Team:Penn State/MSC Design

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     <span class="hide"> | </span> <a class="navitab" href="#http://2012.igem.org/Team:Penn_State/Modeling">Modeling</a>
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     <span class="hide"> | </span> <a class="navitab" href="http://2012.igem.org/Team:Penn_State/Modeling">Main Results</a>
     <span class="hide"> | </span> <a class="navitab" href="http://2012.igem.org/Team:Penn_State/Notebook">Notebook</a>
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   <div id="desc">
   <div id="desc">
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     <h2>Multiple Start Codons</h2>
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     <h3>Multiple Start Codons</h3>
 +
    </br>
     <p>A frustrating yet commonly observed phenomenon in the lab is the production of unexpected proteins. These occurrences may be explainable by multiple start codons in the mRNA strand. Codon slippage is a theory practically untouched by research, and this project aspires to shed some light on the issue.</p>
     <p>A frustrating yet commonly observed phenomenon in the lab is the production of unexpected proteins. These occurrences may be explainable by multiple start codons in the mRNA strand. Codon slippage is a theory practically untouched by research, and this project aspires to shed some light on the issue.</p>
   </div>
   </div>
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   <div id="main">
   <div id="main">
     <h2>Multiple Start Codons</h2>
     <h2>Multiple Start Codons</h2>
 +
<div id="prezi"><iframe width="560" height="315" src="http://www.youtube.com/embed/WTr8n0TCu6s?rel=0" frameborder="0" allowfullscreen></iframe></div>
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     <a class="maintab" href="http://2012.igem.org/Team:Penn_State/Multiple_Start_Codons">Overview</a>
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     <a class="maintab" href="http://2012.igem.org/Team:Penn_State/MultipleStartCodons">Overview</a>
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     <span class="hide"> | </span> <a class="activemaintab" href="http://2012.igem.org/Team:Penn_State/MSC_Design">Design</a>
     <span class="hide"> | </span> <a class="activemaintab" href="http://2012.igem.org/Team:Penn_State/MSC_Design">Design</a>
     <span class="hide"> | </span> <a class="maintab" href="http://2012.igem.org/Team:Penn_State/MSC_Results">Results</a>
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<h3>Initial Construct</h3>
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<h3>Circuit Model</h3><p><img src="http://2012.igem.org/wiki/images/7/76/CodOpt_design.png"></P>
-
    <p>The initial construct for this project was designed using a basic vector and a relatively new product available from IDT (Integrated DNA Technologies), called gBlocks. gBlocks are synthesized dsDNA oligos with adjacent 40 base-pair overlap sequences. These oligos are particularly useful in multi-part constructions within a CBAR or Gibson Assembly reaction protocol.  
+
<h3>Circuit Design</h3>
-
</p>
+
<p>To begin construction of this plasmid, the DNA sequence was generated and edited in ApE, a DNA editing software available through the University of Utah, from several existing plasmid sequences available through other labs on the Penn State campus. The construct was rationally designed to include two start codons within 7 nucleotides of each other, and thus in different open reading frames. <p>    Each codon, labeled RFP-coding ATG and sfGFP-coding ATG for simplicity, codes for a different reporter in the respective reading frame of each start codon. The first start codon, RFP-coding ATG, if recognized by the ribosome, will translate the reporter RFP. The second start codon, sfGFP-codon ATG, will translate the reporter sfGFP if recognized by the ribosome. Super-folder GFP (sfGFP) is used to allow for the nonsense codons of the out-of-frame RFP sequence preceding the sfGFP reporter which would inevitably be translated prior to the green reporter sequence should the ribosome recognize the sfGFP-coding ATG. The super-folder protein will fold regardless of this nonsense sequence, facilitating the measurement of codon slippage through the measurement of fluorescence</p>
-
<h3>Assembly</h3>
+
<p>To prevent premature termination of the translation of either reporter, all stop codons in the respective reading frames of each codon were eliminated through amino acid sequence substitution.</p>
-
<p>The oligos were ordered based on a digital representation of the desired plasmid sequence. The DNA sequence was manipulated through the free software ApE (A plasmid Editor) available through the University of Utah</p>  
+
<h3>Circuit Construction</h3>
-
<p>Initial construction proved difficult. Whether this was due to inexperience or poor design, we cannot be sure. The final construct displaying a correct sequence has been assembled, however, and is giving us new insight into the codon slippage pheomenon.</p>
+
<p>Initially, 4 gBlocks, available through IDT as oligos with overlapping 40bp sequences complimentary to the adjacent oligo, were used to perform a Gibson Assembly reaction with a digested dRBS1 vector. After several failed attempts, a second approach was implemented. Oligos for the missing circuit sequence were designed and annealed, according to their complimentary 20bp overlaps, and inserted into the nearly-complete vector using Gibson Assembly. This attempt was successful, and the initial construct was verified with sequencing.</P>  
   </div>
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       <li><a href="http://2012.igem.org/Team:Penn_State/Human_Practices">Human Practices</a></li>
       <li><a href="http://2012.igem.org/Team:Penn_State/Human_Practices">Human Practices</a></li>
     </ul>
     </ul>
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    <h3>Picture Gallery</h3>
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    <img class="photo" src="../images/test.jpg" height="100" width="130" alt="" />
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     <h3>Projects</h3>
     <h3>Projects</h3>
     <ul>
     <ul>

Latest revision as of 03:17, 27 October 2012

Multiple Start Codons Overview

Multiple Start Codons


A frustrating yet commonly observed phenomenon in the lab is the production of unexpected proteins. These occurrences may be explainable by multiple start codons in the mRNA strand. Codon slippage is a theory practically untouched by research, and this project aspires to shed some light on the issue.

Multiple Start Codons

Circuit Model

Circuit Design

To begin construction of this plasmid, the DNA sequence was generated and edited in ApE, a DNA editing software available through the University of Utah, from several existing plasmid sequences available through other labs on the Penn State campus. The construct was rationally designed to include two start codons within 7 nucleotides of each other, and thus in different open reading frames.

Each codon, labeled RFP-coding ATG and sfGFP-coding ATG for simplicity, codes for a different reporter in the respective reading frame of each start codon. The first start codon, RFP-coding ATG, if recognized by the ribosome, will translate the reporter RFP. The second start codon, sfGFP-codon ATG, will translate the reporter sfGFP if recognized by the ribosome. Super-folder GFP (sfGFP) is used to allow for the nonsense codons of the out-of-frame RFP sequence preceding the sfGFP reporter which would inevitably be translated prior to the green reporter sequence should the ribosome recognize the sfGFP-coding ATG. The super-folder protein will fold regardless of this nonsense sequence, facilitating the measurement of codon slippage through the measurement of fluorescence

To prevent premature termination of the translation of either reporter, all stop codons in the respective reading frames of each codon were eliminated through amino acid sequence substitution.

Circuit Construction

Initially, 4 gBlocks, available through IDT as oligos with overlapping 40bp sequences complimentary to the adjacent oligo, were used to perform a Gibson Assembly reaction with a digested dRBS1 vector. After several failed attempts, a second approach was implemented. Oligos for the missing circuit sequence were designed and annealed, according to their complimentary 20bp overlaps, and inserted into the nearly-complete vector using Gibson Assembly. This attempt was successful, and the initial construct was verified with sequencing.