Cloning of Gene Coding Glyceraldehyde 3-Phosphate Dehydrogenase

Glyceraldehyde-3-phosphate dehydrogenase (GPD, E.C. 1.2.1.12) is one of the key enzymes in the Embden Meyerhof Parnas or glocolysis pathway. It catalyzes phosphorylation of glyceraldehyde-3-phosphate to produce 1, 3-diphosphoglycerate. GPD is a tetrameric enzyme composed of four identical subunits. It is an essential enzyme used to maintain life activities through contributing in thisway to the formation of ATP and providing additional energy to the cell by reducing NADH to NAD+ and H+ upon its action. Moreover, GPD protein also has many other important functions, such as abiotic stress tolerance (Liu and Yang, 2005).The pUC18 vector was isolated from E.coli Top10 strain. It is a prokaryotic vector carrying a multiple cloning site with 13 unique restriction sites. This multiple cloning sites are located within the lacZ’ gene resulting in the disruption of β-galactosidase activity by cloned inserts allowing blue/white selection. The vector pUC18 was restricted using the restriction enzyme Eco.RI. Further it was thymidized to provide sticky ends so that desired gene can be inserted in it. The GAPDH gene was amplified using PCR. Then it was inserted/ligated into cloning vector pUC18.It was then transformed into the suitable competent cells which were freshly prepared using JM107 strain of E.coli which is devoid of plasmid. Inside the host cell the recombinant DNA had undergone replication; thus, a bacterial host gave rise to a colony of cells containing the cloned target gene i.e. GAPDH. Various screening methods could be used to identify such colonies, enabling them to be selected and cultured.