Tuning Expression Output Using Non-Native Sigma Factors and Variable Length Spacers

Among the primary goals of synthetic biology is to establish a greater control of genetic systems. To be successful, genetic parts must offer predictable and robust control of output expression that is also orthogonal to native metabolic networks. Non-native sigma factors, which initiate the transcription process through interaction of the RNA apoenzyme with their cognate promoter, are an attractive method of control; however, it is still unclear how the recognition of sigma factor binding elements influences transcriptional output, specifically the spacer length between sigma factor binding sites. Our work characterizes the expression of a library of nine synthetic promoters in response to three non-native sigma factors. The designed promoters were derived from the wild type general stress response protein genes from B. subtilis and contained a variable spacer length between 10 to 18 base pairs. By altering the spacer length, we observed a 46-fold variation in expression output for sigma factor B, with the highest fluorescence observed at 13-14 base pairs. We next evaluated the orthogonality of our promoters by testing them with sigma factors F and G from B. subtilis, finding similar distributions with a 38- and 52-fold change respectively. We are currently testing libraries of spacers that naturally respond to sigma factors F and G to assess the universality of our hypothesis. Ultimately, our findings indicate that spacer length is an important factor when tuning expression to desired levels, providing another control point for synthetic biologists.