Insertion of the Arabidopsis cao gene into Synechocystis PCC 6803 for the conversion of Chlorophyll a to Chlorophyll b
In Arabidopsis thaliana, the conversion of Chlorophyll a to Chlorophyll b is carried out by an oxygenase (CAO). Cyanobacteria are unable to convert Chlorophyll a to b due to the absence of the cao gene. Work described here focuses on conversion of Chlorophyll a to b using homologous recombination of the cao gene of Arabidopsis thaliana into the Cyanobacterium Synechocystis sp. PCC 6803 genomic DNA. Efficient expression of the cao gene in Synechocystis has been shown to require the promoter Ppsa. In order to achieve homologous recombination of the cao gene into Synechocystis genomic DNA, a 3000 bp region of genomic DNA containing an internal BmgBI site was identified, PCR amplified, and cloned into pCR2.1 cloning vector. The SalI site was then used to clone the cao gene under control of Ppsa along with the antibiotic resistance cassette (aadA, specR) affording selection of transformants. Transformation was confirmed by PCR and Western blot analysis. Whole cell acetone extracts of these transformants were subjected to thin layer chromatography on silica gel 60 plates using 60% (v/v) petroleum ether/16% (v/v) cyclohexane/10% (v/v) ethyl acetate/10% (v/v) acetone/4% (v/v) methanol as the mobile phase. Pigment bands with Rf values identical to Chlorophyll a and b were scraped, re-extracted with acetone and subjected to mass spectroscopic analysis confirming the conversion of Chlorophyll a to b.