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Transcriptional repressors provide widespread biological significance in the regulation of gene expression. However, in prokaryotes, it is particularly difficult to find transcriptional repressors that recognize specific target promoters on genome-scale. To address this need, a genetic biosensor for identifying repressors of target promoters was developed in  Escherichia coli  from a  de novo  designed genetic circuit. This circuit can convert the negative input of repressors into positive output of reporters, thereby facilitating the selection and identification of repressors. After evaluating the sensitivity and bias, the biosensor was used to identify the repressors of  scbA  and  aco  promoters (P scbA  and P aco ), which control the transcription of signalling molecule synthase genes in  Streptomyces coelicolor  and  Streptomyces avermitilis,  respectively. Two previously unknown repressors of P scbA  were identified from a library of TetR family regulators in  S. coelicolor , and three novel repressors of P aco  were identified from a genomic library of  S. avermitilis . Further  in vivo  and  in vitro  experiments confirmed that these newly identified repressors attenuated the transcription of their target promoters by direct binding. Overall, the genetic biosensor developed here presents an innovative and powerful strategy that could be applied for identifying genome-wide unknown repressors of promoters in bacteria.

A genetic biosensor for identification of transcriptional repressors of target promoters