Journal: bioRxiv

Article Title: Synthetic Biology Tool Development Advances Predictable Gene Expression in the Metabolically Versatile Soil Bacterium Rhodopseudomonas palustris

doi: 10.1101/2021.11.01.466785

Figure Lengend Snippet: pRPA as a heterologous gene expression vector. A) Average pRPA copy number for wild type R. palustris grown aerobically in the dark and anaerobically in the light (100 μE white light) in duplicate. Copies of the plasmid’s replication protein gene (TX73_RS24750) was compared to the single copy dxs gene in the chromosome by qPCR and an external standard curve . Error bars indicate one standard deviation. B) Schematic of the P Lac -mrfp expression cassette and a gentamicin selection marker integrated into one of three locations, TX73_RS25310, TX73_RS24785, TX73_RS24780, in R. palustris ’ endogenous plasmid, pRPA, by double homologous recombination. C) Fluorescent fold change average of R. palustris pRPA 25310 mRFP Gent, pRPA 24785 mRFP Gent, and pRPA 24780 Gent strains grown in triplicate either without any antibiotic or with 0.3 mg/mL gentamicin sulfate. The data is representative of the first test with new cultures as well as the test after three rounds of dilution and regrowth . Error bars indicate one standard deviation. D) Comparison of the percent complete mutants (those that do not produce an amplicon from PCR with primers binding to the DNA that is removed and also produce the correct sequence from PCR with primers outside of the homology arms) for the three pRPA locations. E) Representative flow cytometry results of mRFP fluorescence from R. palustris wild type, BBR1 gent mRFP, pRPA 25310 mRFP Gent, pRPA 24785 mRFP Gent, and pRPA 24780 Gent strains grown in triplicate. The growth procedure, media, and antibiotics are the same as for the fluorescent fold change test in . F) Average percent non-fluorescent cells for R. palustris strains grown in triplicate as determined by flow cytometry . Error bars indicate one standard deviation.

Article Snippet: qPCR with gDNA was used to determine the absolute plasmid copy number ratio of both native and non-native plasmids relative to the chromosome as described previously ( ). gDNA was extracted from R. palustris cultures at mid-exponential phase in duplicate using the Monarch® Genomic DNA Purification Kit (New England Biolabs® Inc.).

Techniques: Expressing, Plasmid Preparation, Standard Deviation, Selection, Marker, Homologous Recombination, Amplification, Binding Assay, Sequencing, Flow Cytometry, Fluorescence