Automated Rational Strain Construction Based on High-Throughput Conjugation
Automated Rational Strain Construction Based on High-Throughput Conjugation
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DOI：
10.1021/acssynbio.0c00599
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发表时间：
2021-02-16
影响因子：
4.7
通讯作者：
Noack, Stephan
中科院分区：
文献类型：
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作者：
Tenhaef, Niklas;Stella, Robert;Noack, Stephan
分子克隆是合成生物学的核心，它包括DNA的组装及其在目标宿主中的表达。然而，目前，克隆通常是一个手动的、耗时的、重复的过程，自动化对它大有好处。一个完整的合理克隆程序的自动化，即从DNA的产生到在目标宿主中的表达，涉及到不同操作和机器的集成。这种工作流程的例子很少，特别是当设计是合理的（即，DNA序列设计是固定的，而不是基于随机文库），目标宿主在遗传上不太容易处理（例如，对热休克转换不敏感）。在这项研究中，提出了一种自动化的工作流程，用于合理构建质粒并随后将其共轭转移到生物技术平台有机体谷氨酸谷杆菌中。整个工作流程配有定制的软件工具。作为应用实例，构建了一个合理设计的转录因子-生物传感器库，并对其进行了表征。获得了动态范围更大的传感器，为谷氨酸酵母具有双重调节功能提供了证据。
Molecular cloning is the core of synthetic biology, as it comprises the assembly of DNA and its expression in target hosts. At present, however, cloning is most often a manual, time-consuming, and repetitive process that highly benefits from automation. The automation of a complete rational cloning procedure, i.e., from DNA creation to expression in the target host, involves the integration of different operations and machines. Examples of such workflows are sparse, especially when the design is rational (i.e., the DNA sequence design is fixed and not based on randomized libraries) and the target host is less genetically tractable (e.g., not sensitive to heat-shock transformation). In this study, an automated workflow for the rational construction of plasmids and their subsequent conjugative transfer into the biotechnological platform organism Cotynebacteriunt glutamicum is presented. The whole workflow is accompanied by a custom-made software tool. As an application example, a rationally designed library of transcription factor-biosensors based on the regulator Lrp was constructed and characterized. A sensor with an improved dynamic range was obtained, and insights from the screening provided evidence for a dual regulator function of C. glutamicum Lip.