Cambridge University Technical Services Limited

Advanced Technologies (cambridge) Limited

John Anthony Charles Archer

David Keith Summers

Herve Jacquiau Roland

Jutta Anna T&uuml;rck

Justin Antoine Christian Powell

Disclosed are methods for generating mycolic acid bacterial biosensors for particular analytes (especially industrial pollutants) by the use of innovative methods for isolating DNA encoding an inducible promoter which is induced in response to the specific analyte (and/or associated operon proteins), the methods generally comprising the steps of: (a) culturing a source of mycolic acid bacteria in a selective medium containing said specific analyte and being selective for oligotriphic bacteria; (b) identifying mycolic acid bacteria capable of subsisting on said medium, especially those which do not display catabolic repression; (c) extracting DNA from said mycolic acid bacteria; (d) incorporating said DNA into vectors, such as various shuttle vectors; (e) cloning said vector into a suitable host cell (which may bestrain carrying one or more of the mcrABC mrr hsdSRM recA and recO mutations); (f) screening that host cell (or a second host cell which is preferably a) for said inducible promoter. The methods are exemplified by the isolation of theohp operon.

Biosensor materials and methods

A chemically inducible gene expression system is described. A chimeric gene having a first sequence comprising a promoter and a regulator polypeptide is linked with a second sequence comprising a promoter and a coding or non-coding sequence. Expression of the target gene of the second sequence is controlled by the regulator polypeptide which is acted upon by an inducer. The inducer is a chemical compound, such as OHP, which acts upon the OHP responsive regulator polypeptide, which can be obtained from Rhodococcus sp. V49. Various domain regions and complementary response elements are also described.

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John Anthony Charles Archer