Lon proteases are enzymes responsible for many functions in both eukaryotic and prokaryotic cells, such as protein degradation, and the breakdown of regulatory proteins. Very little is known about the function of Lon in Borrelia burgdorferi, the causative agent of Lyme disease. We are interested in finding the functions of this protein in this organism. We are attempting to study Lon in two separate ways simultaneously. First by purifying the enzyme to see the effect it has on other proteins in B. burgdorferi and second by inactivating it and observing the downstream effects in B. burgdorferi due to the lack of this protein.
In order to purify the protein we need to express it in its native form in E. coli. As a first step, we amplified the B. burgdorferi lon coding sequence by PCR and inserted it by a ligation reaction into a pET28a vector which contains a kanamycin resistance gene and a His tag region. The plasmid was then transformed into competent E-coli cells, which were then plated on agar plates containing kanamycin and incubated overnight. The protein will be induced by IPTG and purified by gel filtration.The second experiment, will help conclude which proteins are the substrates of Lon in B.burgdorferi. To that end we are trying to inactivate this gene in B. burgdorferi by disrupting the coding region of the gene. A plasmid was constructed with a kanamycin cassette inserted into the middle of the B. burgdorferi lon coding sequence. The plasmid was then electroporated into competent B. burgdorferi cells. The electroporated bacteria were then suspended in media and placed to grow both in a 96 well tray in liquid media with kanamycin and on soft agar plates with kanamycin. PCR will be utilized to screen for lon mutants.Supported with funding from the Simons Foundation.