使用CyanoGate模块化克隆工具包通过接合作用对蓝细菌进行基因改造。
Genetic Modification of Cyanobacteria by Conjugation Using the CyanoGate Modular Cloning Toolkit.
作者信息
Gale Grant A R, Schiavon Osorio Alejandra A, Puzorjov Anton, Wang Baojun, McCormick Alistair J
机构信息
Institute of Molecular Plant Sciences, School of Biological Sciences, University of Edinburgh; Centre for Synthetic and Systems Biology, University of Edinburgh; Institute of Quantitative Biology, Biochemistry and Biotechnology, School of Biological Sciences, University of Edinburgh.
Institute of Molecular Plant Sciences, School of Biological Sciences, University of Edinburgh; Centre for Synthetic and Systems Biology, University of Edinburgh.
出版信息
J Vis Exp. 2019 Oct 31(152). doi: 10.3791/60451.
Cyanobacteria are a diverse group of prokaryotic photosynthetic organisms that can be genetically modified for the renewable production of useful industrial commodities. Recent advances in synthetic biology have led to development of several cloning toolkits such as CyanoGate, a standardized modular cloning system for building plasmid vectors for subsequent transformation or conjugal transfer into cyanobacteria. Here we outline a detailed method for assembling a self-replicating vector (e.g., carrying a fluorescent marker expression cassette) and conjugal transfer of the vector into the cyanobacterial strains Synechocystis sp. PCC 6803 or Synechococcus elongatus UTEX 2973. In addition, we outline how to characterize the performance of a genetic part (e.g., a promoter) using a plate reader or flow cytometry.
蓝细菌是一类多样的原核光合生物，可通过基因改造实现有用工业产品的可再生生产。合成生物学的最新进展已促成了多种克隆工具包的开发，如CyanoGate，这是一种标准化的模块化克隆系统，用于构建质粒载体，以便随后转化或接合转移到蓝细菌中。在此，我们概述了一种详细方法，用于组装自我复制载体（如携带荧光标记表达盒），并将该载体接合转移到蓝细菌菌株聚球藻属PCC 6803或聚球藻UTEX 2973中。此外，我们还概述了如何使用酶标仪或流式细胞仪来表征遗传元件（如启动子）的性能。
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