Multigene expression in vivo: supremacy of large versus small terminators for T7 RNA polymerase.
Multigene expression in vivo: supremacy of large versus small terminators for T7 RNA polymerase.
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DOI：
10.1002/bit.24379
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发表时间：
2012-04
影响因子：
3.8
通讯作者：
Forster, Anthony C.
中科院分区：
文献类型：
--
作者：
Du, Liping;Villarreal, Seth;Forster, Anthony C.
设计和构建多基因构建体在合成生物学中很常见。然而，由于基因部分不是完全模块化的，因此第一次尝试的功能成功是罕见的。为了提高转录的模块性，我们先前表明，通过噬菌体T7 RNA聚合酶体外转录终止可以通过用水泡性口炎病毒（VSV）小（II类）终止子的相邻拷贝取代标准的单个TΦ大（I类）终止子而变得更有效。然而，在体外终止下游VSV终止子的效率低于上游VSV终止子，并且体内多基因过表达由于在E.大肠杆菌细胞。在这里，我们解决的假设提出的研究表明，VSV或前甲状旁腺激素（PTH）的小终止子间隔更远，可以独立工作（即更有效地）在体外，VSV和PTH终止在体内受到严重抑制。令人惊讶的是，体内与体外II类终止子功能之间的差异不是由于质粒超螺旋的差异，因为超螺旋对体外终止的影响最小。因此，我们转向TΦ终止子用于适合于体内过表达的五聚体基因构建体的“BioBrick”合成。这确实能够使用尝试的第一个构建体协调过表达和共纯化五种His标记的蛋白质，表明这种策略比其他策略更模块化。这种多基因过表达和蛋白质共纯化方法的应用通过提供六个E.通过共纯化从单个细胞系重构翻译所需的大肠杆菌翻译因子，极大地简化了重构。
Designing and building multigene constructs is commonplace in synthetic biology. Yet functional successes at first attempts are rare because the genetic parts are not fully modular. In order to improve the modularity of transcription, we previously showed that transcription termination in vitro by bacteriophage T7 RNA polymerase could be made more efficient by substituting the standard, single, TΦ large (class I) terminator with adjacent copies of the Vesicular Stomatitis Virus (VSV) small (class II) terminator. However, in vitro termination at the downstream VSV terminator was less efficient than at the upstream VSV terminator, and multigene overexpression in vivo was complicated by unexpectedly inefficient VSV termination within E. coli cells. Here, we address hypotheses raised in that study by showing that VSV or preproparathyroid hormone (PTH) small terminators spaced further apart can work independently (i.e. more efficiently) in vitro, and that VSV and PTH terminations are severely inhibited in vivo. Surprisingly, the difference between class II terminator function in vivo versus in vitro is not due to differences in plasmid supercoiling, as supercoiling had a minimal effect on termination in vitro. We therefore turned to TΦ terminators for “BioBrick” synthesis of a pentameric gene construct suitable for overexpression in vivo. This indeed enabled coordinated overexpression and copurification of five His-tagged proteins using the first construct attempted, indicating that this strategy is more modular than other strategies. An application of this multigene overexpression and protein copurification method is demonstrated by supplying five of the six E. coli translation factors required for reconstitution of translation from a single cell line via copurification, greatly simplifying the reconstitution.
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