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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="#Portability">Portable T7 expression system</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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| − | <h1>Parts Construction</h1>
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| − | <p>text</p>
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| − | <h1>Part Characterization </h1>
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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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| − | <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>Controllability</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 <cite><a href="https://www.nature.com/articles/nchembio.2554"> (Liu et al., 2018) </a></cite>. Interspecies variations (Adams, 2016), such as copy number of plasmids (De Gelder, Ponciano, Joyce, & Top, 2007), transcription rates of promoters (Meysman, et al., 2014), translation initiation rates of ribosome binding sites (RBS) (Omotajo, Tate, Cho, & Choudhary, 2015) and the codon usage of coding sequences (Sharp, Bailes, Grocock, Peden, & Sockett, 2005) influence the functioning of genetic parts. We implemented a unique control system motif (incoherent feed forward loop) into a genetic circuit to achieve gene expression independent of these variables. Furthermore, the iFFL loop was demonstrated to show similar expression across <i> E.coli </i> and <i>P.putida </i>.</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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| − | <a class="toggle " href="javascript:void(0);" ><b>Results</b><span style="float:right;"><b>﹀</b></span></a>
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| − | <ul class="inner accordion">
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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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| − | <div id="CopyNumber">
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| − | <h2>Copy number</h2>
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| − | Copy number of plasmids vary when used in different bacterial hosts and this significantly alters behaviour of parts. We used a modeling approach to study the behavior of a genetic implementation of an iFFL. This model shows complete independence to copy number of the steady-state gene expression.
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| − | <ul class="accordion">
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| − | <a class="toggle " href="javascript:void(0);" ><b>Results -- Copy number independence</b><span style="float:right;"><b>﹀</b></span></a>
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| − | <ul class="inner accordion">
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| − | <p>
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| − | We have modeled the genetic implementation of the iFFL for a wide range of copy numbers. For all of these simulations the steady-state GFP expression was taken.
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| − | <h3>Results</h3>
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| − | <p>As can be seen in figure ..., the system is completely independent to copy number. </p>
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| − | <div id="Portability">
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| − | <h2>Portable T7 expression system</h2>
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| − | 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> <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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| − | <h3>Results</h3>
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| − | <p>We .... </p>
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| − | <div id="Transcription">
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| − | <h2>Transcriptional variation</h2>
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| − | Behavior of promoters (transcriptional rates) significantly changes across different bacterial hosts <a href=”https://www.ncbi.nlm.nih.gov/pubmed/29061047”> (Yang S et al., 2017) </a>. Hence, promoters either need to be re-characterized for each bacterial hosts or promoters specific to the host need to be identified. Using iFFL, we demonstrated gene expression independent of transcriptional rates when the transcription rate of both genes (TALE and GOI) maintain the same ratio, as predicted by <a href="https://2019.igem.org/Team:TUDelft/Model#TranscriptionalVariations" ><b> modeling </b></a>.
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| − | <a class="toggle " href="javascript:void(0);" ><b>Results -- Independence to promoter strengths</b><span style="float:right;"><b>﹀</b></span></a>
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| − | <ul class="inner accordion">
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| − | <p> To validate our model prediction, we designed T7 promoters based iFFL systems with varying promoter strengths. We compared our <a href="http://parts.igem.org/Part:BBa_K2918040">wild-type T7 promoter based iFFL system</a> (figure 1) to a iFFL system based on a T7 promoter variant with 50% strength compared to the wild-type (figure 2) (Ryo Komura et al., 2018) (<a href="http://parts.igem.org/Part:BBa_K2918048">medium T7 based iFFL system</a>).
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| − | As a control we express GFP without any TALE (figure 3). </p>
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| − | <img src="https://static.igem.org/mediawiki/2019/d/d7/T--TUDelft--T7iFFL.png" style="width:60%;border:1px solid #00a6d6;" class="centermodel"
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| − | alt="TALE system">
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| − | <figcaption class="centermodel"><b>Figure 1</b>: T7 based iFFL. Both genes are controlled by a T7 promoter. </figcaption>
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| − | <br>
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| − | <img src="https://static.igem.org/mediawiki/2019/6/6f/T--TUDelft--mediumt7.png" style="width:60%;border:1px solid #00a6d6;" class="centermodel"
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| − | alt="TALE system">
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| − | <figcaption class="centermodel"><b>Figure 2</b>: Medium T7 based iFFL. Both genes are controlled by a medium strength version of a T7 promoter. </figcaption>
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| − | <br>
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| − | <img src="https://static.igem.org/mediawiki/2019/4/4e/T--TUDelft--notale.png#00a6d6;" class="centermodel"
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| − | alt="TALE system">
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| − | <figcaption class="centermodel"><b>Figure 3</b>: Negative control, T7 promoter controlling GFP. </figcaption>
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| − | <br> <br>
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| − | <p>The output GFP fluorescence was measured using flow cytometry during logarithmic growth phase after induction with 1mM IPTG. As a reference for background fluorescence we use <i>E. coli</i> BL21DE(3) cells without any plasmid. The most dense region (determined by eye) in the scatter plot (forward and side-scatter) is selected for gating in order to only compare cells of similar morphology. Furthermore, the fluorescence histogram is gated to discern between cells that are "off" or "on" (expressing GFP or not), by gating the <i>E. coli</i> BL21DE(3) cells without any plasmid.</p> <br>
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| − | <p>The median of the background is subtracted from the median of the samples and the resulting values are plotted (figure ...). </p>
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| − | <h3>Results</h3>
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| − | <p>
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| − | <img src="https://static.igem.org/mediawiki/2019/5/5c/T--TUDelft--promoter_variation_wetlab_model.svg" style="width:60%;border:1px solid #00a6d6;" class="centermodel"
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| − | alt="TALE system">
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| − | <figcaption class="centermodel"><b>Figure 6</b>: Steady-state GFP fluorescence measurement of promoter variation using flow cytometry. The graph depicts T7 and medium T7 iFFL systems, expected to give the same fluorescence according to the model. As a control, GFP under control of an unrepressed T7 promoter was used. </figcaption>
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| − | <br>
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| − | <p>In figure ..., similar GFP fluorescence can be observed for the iFFL systems while the unrepressed control system shows high fluorescence. This suggests successful insulation of gene expression from change in promoter strengths.</p>
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| − | <ul class="accordion">
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| − | <a class="toggle " href="javascript:void(0);" ><b>Results -- Independence to IPTG concentration</b><span style="float:right;"><b>﹀</b></span></a>
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| − | <ul class="inner accordion">
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| − | <p>
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| − | Aside from testing gene expression independent of transcriptional variation by using promoters of different strengths, the effect of different concentrations of IPTG on the iFFL loop was tested. Change in IPTG concentrations, changes in-vivo concentrations of T7 RNAP and this contributes to variations in transcriptional rates.
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| − | In unrepressed systems, the expression of the GOI is a function of IPTG concentrations. However, in iFFL systems, since the transcriptional rates of TALE and GFP are under control of T7 promoters, similar GOI expression is expected (figure 1). As a control we expressed GFP under the control of T7sp1 promoter was used (figure 2).
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| − | <img src="https://static.igem.org/mediawiki/2019/6/6b/T--TUDelft--weakt7.png" style="width:60%;border:1px solid #00a6d6;" class="centermodel"
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| − | alt="TALE system">
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| − | <figcaption class="centermodel"><b>Figure 1</b>: Optimized T7 based iFFL. TALE is under cotntrol of weak T7 promtoer. GFP is controlled by a T7 promoter. </figcaption>
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| − | <br>
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| − | <img src="https://static.igem.org/mediawiki/2019/4/4e/T--TUDelft--notale.png#00a6d6;" class="centermodel"
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| − | alt="TALE system">
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| − | <figcaption class="centermodel"><b>Figure 2</b>: Negative control, T7 promoter controlling GFP. </figcaption>
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| − | </p>
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| − | <p>The output GFP fluorescence was measured using flow cytometry during logarithmic growth phase after induction with 1mM IPTG. As a reference for background fluorescence we use <i>E. coli</i> BL21DE(3) cells without any plasmid. The most dense region (determined by eye) in the scatter plot (forward and side-scatter) is selected for gating in order to only compare cells of similar morphology. Furthermore, the fluorescence histogram is gated to discern between cells that are "off" or "on" (expressing GFP or not), by gating the <i>E. coli</i> BL21DE(3) cells without any plasmid. </p> <br>
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| − | <p>The median of the background is subtracted from the samples and are compared. </p>
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| − | <h3>Results</h3>
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| − | <p>
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| − | <p> Figure ... the GFP fluorescence of the unrepressed control changes with changing concentrations of IPTG while the iFFL system shows the same GFP expression across different IPTG concentrations. Thus, the iFFL system has been shown to insulate gene expression against changes in transcription rates (achieved by varying IPTG concentrations).
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| − | . </p>
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| − | <img src="https://static.igem.org/mediawiki/2019/a/a9/T--TUDelft--IPTGtitration.svg" style="width:60%;border:1px solid #00a6d6;" class="centermodel"
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| − | alt="TALE system">
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| − | <figcaption class="centermodel"><b>Figure ...</b>: Steady-state GFP fluorescence measurement of IPTG titration using FACS. The graph depicts a T7 iFFL system induced using different levels of IPTG, which according to the model should give the same result. As a control GFP under control of an unrepressed T7 promoter was used. </figcaption>
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| − | <ul class="accordion">
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| − | <a class="toggle " href="javascript:void(0);" ><b>Results -- Tunability</b><span style="float:right;"><b>﹀</b></span></a>
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| − | <ul class="inner accordion">
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| − | <p>
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| − | The predictions made by modeling not only tell us that we can maintain the same level of gene expression but also that we can easily tune the expression levels by changing one of the promoters. By changing one of the promoters to another variant of T7 we can expect a different level of expression, while at the same time expect it to behave similarly when transferred between organisms. Through the use of T7 variants we could achieve wide ranges of expression levels, which can be used to establish complex genetic circuits, while also expecting it to work similarly in different biological contexts.
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| − | We tested the prediction by changing the promoter controlling the TALE protein to a variant of T7 which has been shown to be about 10% in strength compared to the wild-type (Ryo Komura et al., 2018). According to the model the expression should increase.
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| − | </p>
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| − | <p>We measure GFP fluorescence using flow cytometry. As a reference for background fluorescence we use <i>E. coli</i> BL21DE(3) cells without any plasmid. The most dense region (determined by eye) in the scatter plot (forward and side-scatter) is selected for gating in order to only compare cells of similar morphology. Furthermore, the fluorescence histogram is gated to discern between cells that are "off" or "on" (expressing GFP or not), by gating the <i>E. coli</i> BL21DE(3) cells without any plasmid. </p> <br>
| + | |
| − | <p>The median of the background is subtracted from the samples and are compared. </p>
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| − | | + | |
| − | | + | |
| − | <h3>Results</h3>
| + | |
| − | <p>
| + | |
| − | <p> Figure ... clearly shows higher fluorescence when a lower T7 promoter version is used to express the TALE protein in comparison to our systems with the same ratio in promoter strengths for both genes. </p>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2019/5/5d/T--TUDelft--tunability.svg" style="width:60%;border:1px solid #00a6d6;" class="centermodel"
| + | |
| − | alt="TALE system">
| + | |
| − | <figcaption class="centermodel"><b>Figure ...</b>: Steady-state GFP fluorescence measurement of <i>E. coli</i> BL21DE(3) cells expressing our iFFL systems. The graph depicts a different T7 iFFL systems, one with both promoters T7, one with both medium strength and one where the promoter controlling of the TALE is weak. As a control GFP under control of an unrepressed T7 promoter was used. </figcaption>
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| − | <br>
| + | |
| − | </ul>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | <h3>Conclusion</h3>
| + | |
| − | <p>Results above indicate successful implementation of the iFFL system to insulate from transcriptional variations. Transcriptional variations were achieved by using T7 promoters of different strengths and by induction at different IPTG concentrations. Furthermore, we demonstrated the tunability of the iFFL system to achieve different levels of gene expression. As predicted by our model, we achieved gene expression independent of changes in transcriptional rates by maintaining constant ratios of transcriptional rates of TALE and GFP genes.</p>
| + | |
| − | <div id="Translation">
| + | |
| − | <h2>Translational variation</h2>
| + | |
| − | In order to see the effect of translational variation on the expression levels of the gene of interest (GOI) we modeled our system for a range of translational rates for both genes (TALE and GOI).
| + | |
| − | <ul class="accordion">
| + | |
| − | <li>
| + | |
| − | <a class="toggle " href="javascript:void(0);" ><b>Results</b><span style="float:right;"><b>﹀</b></span></a>
| + | |
| − | <ul class="inner accordion">
| + | |
| − | <p>As can be seen in figure … the steady-state expression levels of GFP remain the same when the translation rates are kept constant.
| + | |
| − | </p>
| + | |
| − | </ul>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | </div>
| + | |
| − | <h3>Conclusion</h3>
| + | |
| − | <p>According to our model solution we can maintain the same level of GOI expression when both translation rates remain constant. We therefore designed our system to contain the same RBS in front of both TALE and the GOI. </p>
| + | |
| − | | + | |
| − | | + | |
| − | <h2>Expression across different organisms</h2>
| + | |
| − | <p>To achieve similar gene expression across different organisms the iFFL system needs to be robust to changes in copy number, transcriptional and translational rates. We experimentally demonstrated gene expression independent of transcriptional variations and through modelling showed adaptation variations in copy number and translational variation of our iFFL system. Through the implementation of the iFFL loop using engineered broad host range promoters, we successfully demonstrated similar GFP expression across <i> E.coli</i> and <i> P.putida</i>. Thereby demonstrating gene expression insulated from variations associated to microbial hosts.</p>
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| − | <ul class="accordion">
| + | |
| − | <li>
| + | |
| − | <a class="toggle " href="javascript:void(0);" ><b>Results -- Expression across organisms</b><span style="float:right;"><b>﹀</b></span></a>
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| − | <ul class="inner accordion">
| + | |
| − | <p>
| + | |
| − | Broad host range promoter (P<sub>BHR</sub>) was designed by combining the conserved -10 and -35 regions from <i> E.coli</i> and <i> B.subtilis </i> <cite><a href=""> (Yang S et al., 2018) </a></cite> and the promoter was engineered to contain a binding site for TALE repressor (P<sub>BHRsp1</sub>). Using the P<sub>BHR</sub> and P<sub>BHRsp1</sub>, we constructed an iFFL genetic circuit driving GFP expression.
| + | |
| − | The circuit was transformed in <i>E.coli</i> and <i> P.putida</i> and, output fluorescence was measured by flow cytometry during logarithmic growth phase. To correct for background fluorescence, <i>E.coli</i> and <i> P.putida</i> without plasmids were used as blanks. GFP under the control of P<sub>BHRsp1</sub> was used as a positive (unrepressed) control. As the cell morphologies of <i>E.coli</i> and <i>P.putida</i> are different they cannot be compared directly, gating was based on the most dense regions in the scatter plot for each organism. In order to compare the GFP expression levels between each organism, the background fluorescence for each organism was subtracted by its respective blank .
| + | |
| − | </p>
| + | |
| − | <h3>Results</h3>
| + | |
| − | <p>
| + | |
| − | <p> Figure ... clearly shows higher fluorescence when a lower T7 promoter version is used to express the TALE protein in comparison to our systems with the same ratio in promoter strengths for both genes. </p>
| + | |
| − | <img src="https://static.igem.org/mediawiki/2019/4/4a/T--TUDelft--difforgs.svg" style="width:60%;border:1px solid #00a6d6;" class="centermodel"
| + | |
| − | alt="TALE system">
| + | |
| − | <figcaption class="centermodel"><b>Figure ...</b>: Steady-state GOI production while translation rates of both TALE and GOI are changed. The lines indicate the constant rate of the translation rates. </figcaption>
| + | |
| − | <br>
| + | |
| − | <p>
| + | |
| − | In figure …., the median fluorescence of the gated populations is plotted. Significantly large difference in expression levels is observed between the unrepressed controls and the broad host range promoter based iFFL systems in <i>E.coli</i> and <i>P.putida</i>. However, similar levels of expression were observed from iFFL systems in <i> E.coli</i> and <i>P.putida </i>. The difference in expression levels between the unrepressed circuit is significantly higher than the difference in expression levels between the iFFL system (578530 and 2351.2 respectively).
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| − | | + | |
| − | </p>
| + | |
| − | </ul>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | <h3>Conclusion</h3>
| + | |
| − | <p>The implementation of the iFFL significantly decreased the differences in expression levels between organisms. Our system sets the basis for controllability across organisms. </p>
| + | |
| − | | + | |
| − | <h2>Cross species codon harmonization</h2>
| + | |
| − | <br>
| + | |
| − | <p>
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| − | When heterologous proteins are expressed in new bacterial host cells, altered protein expression levels is observed due to the variance in codon usage between the original organism and the new host cell (Angov et al, 2008). To increase the expression level of the heterologous protein in the host cell, new codon optimization tools were developed. The current limitation of both codon adaptation tools is that the adaptation of the DNA coding sequence can be perform for one single organism at the time. Therefore we created the first cross-species codon harmonization tool. The tool provides the user with a single DNA coding sequence that will yield the same protein expression level in different bacterial host cells. We demonstrated functional protein expression using our own harmonized coding sequence for <i>E. coli , B. Subtilus,</i> and <i>V. Natrigen</i>.
| + | |
| − | </p>
| + | |
| − | <ul class="accordion">
| + | |
| − | <li>
| + | |
| − | <a class="toggle " href="javascript:void(0);" ><b>Results -- functional protein production</b><span style="float:right;"><b>﹀</b></span></a>
| + | |
| − | <ul class="inner accordion">
| + | |
| − | <li>
| + | |
| − | To validate whether the cross-species codon harmonization results in a functional protein, we designed a cross-species codon harmonized GFP coding sequence. The sequence was harmonized using <i>E. coli BL21(DE3)</I> as reference organism, and <I>V. natriegens</i> NBRC 15636 = ATCC 14048 = DSM 759) and <I>B. Subtilis</i> subsp. <I>subtilis</i> str. 16 <br>
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| − | | + | |
| − | <img src="https://static.igem.org/mediawiki/2019/9/95/T--TUDelft--harmoinzationGFP.png'' style="width:60%;border:1px solid #00a6d6;" class="centermodel"
| + | |
| − | alt="TALE system">
| + | |
| − | <figcaption class="centermodel"><b>Figure ...</b>: Fluorescence measurement from the IPTG induced cells using the gel doc. From left to right <i>E. coli</I> BL21(DE3) <I>pLysS</I> with no transformed plasmid (negative control), <i>E. coli</i> BL21(DE3) <I>pLysS</I> with JuniperGFP (positive control), <i>E. coli</I> BL21(DE3) <I>pLysS</i> with harmonized GFP, and <i>E. coli </i>BL21(DE3) <I>pLysS</I> with no transformed plasmid (negative control). A) The fluorescing image was taken using the gel doc. As seen the cell pellets are all fluorescing. B) After removing the background to visualize the GFP fluorescence cells. The still fluorescing cell pellets are marked with a green circle. C) A 3D plot of the fluorescing cells. The height of the graph corresponds with the intensity of the measured GFP.
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| − | </figcaption>
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| − | | + | |
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| − | The harmonized GFP was constructed throug MoClo assembling with the WT T7 promoter, universal RBS, harmonized GFP, and WT7 terminator. The assembled plasmid is transformed into <I>E. coli </I> BL21(DE3). <br>
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| − | We measure the GFP fluorescence using the Gel doc. As reference for background fluorescence we use <i>E. coli</i> BL21(DE3) <I>pLysS</i>) cell without plasmid. As reference for fluorescence we use <i>E. coli</i> BL21(DE3) <I>pLysS</i> with ……….30 plasmid
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| − |
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| − | | + | |
| − | <h4> Results</h4>
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| − | | + | |
| − | Both the positive control and the harmonized GFP showed some level of fluorescence expression. The level of fluorescence of the harmonized GFP is higher compared to the positive control, after subtracting the autofluorescence. Since the negative control did not show any fluorescence after subtracting the background.
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| − |
| + | |
| − | </li>
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| − | </ul>
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| − | </li>
| + | |
| − | </ul>
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| − | <br>
| + | |
| − | <p>
| + | |
| − | <h4> Conclusion </h4>
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| − | Both in harmonized GFP and the positive control GFP fluorescence is observed. This indicates that functional GFP protein is produced in E. coli BL21(DE3) cells.
| + | |
| − | Higher GFP expression in the harmonized GFP might occur due to the codon adaption. However, the experimental data obtained from this experiment is insufficient to explain the observed difference in expression.
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| − | </p>
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| − | </div>
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| − | <br>
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| − | <div id="Future">
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| − | <h1>Future Plan </h1>
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| − | <p>text</p>
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| − | </div>
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| − | <br>
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| − | <h3>References</h3>
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| − | <div class="reftu">
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| − | <ul style="list-style:none;">
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| − | <li>
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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>
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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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