Approximately 3% of potential colony-forming units were transformed by conjugal transfer in bi- or tri-parental matings, which was increased about two-fold when a methylase-expressing helper plasmid was included . We did not observe significant differences between bi-parental and tri-parental matings or between the conjugal plasmids pRL443 and pRK2013 . The integration of the Tn5-692 transposon into the Leptolyngbya BL0902 chromosome was confirmed by a set of PCR assays carried out on three putative transconjugant clones. The clones were grown in BG-11 liquid culture, which resulted in the loss of all viable donor E. coli cells. The absence of E. coli was confirmed by a lack of colony formation when transconjugant cyanobacterial samples were inoculated on BG-11 AZD2281 plates supplemented with 0.04% glucose and 5% LB broth and incubated in the dark at 30uC, or on LB plates incubated at 37uC. Two pairs of primers were used for the PCR assays. The primer pair pRL692-6976F/7350R amplifies a 421-bp fragment within the origin of transfer of the plasmid backbone from RAD001 position 6953 to position 7373 of pRL692. The primer pair pRL692-2118F/2418R amplifies a 347-bp fragment within the transposon Tn5-692 from position 2095 to position 2441 of pRL692. The OriT primer pair produced PCR products in the positive-control samples only , indicating the absence of the suicide plasmid in any of the three transconjugants and confirming the loss of all E. coli cells. The Tn5-692 primer pair produced PCR products from all three transconjugant strains and the positive controls, but not from WT Leptolyngbya BL0902. These data show that the Tn5-692 transposon can be used for transposon tagging in Leptolyngbya BL0902. To facilitate the ability to introduce and express genes or noncoding and antisense RNAs in Leptolyngbya BL0902 and other cyanobacterial strains, we constructed plasmid pAM4418 based on the conjugal vector pRL1383a . pAM4418 contains an E. coli lacIq gene and the inducible trc promoter upstream of a Gateway recombination cassette. Genes of interest that are cloned in a pENTR vector can be introduced into pAM4418 by an LR recombination reaction. We monitored the expression of yemGFP as fluorescence emission intensity in Leptolyngbya BL0902 harboring pAM4418-yemGFP for two days following induction with IPTG. The reporter was constitutively expressed at moderately high levels, but there was no significant increase in yemGFP fluorescence intensity with IPTG addition at final concentrations ranging from 0.1 to 10 mM. We conclude that the trc promoter functions well in Leptolyngbya BL0902, but that either the lacIq gene is not expressed or the LacI protein fails to repress expression from the trc promoter on pAM4418. Leptolyngbya BL0902 provides a new experimental model for cyanobacterial research that is focused on the goal of outdoor commercial production.