Homozygous mutants were obtained in both the <i>acsA</i> and <i>glpK</i> loci.

<p>(A) Diagram of the primers used to screen for homozygous strains in the <i>acsA</i> locus. Primer set A amplifies from the up and down5' and 3' stream flanking regions of <i>acsA</i> and should be amplify 3.2 kb, 1.1 kb, and 2.6 kb in fragments for WT 7002, BPSyn_014, and BPSyn_015, respectively. Primer set B amplifies 1.2 kb from inside <i>acsA</i> to the region downstream of <i>acsA</i>. Primer set C amplifies 0.9 kb from the barcode (BC) region to downstream of <i>acsA</i>. Primer set D amplifies a 0.25 kb region internal to YFP. (B) 1% agarose gel showing colony PCR products amplified using the primer sets A, B, C, and D on WT PCC 7002, BPSyn_014, and BPSyn_015. Lane 1 contains a 2-log ladder from New England Biolabs. Lanes 2-5, 6-9, and 10-13 are primers sets A, B, C, and D amplifying WT PCC 7002, BPSyn_014, and BPSyn_015, respectively. (C) Diagram of the primers used to screen for homozygous strains in the <i>glpK</i> locus. Primer set E amplifies from the up and down stream5' and 3' flanking regions of <i>glpK</i> and should be amplify 2.7 kb, 4.8 kb, and 2.6 kb fragments from BPSyn_014, BPSyn_022, and BPSyn_027, respectively. Primer set F amplifies 0.72 kb from inside <i>glpK</i> to the region downstream of <i>glpK</i>. Primer set G amplifies a 2.3 kb region of the <i>acsA</i> cassette that was introduced into the <i>glpK</i> locus. (D) 1% agarose gel showing colony PCR products amplified using the primer sets E, F, G, and D on BPSyn_014, BPSyn_022, and BPSyn_027. Lane 1 contains a 2-log ladder from New England Biolabs. Lanes 2-5, 6-9, and 10-13 are primers sets E, F, G, and D amplifying BPSyn_014, BPSyn_022, and BPSyn_027, respectively.