Chemical and Physical Sciences, University of Toronto Mississauga, Mississauga, Ontario L5L 1C6, Canada.
Cell and Systems Biology, University of Toronto Mississauga, Mississauga, Ontario L5L 1C6, Canada.
Nucleic Acids Res. 2018 Oct 12;46(18):9842-9854. doi: 10.1093/nar/gky785.
Despite recent progress on synthetic transcription factor generation in eukaryotes, there remains a need for high-activity bacterial versions of these systems. In synthetic biology applications, it is useful for transcription factors to have two key features: they should be orthogonal (influencing only their own targets, with minimal off-target effects), and programmable (able to be directed to a wide range of user-specified transcriptional start sites). The RNA polymerase of the bacteriophage T7 has a number of appealing properties for synthetic biological designs: it can produce high transcription rates; it is a compact, single-subunit polymerase that has been functionally expressed in a variety of organisms; and its viral origin reduces the connection between its activity and that of its host's transcriptional machinery. We have created a system where a T7 RNA polymerase is recruited to transcriptional start sites by DNA binding proteins, either directly or bridged through protein-protein interactions, yielding a modular and programmable system for strong transcriptional activation of multiple orthogonal synthetic transcription factor variants in Escherichia coli. To our knowledge this is the first exogenous, programmable activator system in bacteria.
尽管在真核生物中合成转录因子的研究取得了一些进展，但仍需要开发具有高活性的细菌版本的转录因子。在合成生物学应用中，转录因子具有两个关键特征是很有用的：它们应该是正交的（只影响其自身的靶标，最小化脱靶效应），并且是可编程的（能够被引导到广泛的用户指定的转录起始位点）。噬菌体 T7 的 RNA 聚合酶在合成生物学设计中有许多吸引人的特性：它可以产生高转录速率；它是一个紧凑的、单亚基聚合酶，已经在多种生物体中得到了功能性表达；并且它的病毒起源降低了其活性与其宿主转录机制之间的联系。我们创建了一个系统，其中 T7 RNA 聚合酶通过 DNA 结合蛋白直接或通过蛋白-蛋白相互作用招募到转录起始位点，从而产生了一个用于在大肠杆菌中强烈转录激活多个正交合成转录因子变体的模块化和可编程系统。据我们所知，这是细菌中第一个外源性、可编程的激活系统。
