AI SearchCloning of fungal chitin deacetylase gene into bacterial Escherichia coli
Call Number: AIT Thesis no. BP-01-05 Material type:
SeriesSeries: Asian Institute of Technology. Thesis ; no. BP-01-05Publication details: Bangkok : Asian Institute of Technology, 2001Description: 40 leavesSubject(s): Online resources: Dissertation note: Thesis (M.Sc.) - Asian Institute of Technology,2001 Summary: Chitin deacetylase(CDA)gene from Colletotrichum lindemuthianum UPS9 has been used in this study. The gene cloned into shuttle vector pPIC9K has been used as a source of CDA gene throughout the study. Large quantity of gene was obtained allowing its multiplication in component E.coli TOPlOF'. QIAGEN plasmid midi Kit was used for the extraction and purification of the DNA. The gene was integrated into a another shuttle vector pPIC3.5K in its multiple cloning site. The plasmid vector pPIC3.5Kb is 9.0 kb long plasmid earring ampicillin and kanamycin resistant markers. It can be multiplied in E.coli as well as in Picia pastoris. In E.coli, it remains extrachromosomally and multiplies independently. It remains as a low copy number plasmid. On the other hand , in Pichia pastoris, it intergrates into genome via homologous recombination and results in stable transfmmation. In this research work, CDA gene has been intergrated in between EcoRI and Natl sites of multiple cloning sites. Recombinant plasmid was transformed into competent E.coli TOPIOF' intermediate host by calcium chloride method in order to obtain the plasmid in large quantity. The integration of CDA gene was confimed by restriction digestion analysis followed by gel electrophoretic migration and size comparison.
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22-AIT Thesis (Replacement)
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A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science, School of Environment, Resources and Development
Thesis (M.Sc.) - Asian Institute of Technology,2001
Chitin deacetylase(CDA)gene from Colletotrichum lindemuthianum UPS9 has been used in this study. The gene cloned into shuttle vector pPIC9K has been used as a source of CDA gene throughout the study. Large quantity of gene was obtained allowing its multiplication in component E.coli TOPlOF'. QIAGEN plasmid midi Kit was used for the extraction and purification of the DNA. The gene was integrated into a another shuttle vector pPIC3.5K in its multiple cloning site. The plasmid vector pPIC3.5Kb is 9.0 kb long plasmid earring ampicillin and kanamycin resistant markers. It can be multiplied in E.coli as well as in Picia pastoris. In E.coli, it remains extrachromosomally and multiplies independently. It remains as a low copy number plasmid. On the other hand , in Pichia pastoris, it intergrates into genome via homologous recombination and results in stable transfmmation. In this research work, CDA gene has been intergrated in between EcoRI and Natl sites of multiple cloning sites. Recombinant plasmid was transformed into competent E.coli TOPIOF' intermediate host by calcium chloride method in order to obtain the plasmid in large quantity. The integration of CDA gene was confimed by restriction digestion analysis followed by gel electrophoretic migration and size comparison.
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