Difference between revisions of "Team:AFCM-Egypt/Design"

(AFCM-Egypt/Design)
Line 1: Line 1:
−
{{AFCM-Egypt}}
+
{{Template:AFCM-Egypt/bootstrap}}
 +
{{Template:AFCM-Egypt/main}}
 +
{{Template:AFCM-Egypt/header}}
 +
 
 
<html>
 
<html>
−
 
+
<head>
−
 
+
    <meta charset="UTF-8">
−
 
+
    <title>A F C M</title>
−
 
+
    <meta name="description" content="">
−
<div class="column full_size">
+
    <meta name="author" content="Web Domus Italia">
−
<h1>Design</h1>
+
    <meta http-equiv="X-UA-Compatible" content="IE=edge,chrome=1">
−
<p>
+
    <meta name="viewport" content="width=device-width, initial-scale=1.0"> 
−
Design is the first step in the design-build-test cycle in engineering and synthetic biology. Use this page to describe the process that you used in the design of your parts. You should clearly explain the engineering principles used to design your project.
+
<style>
−
</p>
+
.Design {border: 1px solid #fff;
−
 
+
    background: rgba(255,255,255, 0.3);}
−
<p>
+
</style>
−
This page is different to the "Applied Design Award" page. Please see the <a href="https://2017.igem.org/Team:AFCM-Egypt/Applied_Design">Applied Design</a> page for more information on how to compete for that award.
+
</head>
−
</p>
+
<body>
−
 
+
<!-- Price Table -->
 +
<section class="price-table">
 +
<div class="container section-wrapper">
 +
<div class="row" style="margin-bottom: 50px;">
 +
<div class="col-md-6 text-center" style="float: none; margin: auto; width: 82%; background-color: rgba(181, 178, 173, 0.4);">
 +
<p style="text-align: center;"><span style="font-size: 28pt; line-height: 107%; font-family: Calibri, sans-serif; background-color: #ffcc00;"><span style="background-color: #999999;"><em><span style="font-family: 'times new roman', times;">Biomarker Filtration</span></em></span><br /><br /></span></p>
 +
<p style="text-align: center;"><em><span style="text-decoration: underline;"><span style="font-size: 19pt; font-family: 'times new roman', times;">Steps Of Our Biomarker filtration&nbsp;</span></span></em></p>
 +
<p style="text-align: center;"><span style="font-size: 28pt; line-height: 107%; font-family: Calibri, sans-serif;"><img src="https://static.igem.org/mediawiki/2017/1/14/AFCM-Egypt-biomarkerfiltration1.jpg" alt="" width="626" height="469" /></span></p>
 +
<p style="text-align: center;"><span style="font-size: 28pt; line-height: 107%; font-family: Calibri, sans-serif;"><img src="https://static.igem.org/mediawiki/2017/c/cc/AFCM-Egypt-biomarkerfiltration2.jpg" alt="" width="626" height="469" /></span></p>
 +
<p style="text-align: center;"><span style="font-size: 28pt; line-height: 107%; font-family: Calibri, sans-serif;"><img src="https://static.igem.org/mediawiki/2017/a/a6/AFCM-Egypt-biomarkerfiltration3.jpg" alt="" width="616" height="61" /></span></p>
 +
<p style="text-align: center;"><span style="font-size: 28pt; line-height: 107%; font-family: Calibri, sans-serif;"><img src="https://static.igem.org/mediawiki/2017/8/84/AFCM-Egypt-biomarkerfiltration4.jpg" alt="" width="616" height="469" /></span></p>
 +
<p style="text-align: center;"><span style="font-size: 28pt; line-height: 107%; font-family: Calibri, sans-serif;"><img src="https://static.igem.org/mediawiki/2017/b/b6/AFCM-Egypt-biomarkerfiltration5.jpg" alt="" width="616" height="469" /></span></p>
 +
<p style="text-align: center;"><span style="font-size: 28pt; line-height: 107%; font-family: Calibri, sans-serif;"><img src="https://static.igem.org/mediawiki/2017/f/ff/AFCM-Egypt-biomarkerfiltration6.jpg" alt="" width="616" height="469" /></span></p>
 +
<p style="text-align: center;"><span style="font-size: 28pt; line-height: 107%; font-family: Calibri, sans-serif;"><img src="https://static.igem.org/mediawiki/2017/a/a2/AFCM-Egypt-biomarkerfiltration7.jpg" alt="" width="616" height="469" /></span></p>
 +
<p style="text-align: center;"><span style="font-size: 28pt; line-height: 107%; font-family: Calibri, sans-serif;"><img src="https://static.igem.org/mediawiki/2017/8/82/AFCM-Egypt-biomarkerfiltration8.jpg" alt="" width="616" height="469" /></span></p>
 +
<p style="text-align: center;"><span style="font-size: 28pt; line-height: 107%; font-family: Calibri, sans-serif;"><img src="https://static.igem.org/mediawiki/2017/6/60/AFCM-Egypt-biomarkerfiltration9.jpg" alt="" width="616" height="469" /></span></p>
 +
<p style="text-align: center;"><span style="font-size: 28pt; line-height: 107%; font-family: Calibri, sans-serif;"><img src="https://static.igem.org/mediawiki/2017/e/ee/AFCM-Egypt-biomarkerfiltration10.jpg" alt="" width="616" height="469" /></span></p>
 +
<p style="text-align: center;"><span style="font-size: 28pt; line-height: 107%; font-family: Calibri, sans-serif;"><img src="https://static.igem.org/mediawiki/2017/3/3b/AFCM-Egypt-biomarkerfiltration11.jpg" alt="" width="616" height="469" /></span></p>
 +
<p style="text-align: center;"><span style="font-size: 28pt; line-height: 107%; font-family: Calibri, sans-serif;"><img src="https://static.igem.org/mediawiki/2017/1/17/AFCM-Egypt-biomarkerfiltration12.jpg" alt="" width="616" height="469" /></span></p>
 +
<p style="text-align: center;"><span style="font-size: 28pt; line-height: 107%; font-family: Calibri, sans-serif;"><img style="font-size: 37.3333px;" src="https://static.igem.org/mediawiki/2017/7/7f/AFCM-Egypt-biomarkerfiltration14.jpg" alt="" width="616" height="469" /><img src="https://static.igem.org/mediawiki/2017/2/2c/AFCM-Egypt-biomarkerfiltration13.jpg" alt="" width="616" height="469" /></span></p>
 +
<p style="text-align: center;"><span style="font-size: 28pt; line-height: 107%; font-family: Calibri, sans-serif;"><br /><br /><span style="background-color: #ffff00;"><!-- pagebreak --></span><br /></span></p>
 +
<p style="text-align: center;"><span style="color: #ffffff;"><strong><span style="font-size: 30pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif;">Design</span></strong></span></p>
 +
<p style="text-align: justify;">&nbsp;</p>
 +
<p style="text-align: center;"><span style="font-size: 15pt; color: #ffffff;"><strong><span style="line-height: 115%; font-family: 'Trebuchet MS', sans-serif; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">Our design aims to compare the function of both circuits at regulating non-coding RNAs</span></strong></span></p>
 +
<p style="text-align: center;"><span style="color: #000000;">&nbsp;</span></p>
 +
<p style="text-align: center;"><span style="font-size: 28pt; line-height: 107%; font-family: Calibri, sans-serif; color: #000000;">&nbsp;</span></p>
 +
<table style="height: 80px; border-color: #f21111; margin-left: auto; margin-right: auto; width: 1001px;">
 +
<tbody>
 +
<tr>
 +
<td style="width: 463px; text-align: center;"><span style="font-family: 'times new roman', times; font-size: 17pt; color: #ffffff;"><strong>CRISPR circuit</strong></span></td>
 +
<td style="width: 534px; text-align: center;"><span style="font-size: 17pt; color: #ffffff;"><strong><span style="font-family: 'times new roman', times;">ceRNA circuit</span></strong></span></td>
 +
</tr>
 +
<tr style="text-align: center;">
 +
<td style="width: 463px;">
 +
<p style="text-align: center;"><span style="color: #ffffff;"><span style="font-size: 14pt; line-height: 115%; font-family: 'times new roman', times; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><strong>BBa_K2217026</strong></span><span style="font-size: 14pt; line-height: 115%; font-family: 'times new roman', times; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><br /></span></span><span style="font-size: 14pt; line-height: 115%; font-family: 'times new roman', times; color: #282828; background: white;"><br /></span></p>
 +
</td>
 +
<td style="width: 534px;">
 +
<p style="text-align: center;"><span style="color: #ffffff;"><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">BBa_K2217025<br /></span></span><span style="font-size: 14.0pt; line-height: 115%; font-family: 'Trebuchet MS','sans-serif'; color: #282828; background: white;"><br /></span></p>
 +
</td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<p style="text-align: center;"><span style="font-size: 28pt; line-height: 107%; font-family: Calibri, sans-serif;"><img src="https://static.igem.org/mediawiki/2017/a/a6/AFCM-Egypt-Design1%262.png" alt="" width="1100" height="687" /><br /><br /><img src="https://static.igem.org/mediawiki/2017/a/a9/AFCM-Egypt-Design3%264.png" alt="" width="1104" height="689" /><br /></span></p>
 +
<p style="text-align: center;"><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; color: #ffffff; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">Fig-1 Composite Part BBa_K2217018</span></p>
 +
<p style="text-align: center;"><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; color: #282828; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><span style="color: #ffffff;">Part BBa_K2217018 was composed of&nbsp; U6</span><span style="color: #000000;"><span style="color: #ffffff;"> (</span><span style="color: #ff9900;">BBa_K2217002</span><span style="color: #ffffff;">) as a promoter for gRNA (</span><span style="color: #ff9900;">BBa_K2217003</span><span style="color: #ffffff;">)(Designed using MIT CRISPR web tool according to the addgene protocol).<sup>[1][2] </sup>The gRNA needs a scaffold to guide the Cas9 (</span><span style="color: #ff9900;">BBa_K2217004</span><span style="color: #ffffff;">) and finally it was terminated by SV40 poly(A) signal Termination (</span><span style="color: #ff9900;">BBa_K2217005</span><span style="color: #ffffff;">).</span></span></span></p>
 +
<p style="text-align: center;"><span style="color: #000000;">&nbsp;</span><br /><br /><img style="background: rgba(0, 0, 0, .0001);" src="https://static.igem.org/mediawiki/2017/f/f7/AFCM-Egypt-Design5%266.png" alt="" width="1100" height="611" /></p>
 +
<p style="text-align: center;"><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; color: #282828; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><span style="color: #ffffff;">Fig-2 Composite Part BBa_K2217019</span><br /></span></p>
 +
<p style="text-align: center;"><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; color: #282828; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><span style="color: #000000;"><span style="color: #ffffff;">Part</span> </span><span style="color: #ff9900;">BBa_K2217019</span><span style="color: #ffffff;"> was composed of CMV Enhancer as well as CMV Promoter (</span><span style="color: #ff9900;">BBa_K2217000</span> <span style="color: #000000;"><span style="color: #ffffff;">and&nbsp;</span> </span><span style="color: #ff9900;">BBa_K2217006</span><span style="color: #ffffff;"> respectively) as constitutive promoters to enhance the transcription process of the Cas9 (</span><span style="color: #ff9900;">BBa_K1218011</span><span style="color: #ffffff;">) part which was transcribed using&nbsp; T7 Promoter (</span><span style="color: #ff9900;">BBa_K2217007</span><span style="color: #ffffff;">) following Protocol<sup>[3]</sup>. The design aimed at generating Homology Directed Repair instead of Non-Homologous end Joining repair of Cas9 by knocking in the circular RNA as a Competing endogenous RNA enhancing its transcription to regulate miRNA action, so we designed Homology repair template (</span><span style="color: #ff9900;">BBa_K2217009</span><span style="color: #ffffff;">) to be transfected on a separate donor vector then terminated with CYC1 terminator(</span><span style="color: #ff9900;">BBa_K2217008</span></span><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; color: #ffffff; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">).</span><span style="font-size: 14.0pt; line-height: 115%; font-family: 'Trebuchet MS','sans-serif'; color: #282828; background: white;"><br /><br /></span></p>
 +
<p style="text-align: center;">&nbsp;</p>
 +
<p style="text-align: center;"><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; color: #282828; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><img src="https://static.igem.org/mediawiki/2017/c/c8/AFCM-Egypt-Design7%268.png" alt="" width="1106" height="690" /></span><span style="font-size: 14.0pt; line-height: 115%; font-family: 'Trebuchet MS','sans-serif'; color: #282828; background: white;"><br /></span></p>
 +
<p style="text-align: center;"><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; color: #282828; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><span style="color: #ffffff;">Fig-3 Composite Part BBa_K2217020</span><br /></span></p>
 +
<p style="text-align: center;"><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; color: #282828; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><span style="color: #ffffff;">In order to enhance transcription of the circular RNA as a competing endogenous RNA in a ceRNA network, we used the CAG promoter composed of CMV enhancer and Chicken B-actin Promoter</span> <span style="color: #ffffff;">(</span><span style="color: #ff9900;">BBa_K2217013</span><span style="color: #ffffff;">) while circular RNA (hsa-circ-0000064) was submitted to the registry after biomarker filtration using bioinformatics databases demonstrated at our </span></span><span style="color: #ffffff;"><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">biomarker filtration</span>&nbsp;<span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"> page</span></span><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; color: #282828; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><span style="color: #ffffff;"> (</span><span style="color: #ff9900;">BBa_K2217001</span><span style="color: #ffffff;">). Finally it was terminated by SV40 poly(A) signal Termination (</span><span style="color: #ff9900;">BBa_K2217005</span><span style="color: #ffffff;">).</span></span></p>
 +
<p style="text-align: center;">&nbsp;<br /><img src="https://static.igem.org/mediawiki/2017/9/99/AFCM-Egypt-Design9%2610.png" alt="" width="1114" height="695" /><br /><br /><br /><span style="color: #ffffff;"><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">Fig-4 Composite Part BBa_K2217022</span><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><br /></span></span><span style="font-size: 14.0pt; line-height: 115%; font-family: 'Trebuchet MS','sans-serif'; color: #282828; background: white;"><br /></span></p>
 +
<p style="text-align: center;"><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; color: #282828; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><span style="color: #ffffff;">Part</span> <span style="color: #ff9900;">BBa_K2217022</span> <span style="color: #ffffff;">was composed of CMV Enhancer as well as CMV Promoter (</span><span style="color: #ff9900;">BBa_K2217000</span> <span style="color: #ffffff;">and&nbsp;</span> <span style="color: #ff9900;">BBa_K2217006</span> <span style="color: #ffffff;">respectively) as constitutive promoters to enhance the transcription process of the laci that was submitted (</span><span style="color: #ff9900;">Ba_K2217012</span><span style="color: #ffffff;">). We also improved the fragment&rsquo;s characterization by adding the miRNA binding site of the miRNA mir-1825 as determined computationally using circ interactome database <sup>[4]</sup> where we hypothesised its function as a trigger for miRNA binding and synthesis. Finally, it was terminated by SV40 poly(A) signal Termination (</span><span style="color: #ff9900;">BBa_K2217005</span><span style="color: #ffffff;">).</span></span><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; color: #282828; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><br /></span><span style="font-size: 14.0pt; line-height: 115%; font-family: 'Trebuchet MS','sans-serif'; color: #282828; background: white;"><br /><br /><br /></span></p>
 +
<p style="text-align: center;"><img src="https://static.igem.org/mediawiki/2017/a/a8/AFCM-Egypt-Design11%2612.png" alt="" width="1114" height="695" /><br /><br /></p>
 +
<p style="text-align: center;"><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; color: #ffffff; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">Fig-5 Composite Part BBa_K2217023</span></p>
 +
<p style="text-align: center;"><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; color: #282828; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><span style="color: #ffffff;">Part</span> <span style="color: #ff9900;">BBa_K2217023</span><span style="color: #ffffff;"> is composed of lac operator (</span><span style="color: #ff9900;">BBa_K2217016</span><span style="color: #ffffff;">) and the lac promoter (</span><span style="color: #ff9900;">BBa_K2217017</span><span style="color: #ffffff;">) as a weak constitutive promoter in a trial to improve characterization of ceRNA network using yfb (</span><span style="color: #ff9900;">BBa_K2217014</span><span style="color: #ffffff;">) detection using flow cytometry. Finally, it was terminated by Poly_gh termination (</span><span style="color: #ff9900;">BBa_K2217015</span><span style="color: #ffffff;">).</span></span></p>
 +
<p><span style="font-size: 14.0pt; line-height: 115%; font-family: 'Trebuchet MS','sans-serif'; color: #282828; background: white;">&nbsp;</span></p>
 +
<p style="text-align: center;"><span style="color: #ffffff;"><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">All Parts were designed in SBOL format using SBOLDesigner.<sup>[5]</sup> Sequence editing was performed on Benchling<sup>[6]</sup> and composites were constructed in synthetic<sup>TM</sup> Language using cytostudio.<sup>[7]</sup></span><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><sup><br /></sup></span></span><span style="font-size: 14.0pt; line-height: 115%; font-family: 'Trebuchet MS','sans-serif'; color: #282828; background: white;"><sup><br /><br /></sup></span></p>
 +
<p style="text-align: center;"><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; color: #ffffff; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;"><span style="text-decoration: underline;"><strong>References</strong></span></span><span style="font-size: 14.0pt; line-height: 115%; font-family: 'Trebuchet MS','sans-serif'; color: #282828; background: white;"><br /></span></p>
 +
<p><span style="font-size: 14.0pt; line-height: 115%; font-family: 'Trebuchet MS','sans-serif'; color: #282828; background: white;">&nbsp;</span></p>
 +
<p><span style="color: #ffffff;"><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">[1]&nbsp; </span><a style="color: #ffffff;" href="http://crispr.mit.edu"><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">http://crispr.mit.edu</span></a></span></p>
 +
<p><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; color: #ffffff; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;</span></p>
 +
<p><span style="color: #ffffff;"><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">[2]&nbsp; </span><a style="color: #ffffff;" href="https://media.addgene.org/cms/files/hCRISPR_gRNA_Synthesis.pdf"><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">https://media.addgene.org/cms/files/hCRISPR_gRNA_Synthesis.pdf</span></a></span></p>
 +
<p><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; color: #ffffff; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;</span></p>
 +
<p><span style="color: #ffffff;"><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">[3] </span><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">Romanienko PJ, Giacalone J, Ingenito J, et al. A Vector with a Single Promoter for <em>In Vitro</em> Transcription and Mammalian Cell Expression of CRISPR gRNAs. Fujii H, ed. <em>PLoS ONE</em>. 2016;11(2):e014836.</span></span></p>
 +
<p><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; color: #ffffff; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;</span></p>
 +
<p><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; color: #ffffff; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;</span></p>
 +
<p><span style="color: #ffffff;"><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">[4] </span><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">Dudekula DB, Panda AC, Grammatikakis I, De S, Abdelmohsen K, Gorospe M. CircInteractome: A web tool for exploring circular RNAs and their interacting proteins and microRNAs. <em>RNA Biology</em>. 2016;13(1):34-42. </span></span></p>
 +
<p><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; color: #ffffff; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;</span></p>
 +
<p><span style="color: #ffffff;"><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">[5] </span><a style="color: #ffffff;" href="http://www.async.ece.utah.edu/SBOLDesigner"><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">www.async.ece.utah.edu/SBOLDesigner</span></a></span></p>
 +
<p><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; color: #ffffff; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;</span></p>
 +
<p><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; color: #ffffff; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;</span></p>
 +
<p><span style="color: #ffffff;"><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">[6] </span><a style="color: #ffffff;" href="https://benchling.com/"><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">https://benchling.com/</span></a></span></p>
 +
<p><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; color: #ffffff; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;</span></p>
 +
<p><span style="color: #ffffff;"><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">[7] </span><a style="color: #ffffff;" href="https://moleculamaxima.com/"><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">https://moleculamaxima.com/</span></a></span></p>
 +
<p><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; color: #ffffff; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;</span></p>
 +
<p><span style="font-size: 14pt; line-height: 115%; font-family: 'Trebuchet MS', sans-serif; color: #ffffff; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&nbsp;<br /><br /><br /></span></p>
 
</div>
 
</div>
−
 
−
<div class="column half_size">
 
−
<h5>What should this page contain?</h5>
 
−
<ul>
 
−
<li>Explanation of the engineering principles your team used in your design</li>
 
−
<li>Discussion of the design iterations your team went through</li>
 
−
<li>Experimental plan to test your designs</li>
 
−
</ul>
 
−
 
 
</div>
 
</div>
−
 
−
<div class="column half_size">
 
−
<h5>Inspiration</h5>
 
−
<ul>
 
−
<li><a href="https://2016.igem.org/Team:MIT/Experiments/Promoters">2016 MIT</a></li>
 
−
<li><a href="https://2016.igem.org/Team:BostonU/Proof">2016 BostonU</a></li>
 
−
<li><a href="https://2016.igem.org/Team:NCTU_Formosa/Design">2016 NCTU Formosa</a></li>
 
−
</ul>
 
 
</div>
 
</div>
−
 
+
</section>
−
 
+
</body>
−
 
+
 
</html>
 
</html>
 +
{{Template:AFCM-Egypt/footer}}

Revision as of 16:09, 26 October 2017

A F C M

A F C M

Biomarker Filtration

Steps Of Our Biomarker filtration 




Design

 

Our design aims to compare the function of both circuits at regulating non-coding RNAs

 

 

CRISPR circuit ceRNA circuit

BBa_K2217026

BBa_K2217025




Fig-1 Composite Part BBa_K2217018

Part BBa_K2217018 was composed of  U6 (BBa_K2217002) as a promoter for gRNA (BBa_K2217003)(Designed using MIT CRISPR web tool according to the addgene protocol).[1][2] The gRNA needs a scaffold to guide the Cas9 (BBa_K2217004) and finally it was terminated by SV40 poly(A) signal Termination (BBa_K2217005).

 

Fig-2 Composite Part BBa_K2217019

Part BBa_K2217019 was composed of CMV Enhancer as well as CMV Promoter (BBa_K2217000 and  BBa_K2217006 respectively) as constitutive promoters to enhance the transcription process of the Cas9 (BBa_K1218011) part which was transcribed using  T7 Promoter (BBa_K2217007) following Protocol[3]. The design aimed at generating Homology Directed Repair instead of Non-Homologous end Joining repair of Cas9 by knocking in the circular RNA as a Competing endogenous RNA enhancing its transcription to regulate miRNA action, so we designed Homology repair template (BBa_K2217009) to be transfected on a separate donor vector then terminated with CYC1 terminator(BBa_K2217008).

 


Fig-3 Composite Part BBa_K2217020

In order to enhance transcription of the circular RNA as a competing endogenous RNA in a ceRNA network, we used the CAG promoter composed of CMV enhancer and Chicken B-actin Promoter (BBa_K2217013) while circular RNA (hsa-circ-0000064) was submitted to the registry after biomarker filtration using bioinformatics databases demonstrated at our biomarker filtration  page (BBa_K2217001). Finally it was terminated by SV40 poly(A) signal Termination (BBa_K2217005).

 



Fig-4 Composite Part BBa_K2217022

Part BBa_K2217022 was composed of CMV Enhancer as well as CMV Promoter (BBa_K2217000 and  BBa_K2217006 respectively) as constitutive promoters to enhance the transcription process of the laci that was submitted (Ba_K2217012). We also improved the fragment’s characterization by adding the miRNA binding site of the miRNA mir-1825 as determined computationally using circ interactome database [4] where we hypothesised its function as a trigger for miRNA binding and synthesis. Finally, it was terminated by SV40 poly(A) signal Termination (BBa_K2217005).





Fig-5 Composite Part BBa_K2217023

Part BBa_K2217023 is composed of lac operator (BBa_K2217016) and the lac promoter (BBa_K2217017) as a weak constitutive promoter in a trial to improve characterization of ceRNA network using yfb (BBa_K2217014) detection using flow cytometry. Finally, it was terminated by Poly_gh termination (BBa_K2217015).

 

All Parts were designed in SBOL format using SBOLDesigner.[5] Sequence editing was performed on Benchling[6] and composites were constructed in syntheticTM Language using cytostudio.[7]


References

 

[1]  http://crispr.mit.edu

 

[2]  https://media.addgene.org/cms/files/hCRISPR_gRNA_Synthesis.pdf

 

[3] Romanienko PJ, Giacalone J, Ingenito J, et al. A Vector with a Single Promoter for In Vitro Transcription and Mammalian Cell Expression of CRISPR gRNAs. Fujii H, ed. PLoS ONE. 2016;11(2):e014836.

 

 

[4] Dudekula DB, Panda AC, Grammatikakis I, De S, Abdelmohsen K, Gorospe M. CircInteractome: A web tool for exploring circular RNAs and their interacting proteins and microRNAs. RNA Biology. 2016;13(1):34-42.

 

[5] www.async.ece.utah.edu/SBOLDesigner

 

 

[6] https://benchling.com/

 

[7] https://moleculamaxima.com/