Authors
Prakash, S., Jaramillo, A.
Abstract
Engineering bacterial promoters to integrate multiple regulatory signals remains a formidable challenge. Juxtaposing operator sites frequently increases basal leakiness, compresses the fully induced state, and introduces severe sequence-context dependencies. Here, we systematically engineered two-input combinatorial promoters in Escherichia coli that integrate signals from multiple transcription factors. To achieve precise operational control over these regulators, we drove the promoters using highly optimised, small-molecule-responsive sensors from the Marionette transcription-factor cassette, allowing us to assemble 19 reporter-specific, four-state truth tables across 12 distinct promoter architectures. We evaluated each design against a stringent statistical criterion for inducer-conditioned coincidence responses. Nine architectures satisfied this criterion, yielding a robust set of operational AND switches. By comparing successful and unsuccessful designs, we reveal that performance hinges primarily on suppressing partially induced states, ensuring structural compatibility between the promoter scaffold and the inserted operator, and precisely managing the orientation of long operators to avoid recreating unintended promoter-like motifs. Furthermore, reciprocal architectures and alternate downstream reporters frequently display divergent behaviours, underscoring profound asymmetries and local genetic-context dependencies. Ultimately, these findings deliver versatile combinatorial switches alongside practical, sequence-aware design rules for engineering multi-input bacterial promoters.
Preprint server:
bioRxiv
The authors list and abstract were imported from bioRxiv on 26 Mar 2026.
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