Department of Biology and Microbiology, South Dakota State University, Brookings, SD, USA.
Methods Mol Biol. 2022;2489:299-313. doi: 10.1007/978-1-0716-2273-5_16.
Anabaena sp. PCC7120 (hereafter Anabaena 7120) is a nitrogen-fixing, filamentous cyanobacterium. Given its diverse metabolism, it serves as an excellent model organism, particularly for studying cell differentiation, nitrogen fixation, photosynthesis, production of high-value chemicals, and synthetic biology. Gene knockout is a common approach to assess the function of gene products through assessing phenotypic loss of function. In the method described here, a double crossover approach is used to inactivate a target gene or target genes in Anabaena 7120. This method involves replicating the gene(s) from the wild-type genomic DNA and inserting them into an integrative plasmid vector. An internal portion of the genes may be removed and replaced with a GFP-Spectinomycin (gfp-sp) cassette. The plasmid is then introduced into Anabaena 7120 where a double crossover event occurs between the wild-type chromosome and the cargo plasmid, effectively replacing the wild-type gene with the disrupted gene from the plasmid. The gfp-sp cassette combined with the sacB gene serve as positive selection to identify double crossover mutants (Cai and Wolk (1990), 172(6):3138-3145, J. Bacteriol). Finally, the functional genes are cloned into another replicating plasmid vector to produce a cargo plasmid, which is conjugatively introduced into the mutant for a complementation test. By comparing the phenotypes among the wild-type, mutant, and complement, one should see a loss of function in the mutant which is recovered in the complement, thereby defining the function of the target gene. The double crossover approach described here for Anabaena PCC 7120 may be broadly applicable to the study of gene function in cyanobacteria and other prokaryotic organisms.
鱼腥藻 PCC7120（以下简称鱼腥藻 7120）是一种固氮丝状蓝藻。由于其具有多样化的代谢途径，它是一种理想的模式生物，特别适合用于研究细胞分化、固氮作用、光合作用、高价值化学物质的生产以及合成生物学。基因敲除是一种通过评估基因产物丧失功能的表型来评估基因功能的常用方法。在本文描述的方法中，采用双交换方法来使鱼腥藻 7120 中的靶基因或靶基因失活。该方法涉及从野生型基因组 DNA 中复制基因，并将其插入到整合质粒载体中。基因的内部部分可能会被删除，并被 GFP-壮观霉素（gfp-sp）盒取代。然后将质粒引入鱼腥藻 7120 中，野生型染色体和携带 cargo 的质粒之间会发生双交换事件，从而有效地将质粒上的破坏基因取代为野生型基因。gfp-sp 盒与 sacB 基因的组合作为阳性选择，用于鉴定双交换突变体（Cai 和 Wolk（1990），172（6）：3138-3145，J. Bacteriol）。最后，将功能基因克隆到另一个可复制的质粒载体中，以产生携带 cargo 的质粒，然后将其通过共轭转移引入突变体中进行互补测试。通过比较野生型、突变体和互补体之间的表型，可以发现突变体丧失了功能，而互补体则恢复了功能，从而确定了靶基因的功能。本文中描述的用于鱼腥藻 PCC7120 的双交换方法可能广泛适用于研究蓝藻和其他原核生物中的基因功能。
