RT Journal Article SR Electronic T1 Layering genetic circuits to build a single cell, bacterial half adder JF bioRxiv FD Cold Spring Harbor Laboratory SP 019257 DO 10.1101/019257 A1 Adison Wong A1 Huijuan Wang A1 Chueh Loo Poh A1 Richard I Kitney YR 2015 UL http://biorxiv.org/content/early/2015/05/12/019257.abstract AB Gene regulation in biological systems is impacted by the cellular and genetic context-dependent effects of the biological parts which comprise the circuit. Here, we have sought to elucidate the limitations of engineering biology from an architectural point of view, with the aim of compiling a set of engineering solutions for overcoming failure modes during the development of complex, synthetic genetic circuits. Using a synthetic biology approach that is supported by computational modelling and rigorous characterisation, AND, OR and NOT biological logic gates were layered in both parallel and serial arrangements to generate a repertoire of Boolean operations that include NIMPLY, XOR, half adder and half subtractor logics in single cell. Subsequent evaluation of these near-digital biological systems revealed critical design pitfalls that triggered genetic context dependent effects, including 5’ UTR interference and uncontrolled switch-on behaviour of σ54 promoter. Importantly, this work provides a representative case study to the debugging of genetic context dependent effects through principles elucidated herein, thereby providing a rational design framework to program single prokaryotic cell with diversified digital operations.