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Team:Penn State/Bidirectional Promoters Design
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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> | <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> | ||
Revision as of 03:19, 27 October 2012
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
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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