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Jaiyanth Daniel Ph.D.
Department of Biology
Indiana University – Purdue University Fort Wayne
Mycobacterium tuberculosis(Mtb) is very hard to target with regular antibiotics because it tends to go into a dormant stage in which it stores fat, like triacylglycerol (TAG), from the human host. In order to be able to combat the dormant Mtb, it is necessary to first discover the pathways and biomolecules involved in the transport of TAG. We report here our study of a gene thought to encode for a mycobacterial Fatty Acid Transport Protein 1 (mFATP1). We cloned the gene from the genomic DNA of Mtb by PCR into ZeroBlunt TOPO plasmid and transformed E.coliTOP10. After confirming that the DNA sequence was correct, the mFATP gene was digested with HindIII and EcoRI restriction enzymes, ligated into the plasmid pBAD Myc/HisA and E.coliTOP10 cells were transformed with the construct. Following DNA sequencing and digestion by restriction enzymes to confirm the integrity of the cloned gene, the pBAD plasmid containing the mFATP1 gene was used to transform mutant E. colicells (LS6164) which lacked their native fatty acid transport protein. Transformants were selected using antibiotic-containing plates. Expression of the mFATP1 protein in LS6164 cells was induced with L-arabinose. Denaturing protein gel electrophoresis confirmed that the mFATP1 protein was being expressed in the cells.
Daniel, Jaiyanth, "Expression of a Mycobacterial Fatty Acid Transport Protein in a Heterologous Bacterial Cell" (2014). 2014 Science and Society at IPFW (SASI). 3.