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<p>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 <i>Escherichia coli</i> 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 &gt; 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.</p>

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

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2026年7月27日

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  • Satya Prakash,
  • Alfonso Jaramillo

摘要

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.