title: Layering genetic circuits to build a single cell, bacterial half adder.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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 a single cell. Subsequent evaluation of these near-digital biological systems revealed critical design pitfalls that triggered genetic context-dependent effects, including 5' UTR interferences and uncontrolled switch-on behaviour of the supercoiled σ54 promoter. In particular, the presence of seven consecutive hairpins immediately downstream of the promoter transcription start site severely impeded gene expression.
  
  
  CONCLUSIONS
  As synthetic biology moves forward with greater focus on scaling the complexity of engineered genetic circuits, studies which thoroughly evaluate failure modes and engineering solutions will serve as important references for future design and development of synthetic biological systems. This work describes a representative case study for the debugging of genetic context-dependent effects through principles elucidated herein, thereby providing a rational design framework to integrate multiple genetic circuits in a single prokaryotic cell.
authors.affiliation: Division of Bioengineering, School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore, 637459, Singapore.|Centre for Synthetic Biology and Innovation, and Department of Bioengineering, Imperial College London, London, SW7 2AZ, UK.|Present Address: NUS Synthetic Biology for Clinical and Technological Innovation, National University of Singapore, Singapore, 117456, Singapore.
authors.forename: Adison
authors.initials: A
authors.lastname: Wong
authors.affiliation: Division of Bioengineering, School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore, 637459, Singapore.
authors.forename: Huijuan
authors.initials: H
authors.lastname: Wang
authors.affiliation: Division of Bioengineering, School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore, 637459, Singapore. clpoh@ntu.edu.sg.
authors.forename: Chueh Loo
authors.initials: CL
authors.lastname: Poh
authors.affiliation: Centre for Synthetic Biology and Innovation, and Department of Bioengineering, Imperial College London, London, SW7 2AZ, UK. r.kitney@imperial.ac.uk.
authors.forename: Richard I
authors.initials: RI
authors.lastname: Kitney
mesh_terms: D020121:5' Untranslated Regions; D039301:Computers, Molecular; D004926:Escherichia coli / Q000235:genetics; D053263:Gene Regulatory Networks; D011401:Promoter Regions, Genetic; D058615:Synthetic Biology / Q000379:methods*
chemical_list: D020121:5' Untranslated Regions