Biosolar Conversion of N2 and H2O to Ammonia by Engineered N2-fixing Cyanobacteria

The cyanobacterium Anabaena sp. PCC 7120 is capable of using solar energy to produce ammonia through conversion of atmospheric dinitrogen in differentiated cells called heterocysts. Harnessing this system could enable cyanobacteria to be used in ammoniafertilizer production, creating a renewable alternative to the current Haber-Bosch process. The genes patA and alr3304 in the Anabaena 7120 genome are important in heterocyst formation; an increase in heterocyst formation is seen with over-expression of patA or inactivation of alr3304. This research aims to create a novel mutant that forms more heterocysts by simultaneously over-expressing patA and inactivating alr3304, producing higher amounts of ammonia. These two genes were isolated using Polymerase Chain Reaction (PCR) amplification. The resulting amplicons were then inserted into pTOPO 2.1 vectors, creating pZR855 and pZR854, respectively. PatA was removed from pZR855 using enzymatic digestion and then ligated into pZR618 to create pZR856, which enabled the successful transformation of NEB 10-beta Escherichia coli competent cells with pZR856 and later plasmids. Plasmid pZR857 was created by fusing patA from pZR856 to the native Anabaena promoter PpsbA in pZR811. The 2.7kb fragment PpsbA-patA was inserted within alr3304 in pZR854 to create pZR858, thereby disrupting alr3304. PCR and sequencing were used to verify different stages of the plasmid constructions. Results from colony PCR and enzymatic digestion indicated that pZR858 was successfully created. Further progression in this research will potentially create a mutant strain of Anabaena 7120 that forms more heterocysts and therefore produces ammonia at a commercially viable level