Cloning of cellulase gene(s) of trichoderma reesei in sacchromyces cerevisiae

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dc.contributor.advisorChaudhary, Kamla
dc.contributor.authorRochika
dc.date.accessioned2016-11-22T11:58:59Z
dc.date.available2016-11-22T11:58:59Z
dc.date.issued2007
dc.description.abstractA total of seven Trichoderma reesei cultures were purified by reculturing on potato dextrose agar medium. Cellulase production by T. reesei cultures varied from 0.73 IU/ml to a maximum of 3.11 IU/ml in cellulose medium. Among all the strains evaluated, T.reesei culture 5A showed the maximum cellulase activity (3.11 IU). Cellulase activity of T. reesei 5A was monitored at different interval of time (12-96 hrs.) and maximum activity was observed after 96 hrs. of growth. Genomic DNA of T. reesei 5A was isolated and checked for its quality by agarose gel VI electrophoresis and by UV spectrophotometer. The quantity of DNA was found to range between 195 μg/ml to 2607.5 μg/ml. Different parameters i. e. growth period of 36 hrs., CTAB concentration 1.5% and incubation period of 90 min., were found to be optimum during DNA isolation. Partial digestion of genomic DNA was done with Sau 3A and the digested DNA was ligated to Bam H1 treated Yeast-E. coli shuttle vector YEpFLAG-1. The construct was used for transformation of E. coli. Recombinant clones of E. coli were screened on carboxy methyl cellulose and ampicillin containing Reese medium. The recombinant plasmids YEpFLAG-1-cel-exo and YEpFLAG-1-cel-endo were isolated from E. coli recombinant clones and checked for amplification of cellulase gene by PCR using the standard conditions. PCR amplification was observed for exoglucanase and endoglucanase gene. Protoplast of yeast Saccharomyces cerevisiae were transformed with the recombinant plasmids YEpFLAG-1-cel-exo and YEpFLAG-1-cel-endo isolated from E.coli. The recombinant clones of yeast were screened on YEP medium containing 1% phosphocellulose and ampicillin. Expression of cellulase gene(s) in S. cerevisiae transformants was checked by monitoring the cellulase activity and ethanol production. CMCase activity was observed in all the three yeast transformants as 0.80 IU/ml, 0.50 IU/ml and 0.70 IU/ml respectively. No FPase activity could be detected in any of the yeast transformants. Different concentrations of phosphocellulose (2% and 5%) were used for ethanol production by the yeast transformants. Yeast transformant-6 produced maximum 1.5% alcohol when inoculated in 5% phosphocellulose containing YEP medium. Yeast transformant-2 and transformant-4 also produced ethanol in rice straw medium.en_US
dc.identifier.urihttp://krishikosh.egranth.ac.in/handle/1/86900
dc.language.isoenen_US
dc.publisherCCSHAUen_US
dc.subBiotechnology and Molecular Biology
dc.subjectTrichoderma reesei, E. coli, Saccharomyces cerevisiae, Cellulase, Cloningen_US
dc.these.typeM.Sc
dc.titleCloning of cellulase gene(s) of trichoderma reesei in sacchromyces cerevisiaeen_US
dc.typeThesisen_US
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