Department of Biology, Institute for Synthetic Microbiology, Heinrich Heine University, Düsseldorf, Germany.
Methods Mol Biol. 2022;2379:67-79. doi: 10.1007/978-1-0716-1791-5_4.
Genetic engineering of cyanobacteria is currently limited to genomic integration via homologous recombination and RSF1010-based conjugative vector systems. Here, we introduce a rationally designed conjugative vector with two BioBrick-based cloning sites which enables facilitated and modular cloning. This streamlined vector is suitable for a variety of synthetic biology applications, such as expression of multiple enzymes from metabolic pathways for the production of biofuels or secondary metabolites, or screening of modular parts such as promoters, further facilitating applications to improve crop plants using synthetic biology. Finally, we present a general approach to cloning of constructs, as well as detailed protocols for conjugation and culturing of strains carrying said constructs.
蓝藻的基因工程目前仅限于通过同源重组和基于 RSF1010 的共轭载体系统进行基因组整合。在这里，我们引入了一种经过合理设计的共轭载体，它具有两个基于 BioBrick 的克隆位点，可实现方便和模块化的克隆。这种简化的载体适用于各种合成生物学应用，如表达代谢途径中的多种酶以生产生物燃料或次生代谢物，或筛选如启动子等模块化部件，进一步促进使用合成生物学来改良作物植物。最后，我们提出了一种构建克隆的一般方法，以及携带这些构建体的菌株的共轭和培养的详细方案。
