Compatibility of lig-sulfur with genetic techniques and engineered strains
We evaluated the effect of selective pressure on strain isolation and maintenance as an application of the media formulated with lig-sulfur. Under a mock selection scheme where AF-GFP cells, which harbor the pJRD plasmid expressing codon-optimized green fluorescent protein (GFP) under the constitutive tac promoter, were diluted hundred-fold with wild-type (WT) cells simulating the loss of the IncQ plasmid. Negligible enrichment of GFP fluorescence was observed over three passages in AFM1 media compared to pure AF-GFP even when kanamycin was present, confirming that iron accelerates the decomposition of aminoglycosides at low pH (Fig. 4A, 4B). When the energy source was switched from ferrous iron to lig-sulfur at pH 1.8, an increase in GFP fluorescence was observed over three passages, suggesting that effective selective pressure by the antibiotic was applied over time (Fig. 4C). By increasing this sulfur-based media to an initial pH of 5.0, the kanamycin was more effective for selection. Increases in fluorescence, similar in level to that of pure AF-GFP, were seen after the first passage (Fig. 4D). Moreover, our data shows that pure AF-GFP cells grown in LSM4 at either pH value had a higher average median fluorescence intensity (MFI) than those grown in AFM1(K) (Fig. 5).