Part:BBa_K2911000:Experience

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Improvement of prma promoter; USAFA 2020

We transformed the composite part BBa_K3347018, containing an 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.

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