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Scientists in THSTI Develop Novel Genetic Tools to speed up Drug Discovery Research

Genetic information of a living organism is pivotal in understanding its biology. Every single gene carries a code, which defines a particular function in the cell. How exciting it would be if the genes of bacteria like the one that causes cholera and tuberculosis can be studied by turning them “on” or “off” at the will of the scientists? This is not an easy task as the disease causing bacteria are often smart and are difficult to manipulate. Scientists have to outwit them by constantly developing novel tools and techniques. Two scientists at the Translational Health Science and Technology Institute (THSTI) have independently created tools that have successfully addressed this challenge. Their research work was majorly funded by the Department of Biotechnology, Ministry of Science and Technology, Government of India.

Dr. Bhabatosh Das, Assistant Professor at the Centre for Human Microbial Ecology, THSTI, and his team have successfully built a delivery tool technically called “vector” for introducing and controlling any gene that they wish to study in a range of bacteria. The scientific intelligence involved in the building of this vector termed as pBD32 (BD is abbreviation for Bhabatosh Das) is the selection of the DNA from various sources and bringing them together into one delivery system. pBD32 has a piece of DNA termed as XBS selected from a virus, which in nature was known to attack the cholera bacteria and with the help of this DNA fragment goes and sits inside the bacterial chromosome. Dr. Das believed that XBS would come handy to place any gene of interest in other bacterial chromosome to turn the gene “on” or “off”. Scientifically this is termed as a stable, broad host range tightly regulated expression system that as described by Dr. Das and his team in their Journal of Bacteriology Publication (Volume: 196, Number: 23, December 2014, Pages – 4071 4080) could be used in several bacteria like Vibrio cholerae (causing Cholera) and Klebsiella pneumoniae (causing Pneumonia), Salmonella enterica (causing Typhoid fever), Escherichia coli (lives in human gut).

Scientists working across the world like USA, Germany, UK and Japan are using this genetic tool for functional studies in the drug discovery field and highly appreciated the development with encouraging complements:

Victor Di Rita, University of Michigan, USA: “This clever new vector will be a great value to many researchers and I am pleased we are presenting it to the field as a paper in Journal of Bacteriology”.

Dhruba Chattoraj, NIH Bethesda, USA: “You have expanded the repertoire Vc genetic toolbox by making some really useful vectors”.

Christian Berens, Institute of Molecular pathogenesis, Germany: “Thank you very much for your efforts and for providing this vector to the community”.

Robert Blumenthal, The University of Toledo College of Medicine, USA: “I read your J. Bacteriol paper with great interest, and believe that pBD62 could be very useful in my research on E. coli recombination”.

Dr. Catherine Monahan, Cubist Pharmaceuticals, Inc. USA: “The work described in your paper is very interesting, particularly the plasmid’s ability to stably integrate as a single copy into the chromosome of different bacterial species. The capability to differentially regulate the expression of a cloned gene using the pBAD promoter in various backgrounds is very exciting”.

When it comes to the tuberculosis causing bacterium Mycobacterium tuberculosis, nothing is ever enough as this bacterium has successfully made it difficult for the scientists’ to study its essential genes. Tuberculosis is a major global killer accounting for two deaths every three minutes killing ~ 2 million people every year. None of the tools developed till date has been proved to be greatly efficient in elucidating essential gene function in TB. Turning “off” the essential genes is a primary approach but is a major challenge as most of the delivery tools do not achieve finding the exact place where the essential gene sits in the mycobacterial genome and do not have efficient blocking mechanism for causing loss of function. Dr. Nisheeth Agarwal, Assistant Professor at the Vaccine and Infectious Disease Research Center, THSTI, and his team have tackled it by improvising the CRISPRi (Clustered Regularly Interspaced Short Palindromic Repeat Interference) tool for disrupting the mycobacterial genes in a cost effective manner. This tool employs a foreign protein dCas9 borrowed from other bacteria and tweaked by his team to work in mycobacteria, and a stretch of oligonucleotide fragment termed as guide RNA (sgRNA) designed to pair with the gene in the mycobacteria that the scientist would like to disrupt. Dr. Agarwal’s team has successfully engineered specific guide RNAs-dCas9 complex for many essential genes of the mycobacteria and have disrupted them from becoming functional. This tool is simple, cost effective and more importantly quicker considering that the tuberculosis causing mycobacteria takes a long time to culture under laboratory conditions and precious time is lost in the absence of quick manipulative tools. More than 50 genes could be inactivated in a short span of six months using this tool in Dr. Agarwal’s laboratory. This will enable identifying new pathways unique to M.tuberculosis growth and design drugs against it in a significantly greater pace. The study was published in Nature Communications on 25th February, 2015 and has received worldwide acclaim from the community of eminent TB researchers:

Dr. Jeff Cox, University of California San Francisco: “it represents an exciting advance for the field”.

Dr. Jiayun Liu, Johns Hopkins University: “I admire you very much for your great achievement in this field”.

Dr. Dirk Schnappinger, Weill Cornell Institute: “Congratulations on your recent paper in Nature communications, very nice work”.

Dr. Sarah Stanley, University of California, Berkeley: “Congratulations on this excellent publication of a breakthrough technology for those of us in the M. tuberculosis field.