Team:Penn State/Bidirectional Promoters Design

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     <h2>Presentation</h2>
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     <h3>Bidirectional Promoters</h3>
     <p>Scientists are frequently confounded by wayward promoters; that is, promoters which do not produce  
     <p>Scientists are frequently confounded by wayward promoters; that is, promoters which do not produce  
the expected proteins. Some bidirectional promoters are known to exist, but which way they promote  
the expected proteins. Some bidirectional promoters are known to exist, but which way they promote  
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     <h2>Bidirectional Promoters</h2>
     <h2>Bidirectional Promoters</h2>
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     <h2 class="hide">Sample navigation menu:</h2>
     <h2 class="hide">Sample navigation menu:</h2>
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     <a class="activemaintab" href="https://2012.igem.org/Team:Penn_State/Bidirectional_Promoters_Overview">Overview</a>
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     <a class="maintab" href="https://2012.igem.org/Team:Penn_State/Bidirectional_Promoters">Overview</a>
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     <span class="hide"> | </span> <a class="maintab" href="https://2012.igem.org/Team:Penn_State/Bidirectional_Promoters_Design">Design</a>
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     <span class="hide"> | </span> <a class="activemaintab" href="https://2012.igem.org/Team:Penn_State/Bidirectional_Promoters_Design">Design</a>
     <span class="hide"> | </span> <a class="maintab" href="https://2012.igem.org/Team:Penn_State/Bidirectional_Promoters_Results">Results</a>
     <span class="hide"> | </span> <a class="maintab" href="https://2012.igem.org/Team:Penn_State/Bidirectional_Promoters_Results">Results</a>
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<h3>Circuit Model</h3><p><img src="https://static.igem.org/mediawiki/2012/6/65/BiDir_design.png"></p>
<h3>Initial Design</h3>
<h3>Initial Design</h3>
<p>This construct tests the directionality of selected promoters. A small non-coding sequence lies between two restriction enzyme sites; this is the area where each promoter will be inserted. Upstream from the promoter sequence lies a RFP reporter. Downstream codes for a GFP reporter. Thus, as promoters are ligated into the promoter placeholder, the cell fluoresces red, green, or both, indicating the promoter directionality.</p>
<p>This construct tests the directionality of selected promoters. A small non-coding sequence lies between two restriction enzyme sites; this is the area where each promoter will be inserted. Upstream from the promoter sequence lies a RFP reporter. Downstream codes for a GFP reporter. Thus, as promoters are ligated into the promoter placeholder, the cell fluoresces red, green, or both, indicating the promoter directionality.</p>
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<p>The initial construct for this project utilized the reporters of non-BioBrick parts available in a neighboring campus lab. The Salis Lab generously allowed us to use samples of the pBAC plasmid and mFTV3 plasmid to assemble the construct. </p>
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<h3>Sources</h3>
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<p>The initial construct for this project utilized the reporters of non-BioBrick parts available in a neighboring campus lab. The Salis Lab generously allowed us to use samples of the pBAC plasmid and mFTV3 plasmid to assemble the construct.</p>
 +
<h3>Assembly</h3>
<p>The pBAC vector was PCR amplified with 40 basepair overlaps complementary to the ends of the reporter inserts. A RFP reporter and GFP reporter were simultaneously PCR amplified from the mFTV3 plasmid, both containing a 40 basepair overlap sequence complementary to the ends of the adjacent piece.  
<p>The pBAC vector was PCR amplified with 40 basepair overlaps complementary to the ends of the reporter inserts. A RFP reporter and GFP reporter were simultaneously PCR amplified from the mFTV3 plasmid, both containing a 40 basepair overlap sequence complementary to the ends of the adjacent piece.  
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<p>These parts were finally assembled using the CBAR, or Gibson Assembly, reaction. Gibson Assembly utilizes three enzymes.  
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<p>These parts were finally assembled using the CBAR, or Gibson Assembly, reaction. Gibson Assembly utilizes three enzymes.</p>
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<ol>
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<li> An exonuclease, which chews back the overlapping sequences of each piece from the 5' to 3' ends</li>
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<li><p>An exonuclease, which chews back the overlapping sequences of each piece from the 5' to 3' ends</p></li>
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<li> A polymerase, which fills in the gaps left in the DNA from the exonuclease</li>
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<li><p>A polymerase, which fills in the gaps left in the DNA from the exonuclease</p></li>
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<li> A ligase, which heals the nicks in the annealed DNA strand</li>
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<li><p>A ligase, which heals the nicks in the annealed DNA strand</p></li>
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</ol></p>
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</ul>
<p>Annealing the pieces together provided a construct devoid of an actual promoter sequence. By digesting the construct with restriction enzymes flanking the promoter placeholder and inserting promoters isolated from BioBrick parts, the construct fulfilled its purpose.</p>  
<p>Annealing the pieces together provided a construct devoid of an actual promoter sequence. By digesting the construct with restriction enzymes flanking the promoter placeholder and inserting promoters isolated from BioBrick parts, the construct fulfilled its purpose.</p>  
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     <h3>Projects</h3>
     <h3>Projects</h3>
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       <li><a href="https://2012.igem.org/Team:Penn_State/MSC_Overview">Multiple Start Codons</a></li>
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       <li><a href="https://2012.igem.org/Team:Penn_State/Multiple_Start_Codons">Multiple Start Codons</a></li>
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       <li><a href="https://2012.igem.org/Team:Penn_State/Bidirectional_Promoters_Overview">Bidirectional Promoters</a></li>
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       <li><a href="https://2012.igem.org/Team:Penn_State/Bidirectional_Promoters">Bidirectional Promoters</a></li>
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       <li><a href="https://2012.igem.org/Team:Penn_State/Codon_Optimization_Overview">Codon Optimization</a></li>
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       <li><a href="https://2012.igem.org/Team:Penn_State/Codon_Optimization">Codon Optimization</a></li>
     </ul>
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Latest revision as of 03:19, 27 October 2012

Bidirectional Promoters Overview

Bidirectional Promoters

Scientists are frequently confounded by wayward promoters; that is, promoters which do not produce the expected proteins. Some bidirectional promoters are known to exist, but which way they promote and the degree of expression has not been quantified. This project will test the directionality of several BioBrick promoters to answer these questions.

Bidirectional Promoters

Sample navigation menu:

Overview | Design | Results

Circuit Model

Initial Design

This construct tests the directionality of selected promoters. A small non-coding sequence lies between two restriction enzyme sites; this is the area where each promoter will be inserted. Upstream from the promoter sequence lies a RFP reporter. Downstream codes for a GFP reporter. Thus, as promoters are ligated into the promoter placeholder, the cell fluoresces red, green, or both, indicating the promoter directionality.

Sources

The initial construct for this project utilized the reporters of non-BioBrick parts available in a neighboring campus lab. The Salis Lab generously allowed us to use samples of the pBAC plasmid and mFTV3 plasmid to assemble the construct.

Assembly

The pBAC vector was PCR amplified with 40 basepair overlaps complementary to the ends of the reporter inserts. A RFP reporter and GFP reporter were simultaneously PCR amplified from the mFTV3 plasmid, both containing a 40 basepair overlap sequence complementary to the ends of the adjacent piece.

These parts were finally assembled using the CBAR, or Gibson Assembly, reaction. Gibson Assembly utilizes three enzymes.

  • An exonuclease, which chews back the overlapping sequences of each piece from the 5' to 3' ends

  • A polymerase, which fills in the gaps left in the DNA from the exonuclease

  • A ligase, which heals the nicks in the annealed DNA strand

Annealing the pieces together provided a construct devoid of an actual promoter sequence. By digesting the construct with restriction enzymes flanking the promoter placeholder and inserting promoters isolated from BioBrick parts, the construct fulfilled its purpose.

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