Gary Fernandes, Phd Email and Phone Number

Much like the adaptive Immune system seen in human beings, prokaryotes, both bacteria and archaea, have been recently shown to possess a similar such system known as the CRISPR/Cas system. The intricacies of the system have been well documented in common bacteria but not much is known about the CRISPR/Cas in the pathogenic spirochete, Leptospira interrogans. Deciphering the CRISPR/Cas architecture in Leptospira interrogans could lead to a more detailed understanding of the pathophysiology of Leptospirosis in Humans. Moreover CRISPR/Cas could be used as a genetic tool to manipulate the genome of Leptospira which is often considered a tedious task. There are five genes implicated with the CRISPR/Cas defense system of Leptospira interrogans serovar Copenhagani, namely Cas1, Cas2, Cas3, Cas4 and Cas5. With the help of primers designed to carry out directional cloning, I carried out the cloning of all five genes of the CRISPR/Cas system into the pET28a expression vector. For analyzing the expression of the recombinant proteins, I used E.coli Bl21 (DE3) as an expression because of its high yield, absence of post translational modification and ease of purification. Initially, I conducted a pilot experiment in 1ml to check the expression of individual genes. On a large scale the expression and protein purification of only one of the recombinant proteins, Cas2, was optimized. With the purified and active form of the protein Cas2, I have conducted been conducting a biochemical nuclease activity assay which has been suspected as its main function. Moreover, I have designed primers for mutation studies and have used the mutant form of Cas2 to throw more light on the amino acids involved in both functional as well as structural analysis. I used isothermal calorimetry to determine the metal dependence of cas2. Moreover I have also carried out co-expression of two proteins from a single host to determine the role of its interaction with cas1.