Team:Harvard/Results
Results
Purpose
In order to show that optimizing the stoichiometry of the curli pathway can affect curli production, we attempt to demonstrate that variations in the RBS strength of csgG can impact levels of curli production. This would give a proof-of-concept foundation for the idea that curli production can be optimized by regulating expression strengths of the pathway proteins.
Generating RBS Variation
We employed a simple screening process in order to identify and select RBSs of varying expression strengths. Using the Salis Lab RBS generator, we identified a degenerate RBS sequence with a wide range of expression strengths based on whether each degenerate base crystallized to adenine, guanine, cytosine, or thymine. We sent that degenerate sequence out for synthesis and got back our library of interest. Then, we used Gibson Assembly to position the RBSs in front of the csgG gene within the pBbB8K-CsgBACEFG plasmid used by the Joshi Lab, which was originally the consolidated curli operons with the wild-type RBSs for each protein under an arabinose promoter. The Gibson Assembly produced a library of plasmids which were identical except for the csgG modified RBS.
