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Construction of synthetic T7 RNA polymerase expression systems
Methods ( IF 4.8 ) Pub Date : 2018-07-01 , DOI: 10.1016/j.ymeth.2018.02.022
Shaunak Kar , Andrew D. Ellington

T7 RNA polymerase (T7 RNAP) is one of the preferred workhorses for recombinant gene expression, owing in part to its high transcriptional activity and the fact that it has a small (17 base-pair), easily manipulated promoter. Furthermore, the fact that T7 RNAP is largely orthogonal to most hosts enables its use in a wide variety of contexts. However, the high activity of the enzyme also often leads to an increased fitness burden on the host, limiting the predictability of its interactions and impact on host physiology, and potentially leading to mutations in the constructs. Here we use a synthetic biology approach to design and characterize a panel of T7 RNAP expression circuits with different modes of regulation that enable the reliable expression of downstream targets under a variety of conditions. First, we describe the construction of a minimal T7 RNAP expression system that is inducible by a small molecule anhydrotetracycline (aTc), and then characterize a self-limiting T7 RNAP expression circuit that provides better control over the amount of T7 RNAP produced upon induction. Finally, we characterize a so-called T7 RNAP homeostasis circuit that leads to constitutive, continuous, and sub-toxic levels of T7 RNAP. Coupled with previously characterized mutant T7 RNAP promoters in vitro, we demonstrate that this modular framework can be used to achieve precise and predictable levels of output (sfGFP) in vivo. This new framework should now allow modeling and construction of T7 RNAP expression constructs that expand the utility of this enzyme for driving a variety of synthetic circuits and constructs.

中文翻译:

合成T7 RNA聚合酶表达系统的构建

T7 RNA 聚合酶 (T7 RNAP) 是重组基因表达的首选主力之一,部分原因在于其高转录活性以及它具有小(17 个碱基对)、易于操作的启动子这一事实。此外,T7 RNAP 在很大程度上与大多数宿主正交的事实使其能够在各种环境中使用。然而,酶的高活性也经常导致宿主的健康负担增加,限制了其相互作用的可预测性和对宿主生理的影响,并可能导致构建体发生突变。在这里,我们使用合成生物学方法来设计和表征一组具有不同调节模式的 T7 RNAP 表达回路,这些回路能够在各种条件下可靠地表达下游目标。第一的,我们描述了一个由小分子脱水四环素 (aTc) 诱导的最小 T7 RNAP 表达系统的构建,然后描述了一个自限性 T7 RNAP 表达电路,它可以更好地控制诱导后产生的 T7 RNAP 的数量。最后,我们描述了一个所谓的 T7 RNAP 稳态回路,该回路导致 T7 RNAP 的组成性、连续性和亚毒性水平。结合先前在体外表征的突变 T7 RNAP 启动子,我们证明这种模块化框架可用于在体内实现精确和可预测的输出水平 (sfGFP)。这个新框架现在应该允许对 T7 RNAP 表达构建体进行建模和构建,从而扩展这种酶在驱动各种合成电路和构建体方面的效用。
更新日期:2018-07-01
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