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. Author manuscript; available in PMC: 2018 Jun 8.
Published in final edited form as: Gene. 2012 Nov 29;514(2):82–90. doi: 10.1016/j.gene.2012.11.011

Figure 4. For strong promoters, TCDS in E. coli topA strain VS111 did not require the expression of a membrane insertion protein.

Figure 4

The in vivo T-S assays for plasmids carrying a GFPUV gene under the control of IPTG-inducible promoters with different strengths were performed as described under Experimental Procedures. DNA topoisomers were resolved by electrophoresis in a 1% agarose gel containing 2.5 µg/ml chloroquine and stained with ethidium bromide. (A) and (C) The time course of the hypernegative supercoiling of plasmid pZXD57 carrying a GFPUV gene under the control of the strong PT7A1/O4 promoter in E. coli strain VS111 after 1 mM of IPTG induction. Lanes 1–4 contained, respectively, DNA samples isolated from VS111 after 0, 5, 10, and 15 min of IPTG induction. (B) and (D) Dependence of TCDS on promoter strength. Lanes 1–5 contained, respectively, DNA samples isolated from E. coli topA strain VS111 containing plasmids pZXD57, 58, 56, 55, and 54, that carry a cytosolic GFPuv gene under the control of IPTG-inducible promoters with different strengths, i.e., promoters PT7A1/O4, Ptac, PlacUV5, Plac, and PlacL8 after 5 min of IPTG (1 mM) induction. Labels: PR, promoter; T7A1, the T7A1/O4 promoter; tac, the tac promoter; lacUV5, the lacUV5 promoter; lac, the lac promoter; lacL8, the lacL8 promoter. (C) The percentage of hypernegatively supercoiled DNA for pZXD57 is a function of IPTG induction time. These results were calculated as described under Experimental Procedures using the TCDS data shown in (A). (D) The percentage of hypernegatively supercoiled DNA is proportional to promoter strength (the values are the average of at least three independent determinations and the standard deviations are shown). These results were calculated as described under Experimental Procedures using the TCDS data shown in (B). The promoter strength in Pbla units was obtained from ref. (Lanzer and Bujard 1988).