The Establishment Of A Chemical Genetic Screening Method For Inhibiting Mycobacterium Smegmatis

Nowadays, the treatment of tuberculosis is facing a serious situation all over the world. On one hand, the drug R&D is costly and slowly, on the other hand, the problem of bacterial resistance to antibiotics is becoming more and more serious. Identification of new drug targets is the key to solve this problem.In this research, based on chemical genomics and "Multicopy Suppression" theory, we try to establish an anti-mycobacterial drug target screening system which intended to quickly identify targets of active compounds through multiplication of individual genes of Mycobacterium smegmalis. The colony harboring target genes is likely to show elevated resistance to a given active compound.We cloned the M. smegmatis oriM and the kanamycin resistance gene aph into fosmid vector to generate new shuttle vector pEMAC, which was used for constructing the genomic library. However, we found that these plasmids were not stable in the M smegmatis. To solve this problem, we constructed a new shuttle vector pMINDcos1, which was used to construct a new genomic library, but these new library plasmids were unstable too. The attempt to construct the genomic library with small fragments (3.5kb to4.5kb) also did not work. Furthermore, we also tried to knock out the recA gene, or demethylate the plasmid before electrotransformate into M. smegmatis to increase the stability of the plasmid.Considering the results of this study, we suspected the plasmid stability in the M. smegmatis may be related to the sequence of the inserts that the library plasmids harbored.