Its response to HDACis could perhaps explain some of the observations described

Research featuring genetically engineered cyanobacteria for the production of liquid biofuels including ethanol , isobutyraldehyde and isobutanol , and free fatty acids has recently flourished. Although using cyanobacteria as cell factories has become more common, studies are still carried out with standard laboratory model organisms rather than with potential production strains. For their desirable growth qualities, much consideration has been given to Axitinib strains of the genus Arthrospira , which emerged from a screen of cyanobacterial strains for superior growth traits, and show that it is amenable to genetic manipulation. Leptolyngbya BL0902 has good growth characteristics when compared to two common outdoor production strains of the genus Arthrospira. We show that Leptolyngbya BL0902 can receive and maintain conjugal shuttle vectors, express an antibiotic resistance gene and a yemGFP reporter gene, and be subjected to transposon-tagging mutagenesis. Conjugation from E. coli donor cells has been used to introduce DNA into a wide variety of cyanobacteria, and broad-host-range plasmids derived from RSF1010 have been shown to replicate in many strains . To determine whether these methods could be used with Leptolyngbya BL0902, we performed biparental matings with Leptolyngbya BL0902 and a conjugal E. coli donor strain that contained the cargo plasmid pRL1383a, the conjugal plasmid pRL443, and the helper plasmid pRL623. Transconjugant colonies became apparent on selective mating plates after about one week and showed robust growth after transfer to fresh selective plates. Control conjugations without the cargo plasmid never showed any antibiotic resistant colonies. The ability to genetically modify Leptolyngbya BL0902 was further demonstrated by the heterologous expression of the yemGFP gene. The recombinant plasmid pAM4413 carrying the yemGFP gene was electroporated into AM1359, and the resulting strain was conjugated with Leptolyngbya BL0902. After one week, Nutlin-3 isolated transconjugant colonies were restreaked on fresh selective plates, and isolated colonies were then patched to fresh selective plates. Liquid cultures were grown in selective BG-11 medium. Expression of yemGFP was observed by fluorescence microscopy . Our initial conjugation experiments were performed with donor strains carrying the helper plasmid pRL623, which carries 3 methylase genes. The methylase genes are required for efficient conjugation into Anabaena recipient strains . To assess the necessity of these genes for Leptolyngbya BL0902 conjugation, we determined the efficiency of conjugal transfers in biparental and triparental matings with and without the helper plasmid pRL623 and with two different conjugal plasmids: pRL443 and pRK2013 . The conjugation protocol was modified as described in the methods to yield more reproducible data for transconjugant colony forming units .

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