Team:ArtScienceBangalore/Experiments

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<h3> Experimental protocol:</h3>
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<p>
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<ol>
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<p>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: </p>  
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<li>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.</li>
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<ol>  
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<li>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.</li>
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  <li>K12z1(pTrc99a-gs)and k12z1 were grown overnight at 37oC and 250rpm in Luria Bertani (LB) medium containing 100uM ampicillin and spectinomycin respectively.</li>  
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<li>The culture was grown for 12h at 37oC and 250rpm.</li>
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  <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>  
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<li>Geosmin analysis was done through smell, and it appeared that k12z1 cells bearing BBa_”--“, are geosmin positive.</li>
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  <li>The cultures were grown for 12h at 37oC and 250rpm.</li>  
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</ol>
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  <li>Geosmin analysis was done through smell but we could not make any difference between IPTG induced K12z1(pTrc99a-gs) and negative control.</li>  
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</ol>  
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<p> 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:
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<ol> 
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<p>
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  <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>  
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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.
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  <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>  
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</p>
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  <li>The cultures were grown for 12h at 37oC and 250rpm.</li>  
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<h3>What did and did not work</h3>
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  <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>  
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<p>
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</ol>  
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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.
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<li>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.</li>
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<li>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.</li>
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<strong><big>Ongoing/Future Work</strong></big> 
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<p> 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:
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<ol> 
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<li>Improving concentration of geosmin. This can be achieved by growing cells in medium containing synthetic farnesyl diphosphate and IPTG .</li> 
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<li> GC-MS characterization of geosmin.</li>
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<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>
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Latest revision as of 22:01, 21 October 2009

Experiments

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:

  1. K12z1(pTrc99a-gs)and k12z1 were grown overnight at 37oC and 250rpm in Luria Bertani (LB) medium containing 100uM ampicillin and spectinomycin respectively.
  2. 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.
  3. The cultures were grown for 12h at 37oC and 250rpm.
  4. Geosmin analysis was done through smell but we could not make any difference between IPTG induced K12z1(pTrc99a-gs) and negative control.

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:

  1. 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.
  2. 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.
  3. The cultures were grown for 12h at 37oC and 250rpm.
  4. 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.
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:

  1. Improving concentration of geosmin. This can be achieved by growing cells in medium containing synthetic farnesyl diphosphate and IPTG .
  2. GC-MS characterization of geosmin.
  3. 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.

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