Biophysical Society Thematic Meeting| Lima 2019

Revisiting the Central Dogma of Molecular Biology at the Single-Molecule Level

Poster Abstracts

1-POS Board 1 DEVELOPMENT OF A STABLE HETEROLOGOUS EXPRESSION SYSTEM OF THE RNA POLYMERASE OF MYCOBACTERIUM TUBERCULOSIS BY GENOMIC INTEGRATION IN E. COLI Rodrigo Alarcón Rodríguez Paiva 1 ; Daniel Guerra Giraldez 1 ; 1 Universidad Peruana Cayetano Heredia, Faculty of Science and Philosophy, Lima, Peru The RNA polymerase of Mycobacterium tuberculosis is an enzyme of high biomedical interest due to its importance as drug target and the occurrence of polymorphisms linked to drug resistance. In order to facilitate its study, an inducible expression system was developed previously in E. coli. This system consists of the 5 genes coding for the holoenzyme subunits distributed among two low-copy plasmids. Unfortunately, this system presents several difficulties, and frequently a low yield of active M. tuberculosis protein. When different colonies of the same expression clone were tested simultaneously, they showed high heterogeneity in their level of protein production and specific activity. Possibly, this heterogeneity is caused by non- constant copy number of the plasmids and consequent variability in the stoichiometry of the subunits in the expressing cell. Moreover, the expression of these heterologous proteins exerts great stress over the bacteria, evidenced by a strong inhibition of its growth. We hypothesize that this stress contributes to the low expression and heterogeneity in protein production. In this project, we integrated the genes for α, β, β’ and ω subunits, which constitute the apoenzyme of M. tuberculosis, into the E. coli genome using a viral recombination site. We hypothesized that with a single copy of the genes and with the superior stability of genomic DNA, the problems presented would be solved. The σA subunit was introduced with a low copy plasmid to allow eventual production of holoenzymes with different σ factors. A second plasmid was introduced, bearing a GFP gene under the control of a promoter exclusively recognized by the ARN polymerase of M. tuberculosis. We observed a strong signal corresponding to the activity of the heterologous enzyme, exclusively when its expression was induced. This new expression system can have applications in detection of inhibitors, transcription and protein regulation studies, among others.

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