Project Summary/ Abstract Leprosy has plagued mankind throughout human history and still afflicts 2 to 3 million people in Developing Countries. Although the leprosy bacillus was the first bacterium reported to be associated with human disease, it has yet never been cultivated on artificial media. It can be grown in the footpads of nude mice or systemically in nine-banded armadillos. With a generation time of 14 days, testing drug regimens are performed by counting acid-fast bacilli which is laborious and time-consuming. The comparison of the M. leprae DNA sequence to M. tuberculosis suggests the inability to culture M. leprae on artificial media might result from the inability to synthesize methionine, pantothenate and mycobactin. Using genetic tools, we have developed for M. tuberculosis, we hypothesize M. leprae can be stably transformed with phage- based integration proficient plasmids. We plan to incorporate a Bioluminescent Resonance Energy Transfer (BRET)-Nanoluc and Near infra-red proteins into the plasmid to detect transformants. Furthermore, we hypothesize that the transformation of M. leprae with the missing methionine and pantothenate biosynthetic genes and the mycobactin transporters will enable M. leprae to grow on artificial media. An M. leprae strain that glows would enhance drug development and the ability to assess the immunological mechanisms that control M. leprae infections. The genetic engineering of M. leprae to grow on artificial media would enhance our understanding of evolutionary processes that lead to obligate intracellular parasitism.