Team:ArtScienceBangalore/Project

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Geosmin - Our Hero 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.

Synthesis of Geosmin 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 lacI repressible  pTrc  promoter present in pTrc99a vector. This is basically an expression vector (that contains a lacI repressor) which can be induced by IPTG.

Wild type strain SF5 was transformed with pTrc99a expressing the full length germacradienol/germacrene D synthase (pTrc99a-gs) to produce Geosmin and eventually the ‘smell’ of rain. This is tested against a knockout strain SF7N ( delta ispA::neo, delta (srl-recA) 306 :: Tn10) that did not contain farnesyl synthase which is the precursor to the production of farnesyl diphosphate. Hence, when SF7N is transformed with the pTrc99a-gs, no Geosmin will be expected to form.

Experimental Protocol Preliminary geosmin screening was done using E.coli strain, K12z1. This strain is spectinomycin resistant and has chromosomal copy of tetR and lacI under some constitutive promoters. We went through following methods: <ol> K12z1(pTrc99a-gs)and k12z1 were grown overnight at 37 degree centigrade and 250rpm in Luria Bertani (LB) medium containing 100uM ampicillin and spectinomycin respectively.</li> K12z1(pTrc99a-gs)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.</li> The cultures were grown for 12h at 37 degree centigrade and 250rpm.</li> Geosmin analysis was done through smell but we could not make any difference between IPTG induced K12z1(pTrc99a-gs) and negative control.</li> </ol> Geosmin was further characterized using strains, SF5 and SF7N. SF5 is wild type strain and SF7N is ispA mutated strain. ispA gene product codes for farnesyl synthase enzyme that synthesizes farnesyl diphosphate, a precursor of geosmin. SF5 and SF7N were transformed with pTrc99a-gs. We went through following methods to charectrize the geosmin:

<ol>

<li>SF5(pTrc99a-gs),SF7N(pTrc99a-gs)SF5 and SF7N were grown overnight at 37 degree centigrade and 250rpm in Luria Bertani (LB) medium containing 100uM ampicillin and spectinomycin respectively.</li> <li>SF5(pTrc99a-gs) and SF7N(pTrc99a-gs)cultures were inoculated in fresh 1X glucose-M9 medium, containing 0, 50, 500 and 1000uM of IPTG. As a negative control SF5 and SF7N were inoculated in fresh 1X glucose-M9 medium.</li> <li>The cultures were grown for 12h at 37 degree centigrade and 250rpm.</li> <li>Geosmin analysis was done through smell but we could not make any difference between IPTG induced SF5(pTrc99a-gs) and SF7N(pTrc99a-gs) and negative controls.</li> </ol>

Summary Of Outcomes Upto This Point After cloning of germacradienol/germacrene D synthase in pTrc99a vector, restriction digestion was done and  we got germacradienol/germacrene D synthase gene insert of specific size.Sequencing was also done and contruct were found to be positive. Germacradienol/germacrene D synthase enzyme was preliminary charectized by SDS-gel electrophoresis,which showed band of correct size in IPTG induced samples. Reason, why we are unable to differentiate between induced samples and negative controls may be due to low synthesis of Geosmin that is out of the detection limit of our olfactory system.

Ongoing/Future Work Presently, we are working on characterization of geosmin. In future, we wish to make a temperature-sensor bug that will allow geosmin expression under some specific temperature conditions. A brief summary of our ongoing and future works are: <ol>

<li>Improving concentration of geosmin. This can be achieved by growing cells in medium containing synthetic farnesyl diphosphate and IPTG .</li>

<li> GC-MS characterization of geosmin.</li>

<li>We have managed to obtain a plasmid pSF27 (ispA+, cm R,pSC 101 Ori ts) from Dr. Fujisaki 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.</li> </ol> </ol>

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