Fig. 2: Design, construction, and optimization of the programmed switch.
From: Reprogramming microbial populations using a programmed lysis system to improve chemical production
a Designing and characterizing the programmed switch. PrpsM and PrpsT are growth phase promoters; Pfic is a stationary phase promoter; RBS is a ribosome binding site; TEVp is tobacco etch virus protease; TVMVp is tobacco vein mottling virus protease; mKate2 is a red fluorescent protein. b Designing of the programmed switch, and characterizing programmed switch by expression unit. c The fluorescence abundance curve of the repression arm was used to characterize the modification effect of different regulation strategies on the programmed switch. (P values = 0.016130; 0.000202; 0.000202; 0.002192; 0.002192; 0.000089; 0.000132; 0.000013; 0.000001; 0.000001; 0.000059; 0.000002.) The increase of fluorescence intensity represented the accumulation of mKate2, and the decrease of fluorescence intensity represented the degradation of mKate2 and indicated repression arm began to take effect (programmed switch was activated). After 24 h, 0.1 mL of 100 mg/mL yeast extract was added into medium every 4 h. All groups were grown at 37 °C and 200 r.p.m. in LB medium for 44 h. The OD600 and fluorescence intensity were measured every 1 h. Statistical significance of switch was determined and was indicated as * for P < 0.05, ** for P < 0.01 and *** for P < 0.001, respectively. d, e Programmed switch inhibited enzyme activity of β-galactosidase. The absorbance at 420 nm was measured after cells were sampled each 6 h and lysed by ultrasound. The left d was the absorbance curve, and the right e was the reaction solution. In strain fic (the control group), TEVp was driven by Pfic. The enzyme activities were measured every 2 h. Values are shown as mean ± s.d. from three (n = 3) biological replicates. Two-tailed t tests were used to determine statistical significance. Source data are provided as a Source Data file.
