Modular control of multiple pathways using engineered orthogonal T7 polymerases.
Modular control of multiple pathways using engineered orthogonal T7 polymerases.
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DOI：
10.1093/nar/gks597
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发表时间：
2012-09-01
影响因子：
14.9
通讯作者：
Voigt CA
中科院分区：
文献类型：
--
作者：
Temme K;Hill R;Segall-Shapiro TH;Moser F;Voigt CA
合成基因传感器和电路能够对基因表达的时间和条件进行可编程控制。它们越来越多地被纳入复杂的多基因途径和细胞功能的控制中。在这里，我们提出了一种设计策略，从基因上分离的传感/电路功能的路径进行控制。这种分离是通过使电路的输出驱动聚合酶的表达来实现的，聚合酶然后从聚合酶特异性启动子激活通路。传感器、电路和聚合酶一起编码在一个“控制器”质粒上。构建了降低毒性的T7 RNA聚合酶的变体，并将其用作支架，用于构建通过部分挖掘鉴定的与独特启动子序列结合的四种正交聚合酶。这一组是高度正交的，诱导同源启动子比脱靶启动子多8至75倍。这些正交聚合酶使四个独立的通道连接电路的输出到不同的细胞功能的控制。作为示范，我们构建了一个控制器质粒，它集成了两个诱导系统，实现了AND逻辑运算，并在改变大肠杆菌绿色（脱氧铬病毒）和红色（番茄红素）的代谢途径之间切换。这种组织的优点是：（i）可以简单地通过引入不同的控制质粒来改变途径的调节，（ii）转录与宿主机制正交，以及（iii）途径基因在没有控制器的情况下不转录，因此更容易携带，而不会引起进化压力。
Synthetic genetic sensors and circuits enable programmable control over the timing and conditions of gene expression. They are being increasingly incorporated into the control of complex, multigene pathways and cellular functions. Here, we propose a design strategy to genetically separate the sensing/circuitry functions from the pathway to be controlled. This separation is achieved by having the output of the circuit drive the expression of a polymerase, which then activates the pathway from polymerase-specific promoters. The sensors, circuits and polymerase are encoded together on a ‘controller’ plasmid. Variants of T7 RNA polymerase that reduce toxicity were constructed and used as scaffolds for the construction of four orthogonal polymerases identified via part mining that bind to unique promoter sequences. This set is highly orthogonal and induces cognate promoters by 8- to 75-fold more than off-target promoters. These orthogonal polymerases enable four independent channels linking the outputs of circuits to the control of different cellular functions. As a demonstration, we constructed a controller plasmid that integrates two inducible systems, implements an AND logic operation and toggles between metabolic pathways that change Escherichia coli green (deoxychromoviridans) and red (lycopene). The advantages of this organization are that (i) the regulation of the pathway can be changed simply by introducing a different controller plasmid, (ii) transcription is orthogonal to host machinery and (iii) the pathway genes are not transcribed in the absence of a controller and are thus more easily carried without invoking evolutionary pressure.
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