| Description |
The isoprenoid biosynthetic pathway is present in some form in all organisms. Two essential enzymes in the pathway are isopentenyl diphosphate (IPP) isomerase and farnesyl diphosphate (FPP) synthase. IPP isomerase catalyzes an essential activation step in the pathway. FPP synthase catalyzes the synthesis of FPP, which is a key intermediate that serves as a substrate for many important branch-point enzymes in the pathway. The purpose of this project was to construct a synthetic operon containing the IPP isomerase gene and the FPP synthase gene. Translational coupling of the two genes was accomplished by inserting bases in the operon which link genes in the tryptophan operon in E. coli. With such an operon, IPP can quickly be converted to FPP. The operon was constructed by cutting the two genes out of plasmids and inserting them both in the same plasmid with the bases of the tryptophan operon between the genes. The plasmid was inserted into E. coli, and the enzymes encoded by the genes were produced. The activity of the enzymes was measured by adding IPP and determining how much FPP was produced. The operon was successfully constructed, and thus FPP can quickly be synthesized from IPP. Additionally, a fusion protein containing IPP isomerase and FPP synthase will be made from one of the plasmids containing the operon. |
