Figure 4.

Engineering of transcription using tetA^OPT. (a) Illustration of how a short constitutive promoter (J23100) and a TIR sequence library were introduced in front of the native E. coli gene lacZ. (b) Growth in M9 medium supplemented with 0.05% glucose and 0.5% lactose. (c) Illustration of YidC-GFP expression using the T7 system. The T7 RNA polymerase (T7 RNAP) is part of the DE3 element on the E. coli chromosome. T7 RNAP is expressed from the lacUV5 promoter, regulated by LacI and therefore inducible by IPTG. T7 RNAP drives expression of YidC-GFP from a T7 promoter on a pET vector. Cells that successfully express YidC will be green fluorescent. (d) Illustration of the exchange of the lacUV5 promoter with the tunable promoter PrhaBAD with a randomized TIR. (e) Absolute production of YidC-GFP (nmol FITC) and growth of associated strains (OD₆₀₀) were measured for 20 h. In b + e, growth of the control strains with the native promoter (blue), the new promoter and the native TIR (dark blue), and the negative control (red) was measured in triplicates. Measurements of the TIR library variants (gray) are based on single measurements. The fluorescein standard curve used for fluorescence normalization can be found in Figure S6. In a + d, gray triangles depict recombination sites.