization </h1>
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| − | <p>text</p>
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| − | <div id="Orthogonality">
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| − | <h1>Orthogonalibity</h1>
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| − | <p>text</p>
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| − | <div id="Replication">
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| − | <h1>Orthogonal Replication</h1>
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| − | <p>text</p>
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| − | <h1>Toxicity Assay</h1>
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| − | <p>text</p>
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| − | <div id="Controllability">
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| − | <h1>Controllabillity</h1>
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| − | <h2>Overview</h2>
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| − | <p>The behavior of genetic parts and circuits in different bacterial species is unpredictable as it is influenced by host-dependent variations. We identified the variables to be: copy number, transcriptional and translational rates. We implemented a unique control system motif (incoherent feed forward loop) into a genetic circuit to achieve gene expression independent of copy number, transcriptional and translational rates.</p>
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| − | <h2>Construction</h2>
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| − | <p>We modeled the genetic implementation of the iFFL loop and varied the identified variables. Based on the results from the modeling, we made design choices. </p>
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| − | <h2>Results</h2>
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| − | We learned through the implementation of the model that constant transcriptional and translational rates of TALE and GFP needs to be maintained to achieve gene expression independent of transcriptional and translational variations respectively.
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| − | <img src="https://static.igem.org/mediawiki/2019/f/f8/T--TUDelft--transcriptionvariation.svg" style="width:85%;border:1px solid #00a6d6;" class="centermodel"
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| − | alt="TALE system">
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| − | <figcaption class="centermodel"><b>Figure 5</b>: Steady-state GFP production while transcription rates of both TALE and GOI are changed (aT/aG = constant). The lines indicate constant ratio of transcription rates </figcaption>
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| − | <p>To achieve constant ratios of transcriptional rates of TALE and GFP, we used the orthogonal T7 promoter and its variants to express TALE and GFP genes. The following constructs were successfully cloned by Golden Gate Assembly.
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| − | To achieve constant ratios of translational rates for TALE and GFP, we used the same ribosome binding sites for the expression of TALE and GFP. Furthermore, to demonstrate expression independent of translational rates, we switched constructed circuits with different RBSs.
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| − | When transcriptional units are placed in series, leaky expression of the gene in the second transcriptional unit can occur. This is due to the efficiency of the terminator of the first transcriptional unit. The model shows that leaky expression significantly affects the ability of the iFFL system to adapt to changes in copy number.</p>
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| − | <img src="https://static.igem.org/mediawiki/2019/0/0e/T--TUDelft--leakyterminator.svg" style="width:70%;border:1px solid #00a6d6;" class="centermodel"
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| − | alt="TALE system">
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| − | <figcaption class="centermodel"><b>Figure 9</b>: Comparison of a perfect terminator and a leaky terminator on the expression level at different plasmid copy number. </figcaption>
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| − | We therefore designed our genetic circuit such that the transcriptional units of TALE and GFP are oriented in opposite directions.
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| − | <h2>Copy number independence</h2>
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| − | Copy number of plasmids vary when used in different bacterial hosts and this significantly alters behaviour of parts. To achieve higher predictability of parts across different bacterial species, we aimed to demonstrate independence to copy number of our iFFL systems, as predicted by our <a href="https://2019.igem.org/Team:TUDelft/Model#PlasmidCopyNumber" ><b> modeling </b>.</a>. Furthermore, to facilitate the development of portable gene expression systems and reduce host dependency, the iFFL system was successfully expressed along with the Universal Bacterial Expression Resource (UBER) system (Kushwaha & Salis, 2015).
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| − | <ul class="accordion">
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| − | <a class="toggle " href="javascript:void(0);" ><b>Experimental design</b><span style="float:right;"><b>﹀</b></span></a>
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| − | <ul class="inner accordion">
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| − | <p> To test the independence to copy number we cloned our <a href="http://parts.igem.org/Part:BBa_K2918010" ><b> T7 promoter based optimized iFFL </b></a> and a control into low and medium copy number backbones (<a href=”https://www.addgene.org/48073/”> pICH82113
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| − | </a>, and <a href=”https://www.addgene.org/48074/”> pICH82094
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| − | </a> respectively) from the MoClo toolkit. </p> <br>
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| − | <p>To reduce dependency on host transcriptional machinery, we co-transformed these constructs with the UBER portable T7 expression system. The UBER system expresses T7 RNAP at a stable level as described on our <a href="https://2019.igem.org/Team:TUDelft/Design" ><b> design page </b></a>.
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| − | Constructs used: </p>
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| − | <h2>Transcriptional variation</h2>
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| − | <h2>Translational variation</h2>
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| − | <h2>Expression across different organisms</h2>
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| − | <h2>Cross species codon harmonization</h2>
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| − | <h1>Future Plan </h1>
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| − | <p>text</p>
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| − | <h3>References</h3>
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| − | <a target="_blank" href="http://doi.org/10.3389/fmicb.2018.02154">Sun, D. (2018). Pull in and Push Out: Mechanisms of Horizontal Gene Transfer in Bacteria. Frontiers in Microbiology, 9, 2154. https://doi.org/10.3389/fmicb.2018.02154</a>
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| − | <li><a target="_blank" id="Weber2011" href="http://doi.org/10.1371/journal.pone.0016765">Weber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011). A modular cloning system for standardized assembly of multigene constructs. PloS One, 6(2), e16765. http://doi.org/10.1371/journal.pone.0016765</a></li>
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