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Isolation of SODA gene from Mycobacterium tuberculosis and its expression through Escherichia coli.


The International Journal of Biological Research (TIJOBR)

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Saad Yousaf Cheema*, Mishaim Khalid, Saba Gulzar and Nimra Malik.

Institute of Molecular Biology and Biotechnology (IMBB),

 The University of Lahore, Defense Road Campus, Lahore, Pakistan.

*Corresponding Author: Email: saadyousafcheema@gmail.com 

Submitted Accepted Published
Oct 27,2019 Dec 12,2019 Jan 13,2020

2020 / Vol: 3 / Issue: 2


Abstract


Mycobacterium tuberculosis is the major cause of tuberculosis in humans. It is a non-motile bacteria requiring high level of oxygen for growth. Infection starts when bacteria enters into lungs through inhalation, it grows very slowly, and the infection is mostly caused in people with compromised immune system. Superoxide dismutase (SOD) is a protein present in Mycobacterium tuberculosis. SOD is a protective protein present in bacteria, which protects bacteria from poisonous impact of reactive oxygen. SOD fixes radicals of free oxygen molecules present in bacteria. There is an iron co-factored SOD, also known as SODA, which is encoded by Rv3846 gene, and is 624bp long with molecular mass of 23 kDa. SODA is necessary for viability, and it is secreted in large amount. For cloning of SODA 624bp, reverse and forward primers were designed, which were 25bp each, then amplified with annealing temperate of 60oC. Further amplified gene was purified from gel with phenol/chloroform method and geneJet column kit method. Then E. coli were grown in LB broth and pet28avector was isolated. Then both vector and amplified PCR product were digested with Hind III and Nde I, and then ligated. The recombinant plasmid having SODA gene was successfully transformed into E. coli cells and their presence was conformed through colony PCR of transformed colonies. The work done in this project would provide easy expression of SODA protein. The SODA is potential candidate for rapid detection of tuberculosis through immunoassay. 

Keywords: Superoxide dismutase, Cloning, Mycobacterium Tuberculosis, Escherichia coli, Polymerase Chain reaction method (PCR). 


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