A lacZ Reporter-Based Strategy for Rapid Expression Analysis and Target Validation of Mycobacterium tuberculosis Latent Infection Genes

Abstract

We report a novel lacZ fusion vector and demonstrate its utility for expression analysis of genes associated with Mycobacterium tuberculosis latent infection. The vector contains E. coli (oriE) and mycobacterial (oriM) origins of replication, a kanamycin resistance gene (Kmr) as selection marker, and a lacZ reporter gene in fusion with MCS for cloning of upstream regulatory sequence of the desired genes. b-galactosidase activity of the vector was standardized for expression analysis under latent mycobacterial conditions using Phsp60, a constitutive mycobacterial promoter, utilizing Mycobacterium smegmatis as model organism. Validation of the vector was done by cloning and expression analysis of PhspX (alpha crystalline) and Picl (isocitrate lyase), promoters from two of the genes shown to be involved in M. tuberculosis persistence.