Fig. 3

Construction of Alr5283-Alr5284 knockout mutant (DR935) and its complemented strains in Anabaena 7120. a 3′ deletion of ado (alr5283) and 5′ deletion of aar (alr5284) created by inserting a gfp-spec cassette between alr5283–84 in Anabaena 7120 chromosome via double recombination with knockout plasmid pZR935. b PCR verification of DR935. Wildtype Anabaena 7120 had the intact alr5283–84 gene sequence, which has a length of 2.7 kb when amplified by ZR241, 242 (lane 3). DR935 contained the gfp-spec cassette inserted in the alr5283–84 gene sequence, making the amplified gene sequence 4.7 kb (lane 2). c Complementing plasmid constructions: pZR2239 contains ado and aar both under control of their native promoters, pZR2248 contains the engineered ado-aar operon under control of the constitutive glnA promoter, a standard ribosome-binding sequence (AAGGAGA) was introduced between ado and aar in pZR2248, and pZR2243 contains ado under control of PglnA and aar under control of its native promoter