Team:USAFA/Future
So Where Do We Go Now?
While the iGEM competition comes to a close, our project keeps moving forward.
Projet DROP, focussing on identification and obliteration of PFAS, resulted in several interesting research lines to continue research into:
Detection via prmA: We worked on production of our novel PFAS biosensor by expressing codon-optimized mRFP under control of an improved prmA promoter. Improvements were made from the previous year by improving the quality of the DNA to eliminate potential mutations and extending our search up- and downstream of the original sequence to possibly encompass the entire promoter. We did identify a faint presence of mRFP expression, however the signal was too low to be an effective biosensor. We are continuing our work by modeling the promoter -10 and -35 sites in order to place the promoter sequence, RBS, and start codon in the most optimal location to drastically increase translation efficiency. Additionally, we are working to identify and characterize alternative promoter sequences with potentially higher activity or greater specificity for PFAS, which will greatly assist the biosensor research front.
Winogradsky Column, Delftia, & Dehalogenases: From analysis of Winogradsky columns, we initially determined that D. acidovorans was a bacterial species capable of PFAS degradation given the presence of potentially defluorinating enzymes (DeHalogenase type I and II). We are continuing our search into these dehalogenases through techniques such as such as high-purity extraction using 6xHis tagged variants, as well as direct determination of PFAS concentration levels over time by HPLC-MS to better quantify the defluorination reaction. We also plan to monitor degradation products to better understand the mechanisms of both enzymes. If time and resources allow, we also plan to determine true crystal structures of these enzymes for improved modeling of the hydrolysis reaction.
Fungi: Fungi was observed growing in the winogradsky columns, from contaminated soil samples from Schriever AFB. Both fungi were successfully regrown on a plate with PFOS present as the sole carbon resource, suggesting that these fungal species are capable of degradation of PFAS. We have sent off samples for sequencing, and in the future multi-omic analysis will hopefully shed light on their survival strategy in PFOS.
