Division of Microbiology, ICAR-Indian Agricultural Research Institute, New Delhi
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Nothing captures the essence of the heroic era of bacterial genetics better than the immortal phrase ‘just toothpicks and logic’ ascribed to the Nobel Prize winner Sydney Brenner (Shuman, 2003). Over the past 50 years genetic analysis in microbiology has relied predominantly on selections and plate assays using chromogenic enzyme substrates — for example, X-gal assays for the detection of â-galactosidase activity. The power of bacterial genetics lies in the ability to rapidly isolate interesting mutants from large populations of clones (Georgopoulos, 2006). Efforts to understand complex problems of evolutionary, ecological or biochemical significance have placed a burden on the methodologies that are needed for the isolation of mutant bacteria (Habibi, 2013). Advances in instrumentation and experimental design are now opening new vistas for the genetic analysis of bacteria and other microorganisms. Recent advances in fluorescent assays and high throughput screening technologies have paved the way for the rapid isolation of mutants that confer complex phenotypes and for the quantitative analysis of the evolution of new traits in bacterial populations by detection of protein expression, catalytic activity, small molecules and subcellular organization at the single-cell level, with exquisite sensitivity. Techniques such as Forster Resonance Energy Transfer (FRET), fluorescence Correlation Spectroscopy (FCS) and Fluorescence Recovery After Photobleaching (FRAP) can be used to probe the subcellular organization and dynamics of proteins within microbial cells, using either microscopy or flow cytometry and interacting proteins can be detected with fluorescence-based two-hybrid systems that use split GFP or fusions to AFP pairs that exhibit FRET.
Keywords: Mutant bacteria, fluorescent assays, GFP
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