Sigma Factor-Controlled Gene Expression in E. coli Using Different Spacer Lengths

In synthetic biology, genetic circuits are desired to precisely control output expression while being orthogonal to native transcription. Non-native sigma factors, which initiate the transcription process through interaction of the RNA apoenzyme with a promoter, can be used to drive expression of heterologous genes while not influencing innate pathways. Sigma factors interact with the DNA upstream of a gene to be transcribed at the -35 and -10 binding regions. Spacer length and identity between sigma factor binding sites are known to affect transcription, but it is unclear how much either contributes to the level of transcriptional output. To decouple these, designed promoter regions recognized by sigma factor B (SigB) from Bacillus subtilis were created with spacer lengths ranging from 10 to 18 base pairs (bp), with the native promoter having a length of 12 bp. The designed promoters were located upstream of a gene encoding green fluorescent protein (gfp), which was used as a reporter of expression. We found that a 14 bp spacer had the highest fluorescence which matched the native distribution of the promoter. Libraries were created with random spacer identities were built to determine the contribution of spacer length and identity. Similar to the designed spacers, we found high fluorescence for 14 bp, medium for 11bp, and low for 17 bp libraries. We are currently working on designing a 15 bp spacer to be orthogonal to native sigma factors. The learning developed through this work will aid our understanding of bacterial transcription and design of gene circuits.