谷氨酸棒杆菌
工作流程
合理设计
计算生物学
合成生物学
自动化
计算机科学
克隆(编程)
转化(遗传学)
质粒
软件
调节器
生物
DNA
遗传学
工程类
程序设计语言
数据库
基因
机械工程
作者
Niklas Tenhaef,Robert Stella,Julia Frunzke,Stephan Noack
标识
DOI:10.1021/acssynbio.0c00599
摘要
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 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.
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