Fluorescence assay to monitor dormancy in M. luteus.

Tuberculosis is one of the most prevalent infectious diseases, afflicting nearly 10 million and killing ~ 1.5 million people in 2021 (2). Mycobacterium tuberculosis cannot be eradicated entirely because it undergoes clinical dormancy during antibiotic challenge. In this state, the bacteria are not susceptible to antibiotics. How Mycobacterium tuberculosis enters dormancy is key to develop drugs that can prevent dormancy and lead to eradication. Micrococcus luteus (M. luteus) is used as a model organism for studying M. tuberculosis because it is a nonpathogenic Actinobacteria with a short doubling time, smaller genome that has a reproducible dormant state like M. tuberculosis (1). Dormancy is described as a Viable But Non Culturable (VBNC) state of bacteria where dormant cells fail to grow on solid media (1). A more sensitive and convenient measure of dormancy is required for rapid determination of when cells enter or exit dormancy. This assay will allow us to monitor the change in fluorescent signals of individual cells as they switch from active state to dormancy. In this subproject, we aim to create a plasmid that allows the transfer and insertion of any gene, specifically the Green Fluorescent Protein (GFP) gene, into the M. luteus genome via homologous recombination. The plasmid consists of the open reading frame of a growth-neutral gene in M. luteus called ctrE, the chloramphenicol resistance gene (CMr), and the flanking sequences that are complementary to the regions surrounding crtE gene in M. luteus genome.