The development of genetic switches and their integrated forms (genetic circuits) with desired specifications/functions is key for success in synthetic biology. Due to the difficulty in rational design, genetic switches and circuits with desirable specifications are mostly obtained by directed evolution. Based on a virus-derived nucleotide kinase as a single-gene dual selector, we constructed a robust, efficient and stringent selection system for genetic switches. This method exhibited unprecedented enrichment efficacy (>30000-fold) of functional switches from non-functional ones in a single selection cycle. In addition, negative (OFF) selection was exceptionally stringent, allowing the rapid and efficient selection of non-leaky from leaky circuits.
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