Layering genetic circuits to build a single cell, bacterial half adder
File(s)
Author(s)
Wong, A
Wang, H
Poh, CL
Kitney, RI
Type
Journal Article
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.
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.
Date Issued
2015-06-16
Date Acceptance
2015-06-03
Citation
BMC Biology, 2015, 13
ISSN
1741-7007
Publisher
BioMed Central
Journal / Book Title
BMC Biology
Volume
13
Copyright Statement
© 2015 Wong et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium,
provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://
creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
(http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium,
provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://
creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
License URL
Subjects
Science & Technology
Life Sciences & Biomedicine
Biology
Life Sciences & Biomedicine - Other Topics
SYNTHETIC BIOLOGY
ESCHERICHIA-COLI
RNA-POLYMERASE
LOGIC GATES
BURKHOLDERIA-CENOCEPACIA
MULTIPLE PATHWAYS
LIVING CELLS
EXPRESSION
TRANSCRIPTION
NETWORKS
Publication Status
Published
Article Number
ARTN 40