Genetic Manipulation of Synechococcus PCC 7002

All plasmids used in this study are listed in Table 2. A PCC 7002 ΔacsA mutant (BPSyn_006) was constructed by double homologous recombination as previously described [25 ]. Briefly, a linear DNA fragment was constructed containing a streptomycin resistance marker derived from pSRA81 (aadA) flanked by approximately 600 bp regions that are homologous to regions directly 5’ and 3’ of the acsA (SYNPCC7002_A1838) gene. The acsA upstream and acsA downstream pieces were individually amplified using the primers in Table 3, digested, and then ligated together with the digestion fragment of pSRA81. The resulting fragment was PCR amplified and transformed into wild type PCC 7002. A similar protocol was used to replace acsA with a 40 bp barcode sequence, resulting in strain BPSyn_014. A linear fragment was created with 600 bp homologous regions and a 40 bp barcode sequence in between. This fragment was transformed into PCC 7002 and mutants were selected on plates containing 50 µM acrylate. Positive clones were streaked out on plates containing 10 mM acrylate to achieve complete chromosomal segregation. The resulting mutant had a deletion of acsA without a residual antibiotic resistance cassette. Complementation of acsA was performed through the introduction of acsA under the native acsA promoter into glpK (SYNPCC7002_A2842). Due to a frameshift mutation, glpK is a pseudogene in PCC 7002. For this reason, glpK was chosen as a neutral insertion site. Plasmids pGLPK_acsA_Sp^R and pGLPK_acsAW49L_Sp^R were constructed with homologous flanking regions to replace glpK with either (1) a wild type copy of acsA or (2) acsAW49L under the native acsA promoter along with a streptomycin resistance marker, using the in vitro recombination method using primers in Table 3 [26 (link)]. Plasmids pACSA_pcpcB_YFP and pGLPK_pcpcB_YFP were constructed in a similar manner using the cpcB promoter from PCC 6803 and yellow florescent protein (YFP) gene from plasmid pAQ1_Exp_YFP built by Xu et al. [27 ]. All cyanobacterial strains were screened by colony PCR for proper integration and full segregation of all chromosomes.