Difference between revisions of "Team:MIT/L7AeRepressingSystem"

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<title> Recombinases Background Information </title>
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<title>L7Ae k-turn repressing system</title>
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<h1 style="color:#0f3d7f; text-align: center; font-size: 40px; line-height: 40px;">Recombinases: <br> Giving Memory to a Genetic Circuit</h1>
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<h1 style="color:#0f3d7f; text-align: center; font-size: 40px; line-height: 40px;">L7Ae - Kink turn</h1>  
  
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<a href = "https://2016.igem.org/Team:MIT/Experiments/Recombinases"> Back to recombinase overview page </a>
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<h1 style="color:#ffffff; background-color:#0f3d7f; -moz-border-radius: 15px; -webkit-border-radius: 15px; padding:15px; text-align: center; font-family: Trebuchet MS"> How can our circuit demonstrate temporal specificity?</h1>
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<img src="https://static.igem.org/mediawiki/2016/0/0f/T--MIT--recombinase_excise_gif.gif" alt="Recombinase excision gif"/ style="width:350px;height:200px; float: left"  margin: 15px;>
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<h1 style="color:#FFFFFF; background-color:#0f3d7f; -moz-border-radius: 15px; -webkit-border-radius: 15px; padding:15px; text-align: center; font-family: Trebuchet MS">RNA-Based Gene Regulation</h1>
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<div style='width: 340px; text-align: center;'><i>A recombinase excises a segment of DNA.<br><b> Source: </b>University of Rochester Introductory Biochemistry.</i></div>
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<p style="font-family:Verdana;">  
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L7Ae, an archaeal ribosomal protein, binds with high affinity to RNA motifs called kink-turns (K-turns), found in both archaeal and eukaryote RNAs [1][2][3]. L7Ae protein sequence is divided into three structural regions consisting of a highly conserved RNA-binding region (RBR) flanked by less conserved N-terminal and C-terminal regions [2]. Variation in the terminal regions could dictate RNA-binding specificity of different homologs of L7Ae protein [2]. When a K-turn motif is inserted into the target mRNA upstream of the open reading frame, L7Ae can be used as a translational regulator [1][2][3]. The binding activity of L7Ae will prevent the ribosome machinery from performing translation. The strength of the repression can be controlled by varying the distance between the K-turns and the 5’-end of the mRNA, or by changing the number of the k-turn motifs [1].
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Endometriosis cells have distinct characteristics at different points in the menstrual cycle, presenting a major challenge in identifying diseased cells. Capturing chronological molecular traits is very important in the diagnosis of many diseases. For our project, we use <b>recombinases</b>, DNA binding proteins, to achieve this <b>temporal specificity</b>. <br>Recombinases are enzymes that can <b>recognize recombination sites</b>, and can either cut out the DNA between these recognition sites or invert the DNA sequence. There are two main families of recombinases: serine recombinases (also sometimes called serine integrases) and tyrosine recombinases. Serine integrases invert sequences, while tyrosine recombinases can either cut or flip sequences depending on the orientation of recognition sites. Some recombinases exhibit <b>unidirectionality</b>, meaning that once they reverse or cut out the sequence, this action cannot be undone. This means that instead of behaving like a switch, capable of turning on or off, <b>unidirectional recombinases behave as latches</b>. Thus, unidirectional recombinases display higher efficacy in DNA modification than bidirectional recombinases. We can use recombinases as biological "latches" in our circuit to gain temporal specificity. Once the abnormal hormone level and the miRNA profile characteristic of a diseased cell have been identified during one phase of the menstrual cycle, the <b>first recombinase</b> can be activated to essentially <b>“lock in”</b> that information. When the second half of the circuit confirms the cell as being diseased in the second phase of the cycle, a <b>second recombinase latch</b> can be triggered, <b>activating the overall circuit</b>.  
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     <img src="https://static.igem.org/mediawiki/2016/a/a3/T--MIT--recombinase_temporal_mechanism.png" alt="" style="width:500px;margin-bottom:10px;">
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     <img src="https://static.igem.org/mediawiki/2016/1/11/T--MIT--L7Ae_Kink_turn_mechanism.png" alt="" style="width:500px;margin-bottom:10px;">
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     <div style="width: 599px; text-align: center;display:inline-block;"><i><b>Figure. </b> Showing the mechanism of recombinase biological latches capturing temporal specificity during the estrogen and progesterone cycles. 1) Disease-related biological traits during the estrogen high phase activate the inducible promoter, leading to 2) expression of recombinase 1. Recombinase 1 would then 3) <b>"lock-in"</b> this information <b>by irreversible gene modification</b>. Similarly, 4) during the progesterone high phase, biomarkers associated with the disease would activate another promoter, leading to 5) expression of the second recombinase. <b>Two irreversible gene modification events</b> performed by recombinases 1 and 2 at different points in time <b>form an AND gate</b>, activating expression of an output gene.</i></div>
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     <div style="width: 599px; text-align: center;display:inline-block;"><i><b>Figure. </b>Binding of L7Ae to kink-turn motifs preveting translation.</i></div>
 
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<h1 style="color:#ffffff; background-color:#0f3d7f; -moz-border-radius: 15px; -webkit-border-radius: 15px; padding:15px; text-align: center; font-family: Trebuchet MS"> Do our recombinases work?</h1>
 
  
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    <!-- Section two -->
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We investigated <b>2 models of recombinase for regulating gene expression</b>:</p>
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<h1 style="color:#FFFFFF; background-color:#0f3d7f; -moz-border-radius: 15px; -webkit-border-radius: 15px; padding:15px; text-align: center; font-family: Trebuchet MS">Recombinase and L7Ae-Kturn</h1> 
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<ol style="Verdana;">
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<h3 style="color: #000000; text-decoration:underline; font-family: Trebuchet MS;">Purpose</h3>
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     <li>Using a unidirectional tyrosine recombinase (Cre or FLP) to excise a transcriptional stop signal, allowing a downstream gene to be expressed.</li>
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    <p style = "font-family:Verdana;">Using recombinases as biological latches giving our genetic circuit the ability to memorize disease temporal specificity. However, since the recombinase is controlled by an inducible promoter, leaky expression of the promoter (activation without input signals - disease biomarkers) could lead to unwanted activation of the output gene. By puting k-turn motifs in front of the recombinase gene, we hope to reduce leaky expression of the recombinase when the system is inactivated.<br>We designed an experiment to examine the repression level of the L7Ae - kink turn system on the expression of an output gene (EYFP - Enhanced yellow flourescent protein), which is regulated by TP901 (a serine recombinase).</p>
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     <li>Using a unidirectional serine recombinase (TP901) to flip gene from an off to an on orientation.</li>
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<h3 style="color: #000000; text-decoration:underline; font-family: Trebuchet MS;">Experimental Setup</h3>
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    <p></p>
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    <table style="width:80%; self-align: center;">
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     <img src="https://static.igem.org/mediawiki/2016/0/00/T--MIT--Recombinase_gene_regulation_models.png" alt="" style="width:555px;margin-bottom:10px;">
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     <div style="width: 499px; text-align: center;display:inline-block;"><i><b>Figure. </b>Regulating gene expression using recombinases models.</i></div>
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        <td>Untransfected Control</td>
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</div>
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       <td>Single color (Y)<br>1000ng hEF1a:eYFP<br>500ng pDONR</td>
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<p style="font-family:Verdana;"> Our experimental data showed that:</p>
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        <td>Single color (R)<br>1000ng hEF1a:mKate<br>500ng pDONR</td>
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<ol style="font-family:Verdana;">  
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        <td>Single color (B)<br>1000ng hEF1a:tagBFP<br>500ng pDONR</td>
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   </tr>
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    <tr>
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        <td>Three colors<br>500ng hEF1a:eYFP<br>500ng hEF1a:mKate<br>500ng hEF1a:BFP</td>
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 +
        <td style="color:blue;"><b>Control no L7Ae</b><br>300ng EGSH-kturn: TP901<br>300ng EGSH-kturn:mKate<br>200ng hEF1a: flipped EYFP<br>100ng hEF1a: VgEcr<br>100ng hEF1a: rtTA<br>0ng TRE: L7Ae<br>200ng hEF1a:BFP<br>300ng pDONR<br>1000nM Dox; 5uM PonA</td>
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        <td style="color:blue;"><b>Control no k-turn</b><br>300ng EGSH-kturn: TP901<br>300ng EGSH-kturn:mKate<br>200ng hEF1a: flipped EYFP<br>100ng hEF1a: VgEcr<br>100ng hEF1a: rtTA<br>100ng TRE: L7Ae<br>200ng hEF1a:BFP<br><br>1000nM Dox; 0uM PonA</td>
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 +
        <td style="color:blue;"><b>Control no k-turn</b><br>300ng EGSH-kturn: TP901<br>300ng EGSH-kturn:mKate<br>200ng hEF1a: flipped EYFP<br>100ng hEF1a: VgEcr<br>100ng hEF1a: rtTA<br>100ng TRE: L7Ae<br>200ng hEF1a:BFP<br><br>1000nM Dox; 5uM PonA</td>
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     </tr>
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    <tr>
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        <td style="color:red;"><b>Experiment 2x k-turn</b><br>300ng EGSH-kturn: TP901<br>300ng EGSH-kturn:mKate<br>200ng hEF1a: flipped EYFP<br>100ng hEF1a: VgEcr<br>100ng hEF1a: rtTA<br>100ng TRE: L7Ae<br>200ng hEF1a:BFP<br><br>0nM Dox; 0uM PonA</td>
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 +
        <td style="color:red;"><b>Experiment 2x k-turn</b><br>300ng EGSH-kturn: TP901<br>300ng EGSH-kturn:mKate<br>200ng hEF1a: flipped EYFP<br>100ng hEF1a: VgEcr<br>100ng hEF1a: rtTA<br>100ng TRE: L7Ae<br>200ng hEF1a:BFP<br><br>0nM Dox; 5uM PonA</td>
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        <td style="color:purple;"><b>Experiment 4x k-turn</b><br>300ng EGSH-kturn: TP901<br>300ng EGSH-kturn:mKate<br>200ng hEF1a: flipped EYFP<br>100ng hEF1a: VgEcr<br>100ng hEF1a: rtTA<br>100ng TRE: L7Ae<br>200ng hEF1a:BFP<br><br>0nM Dox; 0uM PonA</td>
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 +
        <td style="color:purple;"><b>Experiment 4x k-turn</b><br>300ng EGSH-kturn: TP901<br>300ng EGSH-kturn:mKate<br>200ng hEF1a: flipped EYFP<br>100ng hEF1a: VgEcr<br>100ng hEF1a: rtTA<br>100ng TRE: L7Ae<br>200ng hEF1a:BFP<br><br>0nM Dox; 5uM PonA</td>
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    </tr>
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    <tr>
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        <td style="color:red;"><b>Experiment 2x k-turn</b><br>300ng EGSH-kturn: TP901<br>300ng EGSH-kturn:mKate<br>200ng hEF1a: flipped EYFP<br>100ng hEF1a: VgEcr<br>100ng hEF1a: rtTA<br>100ng TRE: L7Ae<br>200ng hEF1a:BFP<br><br>100nM Dox; 0uM PonA</td>
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 +
        <td style="color:red;"><b>Experiment 2x k-turn</b><br>300ng EGSH-kturn: TP901<br>300ng EGSH-kturn:mKate<br>200ng hEF1a: flipped EYFP<br>100ng hEF1a: VgEcr<br>100ng hEF1a: rtTA<br>100ng TRE: L7Ae<br>200ng hEF1a:BFP<br><br>100nM Dox; 5uM PonA</td>
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 +
        <td style="color:purple;"><b>Experiment 4x k-turn</b><br>300ng EGSH-kturn: TP901<br>300ng EGSH-kturn:mKate<br>200ng hEF1a: flipped EYFP<br>100ng hEF1a: VgEcr<br>100ng hEF1a: rtTA<br>100ng TRE: L7Ae<br>200ng hEF1a:BFP<br><br>100nM Dox; 0uM PonA</td>
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 +
        <td style="color:purple;"><b>Experiment 4x k-turn</b><br>300ng EGSH-kturn: TP901<br>300ng EGSH-kturn:mKate<br>200ng hEF1a: flipped EYFP<br>100ng hEF1a: VgEcr<br>100ng hEF1a: rtTA<br>100ng TRE: L7Ae<br>200ng hEF1a:BFP<br><br>100nM Dox; 5uM PonA</td>
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     </tr>
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    <tr>
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        <td style="color:red;"><b>Experiment 2x k-turn</b><br>300ng EGSH-kturn: TP901<br>300ng EGSH-kturn:mKate<br>200ng hEF1a: flipped EYFP<br>100ng hEF1a: VgEcr<br>100ng hEF1a: rtTA<br>100ng TRE: L7Ae<br>200ng hEF1a:BFP<br><br>500nM Dox; 0uM PonA</td>
 +
       
 +
        <td style="color:red;"><b>Experiment 2x k-turn</b><br>300ng EGSH-kturn: TP901<br>300ng EGSH-kturn:mKate<br>200ng hEF1a: flipped EYFP<br>100ng hEF1a: VgEcr<br>100ng hEF1a: rtTA<br>100ng TRE: L7Ae<br>200ng hEF1a:BFP<br><br>500nM Dox; 5uM PonA</td>
 +
       
 +
        <td style="color:purple;"><b>Experiment 4x k-turn</b><br>300ng EGSH-kturn: TP901<br>300ng EGSH-kturn:mKate<br>200ng hEF1a: flipped EYFP<br>100ng hEF1a: VgEcr<br>100ng hEF1a: rtTA<br>100ng TRE: L7Ae<br>200ng hEF1a:BFP<br><br>500nM Dox; 0uM PonA</td>
 +
       
 +
        <td style="color:purple;"><b>Experiment 4x k-turn</b><br>300ng EGSH-kturn: TP901<br>300ng EGSH-kturn:mKate<br>200ng hEF1a: flipped EYFP<br>100ng hEF1a: VgEcr<br>100ng hEF1a: rtTA<br>100ng TRE: L7Ae<br>200ng hEF1a:BFP<br><br>500nM Dox; 5uM PonA</td>
 +
    </tr>
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    <tr>
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        <td style="color:red;"><b>Experiment 2x k-turn</b><br>300ng EGSH-kturn: TP901<br>300ng EGSH-kturn:mKate<br>200ng hEF1a: flipped EYFP<br>100ng hEF1a: VgEcr<br>100ng hEF1a: rtTA<br>100ng TRE: L7Ae<br>200ng hEF1a:BFP<br><br>1000nM Dox; 0uM PonA</td>
 +
       
 +
        <td style="color:red;"><b>Experiment 2x k-turn</b><br>300ng EGSH-kturn: TP901<br>300ng EGSH-kturn:mKate<br>200ng hEF1a: flipped EYFP<br>100ng hEF1a: VgEcr<br>100ng hEF1a: rtTA<br>100ng TRE: L7Ae<br>200ng hEF1a:BFP<br><br>1000nM Dox; 5uM PonA</td>
 +
       
 +
        <td style="color:purple;"><b>Experiment 4x k-turn</b><br>300ng EGSH-kturn: TP901<br>300ng EGSH-kturn:mKate<br>200ng hEF1a: flipped EYFP<br>100ng hEF1a: VgEcr<br>100ng hEF1a: rtTA<br>100ng TRE: L7Ae<br>200ng hEF1a:BFP<br><br>1000nM Dox; 0uM PonA</td>
 +
       
 +
        <td style="color:purple;"><b>Experiment 4x k-turn</b><br>300ng EGSH-kturn: TP901<br>300ng EGSH-kturn:mKate<br>200ng hEF1a: flipped EYFP<br>100ng hEF1a: VgEcr<br>100ng hEF1a: rtTA<br>100ng TRE: L7Ae<br>200ng hEF1a:BFP<br><br>1000nM Dox; 5uM PonA</td>
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    </tr>       
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    </table>
 
      
 
      
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    <li>The <b>flipped gene system (2nd model)</b> successfully <b>knocked down the expression of the gene</b>, while the transcriptional stop signal (1st model) did not.</li> 
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     <a href="#"><p style="font-family: Trebuchet MS;font-color:#7ECEFD; font-size:15px;"><i><b>Read more about building kturn constructs here.</b></i></p></a>
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    <li>The expression level of the flipped gene can be indirectly controlled by expression of the <b>recombinases (TP901) under an inducible promoter</b>.</li> 
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<h3 style="color: #000000; text-decoration:underline; font-family: Trebuchet MS;">Result</h3>
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    <p></p>
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     <a href="https://2016.igem.org/Team:MIT/Experiments/EGSH_TP901_Experiment"><p style="font-family: Trebuchet MS;font-color:#7ECEFD; font-size:15px;"><i><b>Read more about recombinase experiments here</b></i></p></a>
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     <!--Fourth section-->
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     <h4 style="text-decoration:underline; font-family: Trebuchet MS;"> <center>Testing the 2x k-turn L7Ae system with varied L7Ae expression level</center></h4>
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<h1 style="color:#ffffff; background-color:#0f3d7f; -moz-border-radius: 15px; -webkit-border-radius: 15px; padding:15px; text-align: center; font-family: Trebuchet MS"> Challenges with High Efficiency of Recombinases</h1>
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<p style="font-family:Verdana;"> Recombinases are highly efficient enzymes. When combined with a high-basal-activity promoter, this presents a challenge. A few copies of the recombinase due to promoter's leaky expression, could lead to significant amount of undesired output gene expression. In order to effectively use of recombinases as biological latches, basal expression must be reduced as much as possible. A strong repression system must be used in order to reduce leaky expression.
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    <h4 style="text-decoration:underline; font-family: Trebuchet MS;"> <center>Testing the effect of varying k-turn sequences</center></h4>
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</p>
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<h2 style="color: #000000; text-decoration:underline; font-family: Trebuchet MS;"> Repressible Promoters</h2>
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<br><br>
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    <div style="font-style: italic;">
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        <p style="text-decoration:underline; font-size: smaller;"><b>REFERENCE:</b></p>   
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        <ol style="font-size: smaller;">
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            <li>Oliwia Andries, Tasuku Kitada, Katie Bodner, Niek N Sanders & Ron Weiss (2015) Synthetic biology devices and circuits for RNA-based ‘smart vaccines’: a propositional review, Expert Review of Vaccines, 14:2, 313-331</li>
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            <li> Gagnon KT, Zhang X, Qu G, et al. Signature amino acids enable the archaeal L7Ae box C/D RNP core protein to recognize and bind the K-loop RNA motif. Rna 2010;16(1):79-90</li>
 +
            <li>Stapleton JA, Endo K, Fujita Y, et al. Feedback control of protein expression in mammalian cells by tunable synthetic translational inhibition. ACS Synth Biol 2012;1(3):83-8</li>
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        </ol>
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    </div>
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<p style="font-family:Verdana;">
 
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In order to gain tighter control of the recombinases, we paired them with repressible promoters that do not allow transcription of the recombinase to take place if a specific repressor protein is present. The three repressors we investigated included BM3R1, TAL14, and TAL21 because of their demonstrated success in literature.
 
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<a href="https://2016.igem.org/Team:MIT/Experiments/Repressors"><p style="font-family: Trebuchet MS;font-color:#7ECEFD; font-size:15px;"><i><b>Read more about repressor experiments here</b></i></p></a>
 
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<h2 style="color: #000000; text-decoration:underline; font-family: Trebuchet MS;"> Translational Regulation: L7Ae/kink-turn</h2>
 
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<p style = "font-family:Verdana;"> We did a lot of research into effective high level repression systems, such as degradation tag RNA-based gene regulation systems. After talking to experts in the Dr. Weiss' lab, we decided to test the L7Ae k-turn system due to the availability of the system's components.
 
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<div style="text-decoration: none; color: #000000; float: center; margin: 15px;text-align:center">
 
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    <img src="https://static.igem.org/mediawiki/2016/0/06/T--MIT--TP901_flipped_vsMarker.png" alt="" style="width:100%;margin-bottom:10px;">
 
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    <div style="width: 90%; text-align: center;display:inline-block;"><i><b>Figure. </b> Dox = 1000nM - activate the L7Ae/k-turn repressing system, and PonA = 5uM - activate the expression of TP901 recombinase. Number of k-turn motifs at the 5'UTR can tune the expression of the regulated gene (TP901). Double the k-turn repeats reduces the expression level of TP901 by half (going from 2x k-turn to 4x k-turn). However, this ratio is not reflected in the expression level of the flipped EYFP, which is directly regulated by the recombinase and indirectly affected by the L7Ae/k-turn system. We do, however, observe a reduction in the activation of the output gene when between with and without k-turn samples.</i></div>
 
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<a href="https://2016.igem.org/Team:MIT/L7AeRepressingSystem"><p style="font-family: Trebuchet MS;font-color:#7ECEFD; font-size:15px;"><i><b>Read more about our L7Ae k-turn experiment here</b></i></p> </a>
 
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Revision as of 04:10, 19 October 2016

L7Ae k-turn repressing system

L7Ae - Kink turn

Back to recombinase overview page

RNA-Based Gene Regulation

L7Ae, an archaeal ribosomal protein, binds with high affinity to RNA motifs called kink-turns (K-turns), found in both archaeal and eukaryote RNAs [1][2][3]. L7Ae protein sequence is divided into three structural regions consisting of a highly conserved RNA-binding region (RBR) flanked by less conserved N-terminal and C-terminal regions [2]. Variation in the terminal regions could dictate RNA-binding specificity of different homologs of L7Ae protein [2]. When a K-turn motif is inserted into the target mRNA upstream of the open reading frame, L7Ae can be used as a translational regulator [1][2][3]. The binding activity of L7Ae will prevent the ribosome machinery from performing translation. The strength of the repression can be controlled by varying the distance between the K-turns and the 5’-end of the mRNA, or by changing the number of the k-turn motifs [1].

Figure. Binding of L7Ae to kink-turn motifs preveting translation.

Recombinase and L7Ae-Kturn

Purpose

Using recombinases as biological latches giving our genetic circuit the ability to memorize disease temporal specificity. However, since the recombinase is controlled by an inducible promoter, leaky expression of the promoter (activation without input signals - disease biomarkers) could lead to unwanted activation of the output gene. By puting k-turn motifs in front of the recombinase gene, we hope to reduce leaky expression of the recombinase when the system is inactivated.
We designed an experiment to examine the repression level of the L7Ae - kink turn system on the expression of an output gene (EYFP - Enhanced yellow flourescent protein), which is regulated by TP901 (a serine recombinase).

Experimental Setup

                   
Untransfected ControlSingle color (Y)
1000ng hEF1a:eYFP
500ng pDONR		Single color (R)
1000ng hEF1a:mKate
500ng pDONR		 Single color (B)
1000ng hEF1a:tagBFP
500ng pDONR
Three colors
500ng hEF1a:eYFP
500ng hEF1a:mKate
500ng hEF1a:BFP		 Control no L7Ae
300ng EGSH-kturn: TP901
300ng EGSH-kturn:mKate
200ng hEF1a: flipped EYFP
100ng hEF1a: VgEcr
100ng hEF1a: rtTA
0ng TRE: L7Ae
200ng hEF1a:BFP
300ng pDONR
1000nM Dox; 5uM PonA		Control no k-turn
300ng EGSH-kturn: TP901
300ng EGSH-kturn:mKate
200ng hEF1a: flipped EYFP
100ng hEF1a: VgEcr
100ng hEF1a: rtTA
100ng TRE: L7Ae
200ng hEF1a:BFP

1000nM Dox; 0uM PonA		 Control no k-turn
300ng EGSH-kturn: TP901
300ng EGSH-kturn:mKate
200ng hEF1a: flipped EYFP
100ng hEF1a: VgEcr
100ng hEF1a: rtTA
100ng TRE: L7Ae
200ng hEF1a:BFP

1000nM Dox; 5uM PonA
Experiment 2x k-turn
300ng EGSH-kturn: TP901
300ng EGSH-kturn:mKate
200ng hEF1a: flipped EYFP
100ng hEF1a: VgEcr
100ng hEF1a: rtTA
100ng TRE: L7Ae
200ng hEF1a:BFP

0nM Dox; 0uM PonA		 Experiment 2x k-turn
300ng EGSH-kturn: TP901
300ng EGSH-kturn:mKate
200ng hEF1a: flipped EYFP
100ng hEF1a: VgEcr
100ng hEF1a: rtTA
100ng TRE: L7Ae
200ng hEF1a:BFP

0nM Dox; 5uM PonA		 Experiment 4x k-turn
300ng EGSH-kturn: TP901
300ng EGSH-kturn:mKate
200ng hEF1a: flipped EYFP
100ng hEF1a: VgEcr
100ng hEF1a: rtTA
100ng TRE: L7Ae
200ng hEF1a:BFP

0nM Dox; 0uM PonA		 Experiment 4x k-turn
300ng EGSH-kturn: TP901
300ng EGSH-kturn:mKate
200ng hEF1a: flipped EYFP
100ng hEF1a: VgEcr
100ng hEF1a: rtTA
100ng TRE: L7Ae
200ng hEF1a:BFP

0nM Dox; 5uM PonA
Experiment 2x k-turn
300ng EGSH-kturn: TP901
300ng EGSH-kturn:mKate
200ng hEF1a: flipped EYFP
100ng hEF1a: VgEcr
100ng hEF1a: rtTA
100ng TRE: L7Ae
200ng hEF1a:BFP

100nM Dox; 0uM PonA		 Experiment 2x k-turn
300ng EGSH-kturn: TP901
300ng EGSH-kturn:mKate
200ng hEF1a: flipped EYFP
100ng hEF1a: VgEcr
100ng hEF1a: rtTA
100ng TRE: L7Ae
200ng hEF1a:BFP

100nM Dox; 5uM PonA		 Experiment 4x k-turn
300ng EGSH-kturn: TP901
300ng EGSH-kturn:mKate
200ng hEF1a: flipped EYFP
100ng hEF1a: VgEcr
100ng hEF1a: rtTA
100ng TRE: L7Ae
200ng hEF1a:BFP

100nM Dox; 0uM PonA		 Experiment 4x k-turn
300ng EGSH-kturn: TP901
300ng EGSH-kturn:mKate
200ng hEF1a: flipped EYFP
100ng hEF1a: VgEcr
100ng hEF1a: rtTA
100ng TRE: L7Ae
200ng hEF1a:BFP

100nM Dox; 5uM PonA
Experiment 2x k-turn
300ng EGSH-kturn: TP901
300ng EGSH-kturn:mKate
200ng hEF1a: flipped EYFP
100ng hEF1a: VgEcr
100ng hEF1a: rtTA
100ng TRE: L7Ae
200ng hEF1a:BFP

500nM Dox; 0uM PonA		 Experiment 2x k-turn
300ng EGSH-kturn: TP901
300ng EGSH-kturn:mKate
200ng hEF1a: flipped EYFP
100ng hEF1a: VgEcr
100ng hEF1a: rtTA
100ng TRE: L7Ae
200ng hEF1a:BFP

500nM Dox; 5uM PonA		 Experiment 4x k-turn
300ng EGSH-kturn: TP901
300ng EGSH-kturn:mKate
200ng hEF1a: flipped EYFP
100ng hEF1a: VgEcr
100ng hEF1a: rtTA
100ng TRE: L7Ae
200ng hEF1a:BFP

500nM Dox; 0uM PonA		 Experiment 4x k-turn
300ng EGSH-kturn: TP901
300ng EGSH-kturn:mKate
200ng hEF1a: flipped EYFP
100ng hEF1a: VgEcr
100ng hEF1a: rtTA
100ng TRE: L7Ae
200ng hEF1a:BFP

500nM Dox; 5uM PonA
Experiment 2x k-turn
300ng EGSH-kturn: TP901
300ng EGSH-kturn:mKate
200ng hEF1a: flipped EYFP
100ng hEF1a: VgEcr
100ng hEF1a: rtTA
100ng TRE: L7Ae
200ng hEF1a:BFP

1000nM Dox; 0uM PonA		 Experiment 2x k-turn
300ng EGSH-kturn: TP901
300ng EGSH-kturn:mKate
200ng hEF1a: flipped EYFP
100ng hEF1a: VgEcr
100ng hEF1a: rtTA
100ng TRE: L7Ae
200ng hEF1a:BFP

1000nM Dox; 5uM PonA		 Experiment 4x k-turn
300ng EGSH-kturn: TP901
300ng EGSH-kturn:mKate
200ng hEF1a: flipped EYFP
100ng hEF1a: VgEcr
100ng hEF1a: rtTA
100ng TRE: L7Ae
200ng hEF1a:BFP

1000nM Dox; 0uM PonA		 Experiment 4x k-turn
300ng EGSH-kturn: TP901
300ng EGSH-kturn:mKate
200ng hEF1a: flipped EYFP
100ng hEF1a: VgEcr
100ng hEF1a: rtTA
100ng TRE: L7Ae
200ng hEF1a:BFP

1000nM Dox; 5uM PonA

Read more about building kturn constructs here.

Result

Testing the 2x k-turn L7Ae system with varied L7Ae expression level

Testing the effect of varying k-turn sequences



REFERENCE:

  1. Oliwia Andries, Tasuku Kitada, Katie Bodner, Niek N Sanders & Ron Weiss (2015) Synthetic biology devices and circuits for RNA-based ‘smart vaccines’: a propositional review, Expert Review of Vaccines, 14:2, 313-331
  2. Gagnon KT, Zhang X, Qu G, et al. Signature amino acids enable the archaeal L7Ae box C/D RNP core protein to recognize and bind the K-loop RNA motif. Rna 2010;16(1):79-90
  3. Stapleton JA, Endo K, Fujita Y, et al. Feedback control of protein expression in mammalian cells by tunable synthetic translational inhibition. ACS Synth Biol 2012;1(3):83-8