Extended Data Fig. 4: Additional persistence test results.
From: Population-level amplification of gene regulation by programmable gene transfer
(A) Extended data from Fig. 3d, including showing two additional samples. The first four samples are identical to the those shown in Fig. 3d, with data from two more samples (Msp2 and Msp3) added. All samples are MG1655 + FHR cells carrying pCas9 and one of six pTargets: (1) pTarget encoding a targeting spacer (sp) that perfectly matches the target region but does not encode oriT; (2) pTarget carrying sp and oriT; (3) pTarget carrying a non-targeting spacer (NT) and oriT; (4) – (6): pTarget carrying sp with 1 or 2 base pair mismatches and oriT. The mismatched spacers are the same as those used in the first three panels of Fig. 3c, containing different sets of mismatches (see Supplementary Table 3 for sequences). Cells carrying pCas9 and one of the six pTargets were cultured in LB with 25 µg/mL Cm and diluted 1/1000 every 24 hours for 6 days. The fraction of cells carrying pTarget (Fp) were measured daily by plating on LB agar with 25 µg/mL Cm or 25 µg/mL Cm + 50 µg/mL Kan for CFU counting. Plasmid abundance (Fp) was calculated as: \({Fp}=\scriptstyle\frac{\frac{{CFU}}{{ml}}\text{counted from LB}+\text{Cm}+\text{Kan plates}}{\frac{{CFU}}{{ml}}\text{counted from LB}+\text{Cm plates}}\). The data shows that not all mismatches ensure plasmid persistence. The line plot represents the mean of four technical replicates. (B) All six maintained FHR after 6 days of passaging. The gray bar represents the mean of four technical replicates. Using Day 6 cultures, we measured the fraction of cells carrying FHR on blank LB agar plates and LB plates supplemented with 10 µg/mL tetracycline, which selects for FHR. Despite variability among replicates, nearly 100 % of samples maintained FHR.
