Supplementary Figure 8: Mutant cysK allele retention and plasmid copy number determination.
From: Custom selenoprotein production enabled by laboratory evolution of recoded bacterial strains
Analysis of mutant cysK allele frequency by qPCR in cell populations passaged for 125 generations in the presence of 10 μM Na2SeO3. At generation 0 (purple bars), cell populations contained 50% wild-type cysK and 50% mutant cysK. Bars represent the mean ± S.E.M. (a) Detection of wild-type cysK (T69) in WT:T69I populations ± Na2SeO3. Wild-type cysK is detected at a similar frequency in both the presence and absence of Na2SeO3. (b) In contrast, mutant T69I undetectable (Cq = ~ 35) after 125 generations in all populations. (c) Detection of wild-type cysK (T73) in WT:T73A populations does not change ± Na2SeO3. (d) The cysK T73A mutant is lost from the populations when cultured in the absence of Na2SeO3 (light blue). In contrast, in the presence of Na2SeO3 (dark blue) the mutant allele is retained. (e) Detection of wild-type cysK (H153) in WT:H153Y populations is not affected by Na2SeO3 treatment. (f) Similar to T73A, the H135Y mutant is undetectable after serial passage in media without selenite (light blue), but is partially retained when selenite is present (dark blue). Abundance of pCDF (g) and pRSF (h) plasmids in RTΔA cells with mutations in poly(A) polymerase I (pcnB) and DNA polymerase I (polA). All mutations significantly decreased plasmid abundance compared to wild-type cells. Bars represent the mean ± S.E.M.
