Engineering compact bacterial combinatorial promoters for two-input chemical AND switching

Abstract Engineering compact bacterial promoters that integrate two chemical inputs would simplify biosensors, synthetic circuits, and dynamic metabolic control, but such promoters remain difficult to build because a second operator can increase partial-state leak, reduce the fully induced state, and create sequence- or reporter-context effects. Here we engineered 12 compact Marionette-derived two-input promoter architectures in Escherichia coli by combining defined template scaffolds with added operators, then quantified 19 reporter-specific four-state truth tables under a predefined operational criterion for chemical-input AND behaviour. Single-input template-promoter controls in matched reporter contexts helped separate weak parent-scaffold output from effects introduced by the second operator. Nine curated architectures passed in at least one reporter, and selected constructs delivered high-utility AND responses, including [11]/max-off separation up to > 1,367. The full pass–fail dataset showed that a statistically higher [11] state does not necessarily create a useful switch. Practical utility depended mainly on suppressing [10] and [01] leakage, matching the template scaffold to the inserted operator, and controlling long-operator orientation and local sequence context. Reciprocal architectures behaved differently, and 4 of 7 dual-reporter architectures changed operational classification with reporter context. Thus, this work delivers a compact set of candidate Marionette-compatible two-input promoter parts and a source-traceable design framework for engineering bacterial chemical-input AND switches.