Genetic circuit characterization by inferring RNA polymerase movement and ribosome usage.
Genetic circuit characterization by inferring RNA polymerase movement and ribosome usage.
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DOI：
10.1038/s41467-020-18630-2
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发表时间：
2020-10-05
影响因子：
16.6
通讯作者：
Voigt CA
中科院分区：
文献类型：
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作者：
Espah Borujeni A;Zhang J;Doosthosseini H;Nielsen AAK;Voigt CA
为了执行其计算功能，基因回路通过一系列调控基因表达开启和关闭的基因元件的协同作用来改变状态。由于无法在回路环境中对元件进行特性描述以及确定故障根源，调试工作受阻。在此，我们获取了一个大型基因回路在不同状态下的快照：利用RNA测序技术将回路功能可视化为RNA聚合酶（RNAP）沿DNA流动的变化模式。结合核糖体图谱分析，所有54个基因元件（启动子、核酶、核糖体结合位点、终止子）都被参数化，并用于构建一个数学模型，该模型能够预测回路的性能、动态和稳健性。回路的运行符合设计要求；然而，它存在大量基因错误，包括隐蔽的有义/反义启动子以及翻译、衰减、错误的起始密码子和一个失效的门控。虽然这些错误不影响预期的布尔逻辑，但它们降低了预测准确性，并且当这些元件用于其他设计时可能导致故障。最后，计算了维持回路状态所需的细胞能量（RNAP和核糖体的使用）。这项工作展示了如何利用少量测量来完全参数化一个调控回路并量化其对宿主的影响。
由于无法在回路环境中对元件进行特性描述，基因回路的调试工作受阻。在此，作者利用RNA测序和核糖体图谱分析对一个大型回路在不同状态下进行“快照”式研究。
To perform their computational function, genetic circuits change states through a symphony of genetic parts that turn regulator expression on and off. Debugging is frustrated by an inability to characterize parts in the context of the circuit and identify the origins of failures. Here, we take snapshots of a large genetic circuit in different states: RNA-seq is used to visualize circuit function as a changing pattern of RNA polymerase (RNAP) flux along the DNA. Together with ribosome profiling, all 54 genetic parts (promoters, ribozymes, RBSs, terminators) are parameterized and used to inform a mathematical model that can predict circuit performance, dynamics, and robustness. The circuit behaves as designed; however, it is riddled with genetic errors, including cryptic sense/antisense promoters and translation, attenuation, incorrect start codons, and a failed gate. While not impacting the expected Boolean logic, they reduce the prediction accuracy and could lead to failures when the parts are used in other designs. Finally, the cellular power (RNAP and ribosome usage) required to maintain a circuit state is calculated. This work demonstrates the use of a small number of measurements to fully parameterize a regulatory circuit and quantify its impact on host. Debugging a genetic circuit is frustrated by the inability to characterize parts in the context of the circuit. Here the authors use RNA-seq and ribosome profiling to take ‘snapshots’ of a large circuit in different states.