Team:Penn State/Bidirectional Promoters Design

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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.