Verification of Plasmid Transformation through Gel Visualization

Abstract

Malaria is an infectious disease affecting over 200 million people annually, wherein the most life-threatening cases are transmitted through the parasite Plasmodium falciparum. P. falciparum, as well as many other pathogenic protozoans and bacteria, utilize the methyl erythritol phosphate (MEP) pathway to synthesize the building blocks of isoprenoids; vital metabolites with key cellular functions such as respiration and cell wall biosynthesis. Due to the absence of the MEP pathway in humans, this pathway and its respective enzymes make attractive targets for the development of novel antibiotics. To facilitate further drug development, binding interactions between the first committed MEP pathway enzyme, 1-deoxy-D-xylulose 5-phosphate reductoisomerase (IspC) and bisubstrate inhibitors are being explored. This research focuses on confirming the transformation of IspC mutant plasmids into E. coli storage strains. Five mutants of P. falciparum IspC were rationally designed to assess the binding interactions between the inhibitors and NADPH binding site. Transformation of the mutant constructs were validated through DNA purification, restriction endonuclease digestion, and agarose gel electrophoresis.