Team:UPO-Sevilla/Project/Improving Flip Flop/asRNA/OmpN GFP

{{:Team:UPO-Sevilla/headerTemplate}}

<html>
                    <div id="principal">
                        <div class="main">


                        <ul id="breadcrumbs" class="xbreadcrumbs">
                                    <li class="">
                                       <a class="home" href="/Team:UPO-Sevilla" style="white-space: nowrap; float: left; overflow: hidden; width: 40px; display: block;" init-width="29">Home</a><ul></ul>
                                    </li>
                                    <li><a href="/Team:UPO-Sevilla/Project/Overview" style="white-space: nowrap; float: left;">Project</a><ul></ul></li>
                                    <li><a href="/Team:UPO-Sevilla/Project/Improving_Flip_Flop/Overview" style="white-space: nowrap; float: left;">Improving Flip Flop</a><ul></ul></li>
                                    <li><a href="/Team:UPO-Sevilla/Project/Improving_Flip_Flop/asRNA" style="white-space: nowrap; float: left;">asRNA</a><ul></ul></li>
                                    <li class="current"><a href="/Team:UPO-Sevilla/Project/Improving_Flip_Flop/asRNA/OmpN_GFP" style="white-space: nowrap; float: left;">OmpN::GFP</a><ul></ul></li>
                        </ul>

                            <h1>OmpN::GFP</h1>
                            
                            <p>In order to validate this inhibition in vivo, <a href="http://www.ncbi.nlm.nih.gov/pubmed/19111662"_blank">Bouvier et al.</a> constructed a translational <strong>OmpN fusion</strong> to the <strong>N terminus of GFP</strong>. <strong>Figure 3</strong> shows the mentioned <strong>OmpN::GFP</strong> fusion. They observed that the <strong>GFP signal</strong> was <strong>inhibited by the asRNA</strong>, probing that the fusion of the beginning of the OmpN gene with the GFP gene was also regulable by the RybB asRNA.</p>
<div clas="center"><img src="https://static.igem.org/mediawiki/2011/6/67/UPOSevilla_figure32.png"></div>
<p><strong>Figure 3.</strong> OmpN::GFP fusion. The fusion includes the nucleotides -75 to 90 relative to the ATG of OmpN. The beginning of the OmpN CDS is shown in red capital letters. Taken from <a href="http://www.cell.com/molecular-cell/supplemental/S1097-2765(08)00807-1" target="_blank">Bouvier et al., Suplemental Data (2008)</a></p><br/>

<p>We will take advantage of this strategy by making an analogue <strong>fusion of OmpN</strong> start sequence with those <strong>proteins</strong> whose translation we want <strong>to regulate</strong>. As we can see in <strong>Figure 1</strong>, the RybB asRNA binds to <strong>nucleotides from 5th to 20th</strong> relative to AUG. Consequently, we have designed a <strong>shorter fusion</strong> in which we fuse the first 7 codons of the OmpN gene CDS upstream of the <strong>RFP</strong> and <strong>Sspb</strong> CDS. As for the upstream nucleotides of the OmpN gene we have chosen those from the <strong>mRNA</strong> (see <strong>Figure 4</strong>) to make sure that <strong>no promoter was included</strong>. As we can see in <strong>Figure 1</strong>, these fusions do include the <strong>OmpN RBS</strong>.</p>
<div clas="center"><img src="https://static.igem.org/mediawiki/2011/c/c9/UPOSevilla_figure42.png"><p><strong>Figure 4.</strong> OmpN in vitro RNA sequence. Taken from <a href="http://www.cell.com/molecular-cell/supplemental/S1097-2765(08)00807-1" target="_blank">Bouvier et al., Suplemental Data (2008)</a>.</p></div><br/>
<p>Our final fusions of <strong>OmpN::SspB</strong> and <strong>OmpN::RFP</strong> are shown in <strong>Figure 5</strong>.</p>
<div clas="center"><img src="https://static.igem.org/mediawiki/2011/4/4f/UPOSevilla_figure52.png"></div>
<p><strong>Figure 5.</strong> Final OmpN::SspB and OmpN::RFP fusions. The OmpN fragment in each fusion is shown in red and the SspB/RFP CDS in black. The complete CDS of our fusion is shown in capital letters, while the upstream UTR, including the RBS, is shown in lower-case letters.</p><br/>
<p>With these fusions, present in the State 2 of our bistable, and expressing the asRNA under the same promoter as in State 1, State 1 machinery will be able to repress State 2 more efficiently (see Figure 6). As in the proteolysis mechanism, we needed bacteria withthe RybB asRNA. We have performed the mentioned deletion to our Sspb- and Clpx- strains by using the lambda red protocol (Wanner et al., 2000).</p>

<div clas="center"><img src="https://static.igem.org/mediawiki/2011/b/b2/UPOSevilla_figure62.png" width="700px" height="300px"></div>
<p>Figure 6. When State 1 of our bistable is being expressed by temperature (42 ºC), we are not only expressing the LacI repressor of the Lac promoter but we also are expressing the asRNA under the same promoter. This RybB asRNA binds to the mRNAs that have the beginning of OmpN mRNA fused, being these SspB and RFP of State 2. When this asRNA binds to SspB and RFP mRNA it inhibits their translation and, therefore, their expression. In conclusion, when the State 1 is active we are repressing State 2 promoter and preventing both State 1 reporter’s and Sspb’s translation. </p>



                        </div>
                        <div class="left">
                              </html>{{:Team:UPO-Sevilla/leftTemplateProject}}<html>
                        </div>
</html>

{{:Team:UPO-Sevilla/footTemplate}}