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Team:ArtScienceBangalore/Project
From 2009.igem.org
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Geosmin, which means ‘earth odor’, is a volatile microbial metabolite that is responsible for the characteristic smell of moist soil or freshly plowed earth. Geosmin is produced by a number of microorganisms, including most Streptomyces and several species of cyanobacteria, myxobacteria and fungi. Besides its pleasant, characteristic earthy aroma, geosmin is also associated with an undesirable musty odor or off flavor in drinking water, as well as in wine, fish, and other food stuffs. The structure of geosmin was first established as trans- 1,10-dimethyl-trans-9 decalol by N.N Gerber who detected the volatile oil in 17 different species of Streptomyces and a blue green alga following its initial isolation from S. griseus.
Biosynthesis of geosmin from farnesyl diphosphate is catalyzed by a single enzyme germacradienol/germacrene D synthase. Escherichia coli: ispA codes for farnesyl diphosphate (FPP) synthase. FPP synthase catalyzes the sequential condensation of isopentenyl diphosphate (IPP) with dimethylallyl diphosphate (C5) and geranyl diphosphate (C10) to form FPP.
E. coli, does not bear a gene that codes for germacradienol/germacrene D synthase. In our project, we are expressing germacradienol/germacrene D synthase gene in different strains of E. coli, under control of pTrc promoter present in pTrc99a vector (BBa_”---“). This is basically an expression vector (that contains a LacI repressor) which can be induced by IPTG. A map of this vector is given below :
This synthase plasmid SF5 ( ispA+, triangle (srl-recA) 306::Tn10) encoding the whole protein is transformed to the full-length wild strain SF5 to produce Geosmin and eventually the ‘smell’ of rain. This is tested against a knockout strain SF7N ( triangle ispA::neo, triangle (srl-recA) 306 :: Tn10) that did not contain farnesyl synthase which is the precursor to the production of farnesyl diphosphate. Hence, when the plasmid is inoculated, no Geosmin will be expected to form.
Experimental protocol:
- E. coli k12z1 bearing BBa_”---“and k12z1 was grown overnight at 37oC and 250rpm in Luria Bertani (LB) medium containing 100uM ampicillin and spectinomycin respectively.
- BBa_”---“culture was inoculated in fresh 1X glucose-M9 medium, containing 0, 50, 500 and 1000uM of IPTG. AS a negative control K12z1 was inoculated in fresh 1X glucose-M9 medium.
