Abstract
Genetically amenable cyanobacteria are most suited for analyzing the mechanism of plant-type oxygen evolution. We have constructed cyanobacterial strains of Synechococcus elongatus PCC 7942 which carried thermostable D1/D2 heterodimer of photosystemII(PSII) from Thermosynechococcus vulcanus. In this study, a Histag was inserted at CP47 subunit of PSII, enabling purification of PSII via affinity chromatography. For this purpose, rps12 -mediated gene replacement scheme was employed to construct recombinant strains carrying psbB gene with Histag sequence at the 3' end. PCR analysis of the recombinant strains confirmed that chromosomal psbB-psbT operon had been replaced with psbB-histag::[rps12-kan]-psbT.
To circumvent the adverse effect on the downstream psbT gene, a surrogate psbB promoter was inserted upstream the psbT gene to make a recombinant strain GRPS801.These strains as well as strains devoid of [rps12-kan ] cassette are now tested for one-step purification of PSII complex.
