Fig. 3

Debugging and optimization of the light-sensing module. a Annotated sequence of the legacy xylose-inducible promoter P_xylA in LSM v0.1, obtained from integration plasmid pAX01^18,81. The −35, −10, and +1 sites⁸² and xylR operators xylO_L and xylO_R⁸³ have been previously identified. The operators are followed by an unwanted untranslated region including the xylA RBS, and a vestigial truncated xylA ORF (xylA’). A second, truncated xylA RBS (xylA^†) is present in pAX01 to enable translation of a gene of interest placed downstream. Brackets indicate the end of the legacy P_xylA promoter, as well as sequential truncations P_xylA(+66) and P_xylA(+47), where problematic parts of P_xylA are eliminated. The start codon of ccaS is shown at the end in bold. b Measurement and optimization of ccaS expression. We first inserted ccaS-sfgfp as in (a). Next, we truncated P_xylA to remove the vestigial elements (resulting in P_xylA(+66)) and placed ccaS-sfgfp directly after the xylA RBS. Then, we switched the xylA RBS with synthetic RBS MF001 (see “Methods” section) and recoded the initial 15 codons of ccaS. Finally, we replaced the vestigial antisense promoter P3 with synthetic terminators L3S1P52 and L3S2P56⁸⁴, shortened the P_xylA(+66) 5’UTR (resulting in P_xylA(+47)), and moved the entire cassette to the xylA chromosomal locus. c Expression of CcaS-sfGFP from each engineered module shown in (b). Bars show the mean of three experiments run on separate days. Dots show values of individual experimental replicates. N.D.: not detected (see “Methods” section). Source data is available in the Source Data file