期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2021
卷号：118
期号：46
DOI：10.1073/pnas.2111450118
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Significance
As the size and complexity of genetic circuits increases, scientists and engineers need to find solutions to rapidly optimize flux and reduce the metabolic burden imposed on chassis cells. In this study, we report synthetic biology tools that imbue chassis cells with advanced signal processing functions akin to electrical devices commonly used in wireless transmitters and receivers (i.e., biological BANDPASS and BANDSTOP devices) that can simultaneously reduce metabolic burden. Moreover, this study presents a set of granular and more complete design rules for engineering allosteric transcription factors in the broader LacI/GalR topology. In addition, this study has improved our fundamental understanding of the plasticity and continuum of allosteric communication from the binding pocket to the protein–DNA interaction.
Signal processing is critical to a myriad of biological phenomena (natural and engineered) that involve gene regulation. Biological signal processing can be achieved by way of allosteric transcription factors. In canonical regulatory systems (e.g., the lactose repressor), an INPUT signal results in the induction of a given transcription factor and objectively switches gene expression from an OFF state to an ON state. In such biological systems, to revert the gene expression back to the OFF state requires the aggressive dilution of the input signal, which can take 1 or more d to achieve in a typical biotic system. In this study, we present a class of engineered allosteric transcription factors capable of processing two-signal INPUTS, such that a sequence of INPUTS can rapidly transition gene expression between alternating OFF and ON states. Here, we present two fundamental biological signal processing filters, BANDPASS and BANDSTOP, that are regulated by D-fucose and isopropyl-β-D-1-thiogalactopyranoside. BANDPASS signal processing filters facilitate OFF–ON–OFF gene regulation. Whereas, BANDSTOP filters facilitate the antithetical gene regulation, ON–OFF–ON. Engineered signal processing filters can be directed to seven orthogonal promoters via adaptive modular DNA binding design. This collection of signal processing filters can be used in collaboration with our established transcriptional programming structure. Kinetic studies show that our collection of signal processing filters can switch between states of gene expression within a few minutes with minimal metabolic burden—representing a paradigm shift in general gene regulation.
关键词：synthetic biology; protein engineering; transcription factors; BANDPASS; BANDSTOP
