Team:USAFA/Contribution

Template:Usafa

>

Contribution

The 2019 USAFA iGEM team developed a plasmid containing a prmA promoter (BBa_K2911000) directly upstream of the mRFP gene(BBa_E1010). The objective of this recombinant plasmid was to express RFP (BBa_E1010) in Rhodococcus bacteria that are exposed to PFAS. The prmA promoter (BBa_K2911000) was selected and engineered because it was previously reported to increase expression three-fold in the presence of PFAS (Weathers et al). The data from the 2019 USAFA team showed no visual expression of functional fluorescent RFP in Rhodococcus, but an increase in gene expression was detected by qRTPCR. To improve the prmA promoter (BBa_K2911000), there were three approaches:

  1. The first was to add more base pairs upstream of the prmA promoter (BBa_K2911000) to identify if a larger sequence of DNA was required to function correctly.
  2. The second approach was to fix any potential mutations within the prmA promoter (BBa_K2911000). A new prmA promoter was ordered and checked for mutations before testing.
  3. The last approach was to codon optimize mRFP (BBa_E1010) for Rhodococcus to provide maximum affinity between the promoter and RFP. There is a possibility that the prmA promoter will not function in Rhodococcus, as it has several differences from the microorganism it was discovered in. Therefore, if the new prmA promoter does not express RFP, there are several other promoters to replace prmA.

References: Weathers, T. S., Higgins, C. P., & Sharp, J. O. (2015). Enhanced biofilm production by a toluene-degrading Rhodococcus observed after exposure to perfluoroalkyl acids. Environmental science & technology, 49(9), 5458–5466. https://doi.org/10.1021/es5060034

Improvement of an Existing Part

To improve on the 2019 USAFA iGEM team's work, this year, we improved BBa_K2911000 which was a prmA promoter and improved it into part BBa_K3347017 which is an improved version of that promoter.



Analysis of Part Functionality

We transformed the composite part BBa_K3347018, containing the improved prma promoter and the codon optimized mRFP into pSRKBB (KanR) and expressed into Rhodococcus jostii RHA1. Three successful colonies were selected and grown in tryptic soy broth with kanamycin and PFOA for 24 hrs before being pelleted and resuspended in PBS and analyzed for mRFP content by UV-Vis spectroscopy. Results are shown below in the figure below. From this information, we can see a very faint signal of mRFP expression. Usually this reporter is expressed strongly, however in the previous year, no expression was seen due to several hypotheses. By correcting the promoter site and codon optimizing mRFP for expression in the RHA1 chassis, we successfully show significant improvement by the presence of any expression at all. We suspect that the promoter-RBS-start codon distances are not effectively optimized, resulting in significant loss in transcription efficiency and very low expression of the reporter.



Figure: Analysis of mRFP content in BBa_K3347018 expressing clones induced with 200 µM PFOA. Absorbance of cell cultures taken at 584 nm and subtracted from absorbance at 600 nm. Background absorbance from matrix and non-induced samples also removed.