Genetic switchboard for synthetic biology applications
Genetic switchboard for synthetic biology applications
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DOI：
10.1073/pnas.1203808109
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发表时间：
2012-04-10
影响因子：
11.1
通讯作者：
Collins, James J.
中科院分区：
文献类型：
--
作者：
Callura, Jarred M.;Cantor, Charles R.;Collins, James J.
合成生物学的下一步关键是将简单的电路联合收割机组合成高阶系统。在这项工作中，我们将我们的合成核糖核酸调控平台扩展为一个遗传开关板，它独立地平行控制多个基因的表达。首先，我们设计并表征了核糖核酸调节子变体，以完成遗传开关板的基础;然后我们构建了开关板传感器，这是一个测试平台，可以报告单细胞中的群体信号分子、DNA损伤、铁饥饿和细胞外镁浓度。为了证明我们的合成装置的生物技术潜力，我们建立了一个代谢开关板，调节四个代谢基因，pgi，zwf，edd和gnd，以控制通过三个大肠杆菌葡萄糖利用途径的碳流：Embden-Meyerhof，Entner-Doudoroff和戊糖磷酸途径。我们通过测量核糖调节基因的mRNA水平、相关酶和途径活性的变化以及大肠杆菌的靶向变化，为开关板介导的代谢流分流提供了直接证据。大肠杆菌代谢组。遗传交换机的设计、测试和实现说明了一个高阶系统的成功构建，该系统可用于合成生物学和生物技术的广泛实际应用。
A key next step in synthetic biology is to combine simple circuits into higher-order systems. In this work, we expanded our synthetic riboregulation platform into a genetic switchboard that independently controls the expression of multiple genes in parallel. First, we designed and characterized riboregulator variants to complete the foundation of the genetic switchboard; then we constructed the switchboard sensor, a testing platform that reported on quorum-signaling molecules, DNA damage, iron starvation, and extracellular magnesium concentration in single cells. As a demonstration of the biotechnological potential of our synthetic device, we built a metabolism switchboard that regulated four metabolic genes, pgi, zwf, edd, and gnd, to control carbon flow through three Escherichia coli glucose-utilization pathways: the Embden-Meyerhof, Entner-Doudoroff, and pentose phosphate pathways. We provide direct evidence for switchboard-mediated shunting of metabolic flux by measuring mRNA levels of the riboregulated genes, shifts in the activities of the relevant enzymes and pathways, and targeted changes to the E. coli metabolome. The design, testing, and implementation of the genetic switchboard illustrate the successful construction of a higher-order systemthat can be used for a broad range of practical applications in synthetic biology and biotechnology.