Promoter analysis in mycobacteria using xyIE reporter assays and its implication in high throughput screening.
Understanding the molecular biology of Mycobacterium tuberculosis has become the focus of recent studies in infectious diseases. Here, we describe a spectrophotometric method for quantitative analysis of promoters in mycobacteria. The assay is a modification of the xyIE reporter assay, which uses an inexpensive substrate, catechol. The distinguishing feature of this protocol is that cells are grown by patching on agar plates, and directly used in the assays. The absorption measurements of the cell suspension at 375 nm in the presence and absence of the substrate are used for obtaining xyIE activity and for normalization of the assay values respectively. These features make the assay system directly amenable for high throughput analyses.
Dastur
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Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560 012, India.
Varshney
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U
D050557:Catechol 2,3-Dioxygenase; D049308:Dioxygenases; D017930:Genes, Reporter; D006801:Humans; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D010105:Oxygenases / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D013053:Spectrophotometry / Q000379:methods
D010105:Oxygenases; D049308:Dioxygenases; D050557:Catechol 2,3-Dioxygenase