Construction Of Gfp Transformation System For Filamentous Cyanobacterium, Anabaena PCC7120

The green fluorescent protein(GFP) gene from the jellyfish Aequorea Victoria has been expressed in a variety of heterologous species and widely used as a novel reporter possessing a number of desirable traits in the research fields of modern cellular and molecular biology. The expression of GFP is cell autonomous and independent of cell type and location, Blue or UV light are required for green fluorescence emission but not any other exogenous substrates or cofactors. In addition, fusion protein with GFP can be easily made to provide a fluorescent tag without disturbing the native protein function and compartmentation. GFP has been a vital marker to investigate into gene expression and protein localization in the living tissues.The mutant gfp65T was cut from pFastBacl and ligated into a Ecoli-cyanobacterium shuttle plasmid pRL25C. The recombinant plasmid wastransformed into E.coli HB101 which bears a helper plasmid pRL528. Then three parents: E.coli HB101 bearing the recombinant plasmid and helper plasmid pRL528; E.coli HB101 bearing RP4; and Anabaena PCC7120, a nitrogen-fixing filamentous cyanobacterium were put together to do triparent matings. Both plasmid extraction and specific PCR amplification demonstrated that the engineering Anabaena PCC7120 was constructed successfully. Onlythe red fluorescence could be visualized with a fluorescent microscope probably for no proper filter was used to filter out the red fluorescence emitted from Anabaena PCC7120 of itself. Less bright green fluorescence observed under a con-focal laser scanning microscope showed that green fluorescent protein(gfp) gene was cloned and expressed well.