Future Directions

In terms of future directions for the FAS Operon, we would like to have synthesized the remaining individual enzyme gene plasmids and validate the synthesized plasmids by sequencing confirmation like we have done for FabD, FabF, FabG, and FabZ. Once all the individual enzyme gene plasmids have been sequence confirmed, we would like to further validate all of the plasmids via Western Blot. After the two-step validation, we would like to synthesize the complete FAS operon by combining all the individual plasmids. Upon completion of this synthesis, validation in Synechococcus sp. PCC 7002 is the ultimate goal to confirm that this operon would function as we would like it to in our desired organism.

For the Alk Operon, we would like to troubleshoot our Gas Chromatography Mass Spectroscopy methods and results, which will validate the production of alkanes. Further, we would like to transform the full Alk Operon upon its synthesis into Synechococcus sp. PCC 7002, our model organism, to validate its functionality and that would be our remaining ultimate goal.

In the future, we hope to obtain more definitive growth curve data. This would involve more controls to account for E. coli’s slower growth in the presence of two antibiotics. In addition, we are looking to perform directed evolution to create a mutant library. We hope that some of these mutants will produce a channel protein that is an exporter instead of an importer.

Looking forward, we also want to adjust our parB-R and parB-F primer design such that we can create the desired PCR product and synthesize the complete parB plasmid. Ultimately, adding the parB plasmid to the system would eliminate the dependency and the cost of antibiotics as positive selective markers. It would also address concerns raised by some of our advisers concerning genetic drift.