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 part 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 Corynebacterium 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 Lrp.

中文翻译：

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基于高通量共轭的自动化合理应变构造

分子克隆是合成生物学的核心，因为它包括DNA的组装及其在靶标宿主中的表达。但是，目前，克隆通常是一种手动，耗时且重复的过程，该过程可从自动化中受益匪浅。完整的合理克隆程序（即从DNA片段的创建到目标宿主中的表达）的自动化涉及不同操作和机器的集成。这种工作流程的例子很少，特别是在设计合理的情况下（即DNA序列设计是固定的，而不是基于随机库）并且目标宿主的遗传易感性较差（例如，对热冲击转变不敏感）。在这项研究中，提出了一种用于合理构建质粒及其随后共轭转移到生物技术平台生物谷氨酸棒杆菌中的自动化工作流程。整个工作流程都配有定制软件工具。作为一个应用实例，构建并表征了基于调控因子Lrp的合理设计的转录因子生物传感器库。获得了具有改善的动态范围的传感器，并且从筛选中获得的见识为谷氨酸棒杆菌Lrp的双重调节功能提供了证据。