期刊
ACS SYNTHETIC BIOLOGY
卷 10, 期 3, 页码 552-565出版社
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.0c00582
关键词
engineered system of transcription factors; synthetic biology; gene regulatory network: protein engineering
资金
- National Science Foundation [GCR 1934836, MCB 1921061, CBET 1844289, CBET 1804639, MCB 1747439]
Recent advancements in synthetic biology and protein engineering have led to an increase in the number of allosteric transcription factors used to regulate independent promoters. These engineered transcription factors are now capable of responding to caffeine while bypassing the native ligand hypoxanthine, creating 38 new transcription factors with alternate DNA binding functions. Additionally, this study successfully demonstrates the integration of NOR logic and feedback operations using the new system of transcription factors.
Recent advances in synthetic biology and protein engineering have increased the number of allosteric transcription factors used to regulate independent promoters. These developments represent an important increase in our biological computing capacity, which will enable us to construct more sophisticated genetic programs for a broad range of biological technologies. However, the majority of these transcription factors are represented by the repressor phenotype (BUFFER), and require layered inversion to confer the antithetical logical function (NOT), requiring additional biological resources. Moreover, these engineered transcription factors typically utilize native ligand binding functions paired with alternate DNA binding functions. In this study, we have advanced the state-of-the-art by engineering and redesigning the PurR topology (a native antirepressor) to be responsive to caffeine, while mitigating responsiveness to the native ligand hypoxanthine-i.e., a deamination product of the input molecule adenine. Importantly, the resulting caffeine responsive transcription factors are not antagonized by the native ligand hypoxanthine. In addition, we conferred alternate DNA binding to the caffeine antirepressors, and to the PurR scaffold, creating 38 new transcription factors that are congruent with our current transcriptional programming structure. Finally, we leveraged this system of transcription factors to create integrated NOR logic and related feedback operations. This study represents the first example of a system of transcription factors (antirepressors) in which both the ligand binding site and the DNA binding functions were successfully engineered in tandem.
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