Team:Penn State/MSC Design

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     <a class="activemaintab" href="href="http://2012.igem.org/Team:Penn_State/MSC_Multiple_Start_Codons">Overview</a>
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     <a class="maintab" href="http://2012.igem.org/Team:Penn_State/Multiple_Start_Codons">Overview</a>
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<h3>Background</h3>
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<h3>Circuit Model</h3><p><img src="http://2012.igem.org/wiki/images/7/76/CodOpt_design.png"></P>
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    <p>mRNA is the molecule that carries information about the sequence of amino acids in a protein. However, much like the lines on a sheet of paper, the protein coding sequence of an mRNA molecule does not start right at the beginning, or top of the page. Instead, once the mRNA is bound by a ribosome, a start codon must first be read before the protein can be translated. This start codon is generally AUG, or Methionine.
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<h3>Circuit Design</h3>
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</p>
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<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>
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<h3>The Problem</h3>
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<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>
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<p>Once this start codon in read the ribosome will continue reading and building the polypeptide (protein) until a stop codon is reached. But what happens if you have two AUG codons close together? That is the question we are attempting to answer.</p>  
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<h3>Circuit Construction</h3>
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<p>We are trying to understand what happens when there are two start codons very close together, but out of frame. Out of frame refers to how the ribosome reads the mRNA. Remember those codons and how they are groups of three bases on the mRNA? The reading frame refers to which group of three. If you start at one base and read the bases in groups of three from that point on, that is one frame of reference. If you then move your start point ahead one base, then you are reading in a new reading frame. If you advance you starting point one more base, that is the third reading frame. If you advance it again you are now back in your first reading frame, but you have skipped the first codon. We are looking into what happens when you have multiple start codons close together, but in different reading frames. Which frame will be preferred?</p>
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<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>  
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<h3>The Objective</h3>
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<p>This construct aims to test the possibility of multiple start codons on a single mRNA strand. A plasmid has been engineered with two start codons slightly out of frame from each other, but close enough to test start codon slippage. Testing the fluorescence of E. coli carrying the plasmid will determine the rate of codon slippage</p>
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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.