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Team:ArtScienceBangalore/Project
From 2009.igem.org
Our Project
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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.
- The culture was grown for 12h at 37oC and 250rpm.
- Geosmin analysis was done through smell, and it appeared that k12z1 cells bearing BBa_”--“, are geosmin positive.
We have managed to obtain a mutated strain pSF27 (ispA+, cm R,pSC 101 Ori ts) from Dr. XX at Toho University, which is temperature- sensitive and using this, a controlled ispA strain is being constructed by transforming SF7N with pSF27 wherein on reducing the temperatures, synthesis of Geosmin takes place and the smell of rain is produced.
What did and did not work
While working with the full length synthase plasmid SF5, and testing it with the knockout strain SF7N, no difference could be observed in the two resultant smells. From this we inferred that geosmin was not being synthesized as per our theoretical analysis.
- One reason for this could be the fact that the concentrations of farnesyl diphosphate may not be accurate and require further trial and error through experimentation.
- It was also discovered that the C- terminal of the synthase contained a restriction enzyme which could well be the reason for the failure of our experiment. We also discovered that the expression of the N-terminal domain of the plasmid pTRC99a gave a fully functional germacradienol synthase with steady-state catalytic parameters similar to those of the full-length protein but the expressed C-terminal domain had no detectable FPP cyclase activity. Hence, it was only the N- terminal that was needed for the synthesis of geosmin and the C- terminal was the half that contained the restriction enzyme and as it played no part in the synthesis process, it could be deleted from the gene.
