Double Crossover Approach to Inactivate Target Gene in Cyanobacteria

Abstract

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.