Team:ArtScienceBangalore/Experiments

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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 37oC 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 37oC 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 a wild type strain and SF7N is an ispA mutated strain. The 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 37oC 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 37oC 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>

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>