Skip to main content
. 2020 Jan 29;202(4):e00672-19. doi: 10.1128/JB.00672-19

FIG 2.

FIG 2

In vivo recombinant reporter assay for assessing the MprAB output as a function of increasing concentration of MprA with constant MprB and vice versa. (A) Schematic diagram of tuning and functioning of the MprAB TCS circuit, which is the same as Fig. 1A except the concentration of MprB is controlled by the ara promoter. (B) Design of the synthetic circuit using a four-plasmid reporter system in E. coli, in which the first two plasmids were used for the expression of the Mtb RNAP-σE holoenzyme, a third plasmid pFPV mCherry with the tetRO-inducible mprA genes and the mCherry reporter gene placed under the control of an MprA∼P-inducible ppk1 promoter as in Fig. 1B, and then a fourth plasmid pCDF has the mprB gene inducible by l-arabinose. (C) (Top) Relative fluorescence intensities of mCherry expression with respect to control for 1 OD cells (average from 3 replicates with standard error) were plotted against various concentrations (0.10, 0.31, 0.60, 0.91, 1.05, 1.15, 1.29, 1.40, and 1.56 μg/OD/ml) of MprA (average from 3 replicates) while keeping MprB fixed at 0.14 μg/OD/ml (solid line). In the control, cells had no induction of MprA but had a fixed concentration of MprB (0.14 μg/OD/ml). The assay was further repeated with a higher value of MprB (0.26 μg/OD/ml) (dashed line) and with a lower value of MprB (0.12 μg/OD/ml) (dotted line) (Kruskal-Wallis test; df = 2, P > 0.05). (Bottom) Western blot analysis of the expression of MprA at each point of induction and corresponding protein concentrations. (D, top) Same as panel C, top, but with various concentrations (0.06, 0.12, 0.14, 0.18, 0.24, 0.26, 0.30, 0.33, and 0.38 μg/OD/ml) of MprB (average from 3 replicates), keeping MprA fixed at 0.96 μg/OD/ml (solid line). Control cells had no induction of MprB but had a fixed concentration of 0.96 μg/OD/ml MprA. The assay was further repeated with MprA fixed at a higher value of 1.15 μg/OD/ml (dashed line) and with a lower value of MprA (0.31 μg/OD/ml) (dotted line). Statistical analysis showed that the MprA~P pool remained unchanged (one-way analysis of variance [ANOVA]; Fcritical > F value) with various concentrations of MprB. (Bottom) Same as panel C, bottom, but for MprB.