Team:Penn State/Bidirectional Promoters Design

From 2012.igem.org

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     <h2 class="hide">Sample navigation menu:</h2>
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     <a class="maintab" href="https://2012.igem.org/Team:Penn_State/Bidirectional_Promoters_Overview">Overview</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>
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     <span class="hide"> | </span> <a class="maintab" 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>
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<h3>Background</h3>
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<h3>Initial Design</h3>
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    <p>All of the proteins around us, with few exceptions, are made up of 20 fundamental building blocks of life - amino acids. Different arrangements and combinations of these basic building blocks give us the diversity of proteins that we see. Messenger RiboNucleic Acids (mRNA) is in essence a "photocopy" of DNA that codes for a gene. mRNA carry codons, which are groups of three bases that code for a single amino acid. There are 64 possible combinations of codons (4 x 4 x 4 = 64), but these combinations are degenerate, so there can be more than one codon that codes for a single amino acid.  
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<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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<h3>The Problem</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>
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<p>Proteins are assembled by ribosomes which read the mRNA and catalyze the binding between Amino Acids; however, they cannot read the codon code themselves and require the help of tRNA (transfer RNA). Each tRNA matches a single codon sequence and can only carry a single specific amino acid at a time. tRNA has complimentary bases to their respective codon sequences on the mRNA strand, and once the tRNA matches the sequence on the mRNA, the tRNA deposits the Amino Acids, which then bind together to form proteins.  
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<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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We have mentioned how these can be a number of codons that can code for the same Amino Acid, and subsequently, a number of tRNA molecules that can carry a given Amino Acid. In nature, and in many organisms, only a select few of these tRNA and codon combinations are used instead of all sequences that code for the same amino acid. This is called codon bias.
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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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<h3>Objective</h3>
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<li> A polymerase, which fills in the gaps left in the DNA from the exonuclease</li>
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<p>Our goal for this project is to see which codons are preferred, or biased. We also want to investigate if this bias can change over time, different circumstances, or stresses.  
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<li> A ligase, which heals the nicks in the annealed DNA strand</li>
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<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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Revision as of 04:15, 3 October 2012

Bidirectional Promoters Overview

Presentation

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

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.

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.

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.

  1. An exonuclease, which chews back the overlapping sequences of each piece from the 5' to 3' ends
  2. A polymerase, which fills in the gaps left in the DNA from the exonuclease
  3. 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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