Team:UT-Dallas/temp

From 2014.igem.org

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<section id="titlechart"></html>{{Header_menu}}<html><div class="page_content"><br><h2>Introduction</H2><p style="display:block">
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<section id="titlechart"></html>{{Header_menu}}<html><div class="page_content"><br><h2>Parts</H2><p style="display:block">
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We envision a new paradigm for treating infections of the human gastrointestinal tract through exploitation of engineered probiotics that produce anti-microbials with high specificity for pathogens. The anti-microbials we are exploring do not utilize a one-fit all therapy mold, but target unique features specific to organisms at the genetic level. Towards this aim, we have utilized a general-purpose system that will be delivered to pathogenic bacteria from an engineered bacterial species found in the GI tract (Escherichia coli), which will cleave pathogenic genes with single nucleotide resolution. To achieve specific genome targeting, we will utilize the CRISPR/Cas9 system with gRNA engineered to recognize genes from infectious bacteria that contribute to pathogenicity in humans. Our CRISPR/Cas9 system will be delivered from the engineered E. coli to infectious bacteria using bacterial specific phages, minimizing any side-effects to native microbiota and human-host cells. As a proof-of-principle for our engineered probiotic, we are starting by targeting Vibrio cholerae, however we hope to expand the system to other pathogens of the GI tract.  
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<br><img src="https://static.igem.org/mediawiki/2014/4/48/GRNA_Library.png" width="1024px">
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<br><h2>Probiotics</H2><br><p style="display:block">
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<br><h2>Parts List</H2><br><p style="display:block">
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We used E. coli as our chassis for our experiments in the lab, but we envision that our system would be used in a common probiotic strain of bacteria, such as Lactobacillus acidophilus. Our probiotic system can be used prophylactically to persons in a region experiencing a cholera outbreak, or it can be administered to someone already exposed to V. cholerae to quickly deliver therapeutic phage directly to the site of the infection. We believe our system would be particularly useful to military personnel or aid workers stationed in a cholera outbreak zone where they would be frequently exposed to V. cholerae. Although an oral cholera vaccine exists, its efficacy is relatively low: 52-62% in healthy adults and as low as 38% for the highest risk age group - children under 5 years of age (1).
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<table style="border:1px solid black;" width="1024px" align="center">
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Once complete, our system will be able to remain as a stable population in a person’s gut until the event of a V. cholerae infection. Detection of V. cholerae will then activate production of the phage delivery system, which will package the gRNA and Cas9 targeting system into a phage coat, exit the probiotic and transfer the system into V. cholerae present in the gut (transmission of a heterologous DNA message via phage was demonstrated by Ortiz and Endy in 2012 and featured in Waterloo’s 2013 iGEM project (2)). Once inside V. cholerae, the targeting system will bind and cleave sites complementary to the gRNA that correspond to selected pathogenicity genes. We proposed using Cas9/gRNA to target and kill pathogens as opposed to traditional phage therapy using a targeted lytic phage because it allowed us to differentiate and kill harmful pathogens of a strain that has both harmful and harmless serotypes. The gRNA in our system, while specific to V. cholerae, can easily be altered with PCR to target unique regions in other gastrointestinal pathogens.
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<td style="border:1px solid black;">Name</td>
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<td style="border:1px solid black;">Type</td>
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<td style="border:1px solid black;">Description</td>
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<td style="border:1px solid black;">Length</td>
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</tr>
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<tr style="border:1px solid black;">
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487021"> BBa_K1487021</a> </td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P-acfA gRNA</td><td style="border:1px solid black;">145</td>
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</tr>
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<tr>
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487022"> BBa_K1487022</a> </td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P-acfB gRNA</td><td style="border:1px solid black;">145</td>
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</tr>
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<tr>
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487023"> BBa_K1487023</a></td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P-acfC gRNA</td><td style="border:1px solid black;">145</td>
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</tr>
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<tr>
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487024"> BBa_K1487024</a></td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P-acfD gRNA</td><td style="border:1px solid black;">145</td>
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</tr>
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<tr>
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487025"> BBa_K1487025</a></td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P-ctxA gRNA</td><td style="border:1px solid black;">145</td>
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</tr>
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<tr>
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487026"> BBa_K1487026</a></td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P-ctxB gRNA</td><td style="border:1px solid black;">145</td>
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</tr>
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487027"> BBa_K1487027</a></td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P-tcpA gRNA</td><td style="border:1px solid black;">145</td>
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</tr>
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487028"> BBa_K1487028</a></td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P-tcpB gRNA</td><td style="border:1px solid black;">145</td>
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</tr>
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<tr>
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487029"> BBa_K1487029</a></td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P-tcpC gRNA</td><td style="border:1px solid black;">145</td>
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</tr>
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487030"> BBa_K1487030</a></td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P-tcpD gRNA</td><td style="border:1px solid black;">145</td>
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</tr>
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487031"> BBa_K1487031</a></td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P-tcpE gRNA</td><td style="border:1px solid black;">145</td>
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</tr>
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487032"> BBa_K1487032</a></td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P-tcpF gRNA</td><td style="border:1px solid black;">145</td>
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</tr>
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<tr>
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487033"> BBa_K1487033</a></td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P-tcpH gRNA</td><td style="border:1px solid black;">145</td>
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</tr>
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<tr>
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487034"> BBa_K1487034</a></td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P-tcpI gRNA</td><td style="border:1px solid black;">145</td>
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</tr>
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487035"> BBa_K1487035</a></td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P-tcpJ gRNA</td><td style="border:1px solid black;">145</td>
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</tr>
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487036"> BBa_K1487036</a></td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P-tcpP gRNA</td><td style="border:1px solid black;">145</td>
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</tr>
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487037"> BBa_K1487037</a></td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P-tcpQ gRNA</td><td style="border:1px solid black;">143</td>
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</tr>
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487038"> BBa_K1487038</a></td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P-tcpR gRNA</td><td style="border:1px solid black;">145</td>
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</tr>
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<tr>
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487039"> BBa_K1487039</a></td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P-tcpS gRNA</td><td style="border:1px solid black;">145</td>
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</tr>
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487040"> BBa_K1487040</a></td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P-tcpT gRNA</td><td style="border:1px solid black;">145</td>
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</tr>
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487041"> BBa_K1487041</a></td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P-toxT gRNA</td><td style="border:1px solid black;">145</td>
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</tr>
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487063"> BBa_K1487063</a></td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P + t.seq(acfA) - YFP</td><td style="border:1px solid black;">807</td>
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</tr>
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<tr>
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487064"> BBa_K1487064</a></td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P + t.seq(acfB) - YFP</td><td style="border:1px solid black;">807</td>
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</tr>
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<tr>
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487065"> BBa_K1487065</a></td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P + t.seq(acfC) - YFP</td><td style="border:1px solid black;">807</td>
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</tr>
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<tr>
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487066"> BBa_K1487066</a></td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P + t.seq(acfD) - YFP</td><td style="border:1px solid black;">807</td>
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</tr>
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<tr>
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487067"> BBa_K1487067</a></td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P + t.seq(ctxA) - YFP</td><td style="border:1px solid black;">807</td>
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</tr>
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<tr>
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487068"> BBa_K1487068</a></td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P + t.seq(ctxB) - YFP</td><td style="border:1px solid black;">807</td>
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</tr>
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<tr>
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487069"> BBa_K1487069</a></td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P + t.seq(tcpA) - YFP</td><td style="border:1px solid black;">807</td>
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</tr>
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<tr>
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487070"> BBa_K1487070</a></td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P + t.seq(tcpB) - YFP</td><td style="border:1px solid black;">807</td>
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</tr>
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<tr>
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487071"> BBa_K1487071</a></td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P + t.seq(tcpC) - YFP</td><td style="border:1px solid black;">807</td>
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</tr>
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<tr>
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487072"> BBa_K1487072</a></td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P + t.seq(tcpD) - YFP</td><td style="border:1px solid black;">807</td>
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</tr>
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<tr>
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487073"> BBa_K1487073</a></td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P + t.seq(tcpE) - YFP</td><td style="border:1px solid black;">807</td>
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</tr>
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<tr>
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487074"> BBa_K1487074</a></td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P + t.seq(tcpF) - YFP</td><td style="border:1px solid black;">807</td>
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</tr>
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<tr>
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487075"> BBa_K1487075</a></td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P + t.seq(tcpH) - YFP</td><td style="border:1px solid black;">807</td>
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</tr>
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<tr>
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487076"> BBa_K1487076</a></td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P + t.seq(tcpI) - YFP</td><td style="border:1px solid black;">807</td>
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</tr>
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<tr>
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487077"> BBa_K1487077</a></td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P + t.seq(tcpJ) - YFP</td><td style="border:1px solid black;">807</td>
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</tr>
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<tr>
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487078"> BBa_K1487078</a></td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P + t.seq(tcpP) - YFP</td><td style="border:1px solid black;">807</td>
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</tr>
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<tr>
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487079"> BBa_K1487079</a></td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P + t.seq(tcpQ) - YFP</td><td style="border:1px solid black;">805</td>
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</tr>
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<tr>
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487080"> BBa_K1487080</a></td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P + t.seq(tcpR) - YFP</td><td style="border:1px solid black;">807</td>
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</tr>
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<tr>
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487081"> BBa_K1487081</a></td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P + t.seq(tcpS) - YFP</td><td style="border:1px solid black;">807</td>
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</tr>
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<tr>
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487082"> BBa_K1487082</a></td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P + t.seq(tcpT) - YFP</td><td style="border:1px solid black;">807</td>
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</tr>
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<tr>
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<td style="border:1px solid black;"><a href="http://parts.igem.org/Part:BBa_K1487083"> BBa_K1487083</a></td>
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<td style="border:1px solid black;">Composite</td><td style="border:1px solid black;">P + t.seq(toxT) - YFP</td><td style="border:1px solid black;">807</td>
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</tr></tbody></table>
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<br><h2>References</H2><br><p style="display:block">
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1. Sinclair D., Abba K., Zaman K., Qadri F., Graves P.M., Oral vaccines for preventing cholera. Cochrane Database Syst Rev. 2011 Mar 16;(3):CD008603.<br>
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2. Ortiz M.E., Endy D. Engineered cell-cell communication via DNA messaging. J Biol Eng. 2012 Sep 7;6(1):16.</p><br><br>
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Latest revision as of 03:48, 18 October 2014


Parts





Parts List


Name Type Description Length
BBa_K1487021 CompositeP-acfA gRNA145
BBa_K1487022 CompositeP-acfB gRNA145
BBa_K1487023 CompositeP-acfC gRNA145
BBa_K1487024 CompositeP-acfD gRNA145
BBa_K1487025 CompositeP-ctxA gRNA145
BBa_K1487026 CompositeP-ctxB gRNA145
BBa_K1487027 CompositeP-tcpA gRNA145
BBa_K1487028 CompositeP-tcpB gRNA145
BBa_K1487029 CompositeP-tcpC gRNA145
BBa_K1487030 CompositeP-tcpD gRNA145
BBa_K1487031 CompositeP-tcpE gRNA145
BBa_K1487032 CompositeP-tcpF gRNA145
BBa_K1487033 CompositeP-tcpH gRNA145
BBa_K1487034 CompositeP-tcpI gRNA145
BBa_K1487035 CompositeP-tcpJ gRNA145
BBa_K1487036 CompositeP-tcpP gRNA145
BBa_K1487037 CompositeP-tcpQ gRNA143
BBa_K1487038 CompositeP-tcpR gRNA145
BBa_K1487039 CompositeP-tcpS gRNA145
BBa_K1487040 CompositeP-tcpT gRNA145
BBa_K1487041 CompositeP-toxT gRNA145
BBa_K1487063 CompositeP + t.seq(acfA) - YFP807
BBa_K1487064 CompositeP + t.seq(acfB) - YFP807
BBa_K1487065 CompositeP + t.seq(acfC) - YFP807
BBa_K1487066 CompositeP + t.seq(acfD) - YFP807
BBa_K1487067 CompositeP + t.seq(ctxA) - YFP807
BBa_K1487068 CompositeP + t.seq(ctxB) - YFP807
BBa_K1487069 CompositeP + t.seq(tcpA) - YFP807
BBa_K1487070 CompositeP + t.seq(tcpB) - YFP807
BBa_K1487071 CompositeP + t.seq(tcpC) - YFP807
BBa_K1487072 CompositeP + t.seq(tcpD) - YFP807
BBa_K1487073 CompositeP + t.seq(tcpE) - YFP807
BBa_K1487074 CompositeP + t.seq(tcpF) - YFP807
BBa_K1487075 CompositeP + t.seq(tcpH) - YFP807
BBa_K1487076 CompositeP + t.seq(tcpI) - YFP807
BBa_K1487077 CompositeP + t.seq(tcpJ) - YFP807
BBa_K1487078 CompositeP + t.seq(tcpP) - YFP807
BBa_K1487079 CompositeP + t.seq(tcpQ) - YFP805
BBa_K1487080 CompositeP + t.seq(tcpR) - YFP807
BBa_K1487081 CompositeP + t.seq(tcpS) - YFP807
BBa_K1487082 CompositeP + t.seq(tcpT) - YFP807
BBa_K1487083 CompositeP + t.seq(toxT) - YFP807



Retrieved from "http://2014.igem.org/Team:UT-Dallas/temp"