Construction of a gene knockout cassette for leucosporidium antarcticum

Construction of a gene knockout cassette for leucosporidium antarcticum

Leucosporidium .antarcticum, a psychrophilic yeast was isolated from Antarctica. It grew optimally at 12°C, and adapted well to the cold temperature in Antarctic. The whole genome of the L. antarcticum has been sequenced using the 454 system. It is ideal to conduct systematic knockout of genes to d...

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Main Author: Koh, Joseph Soon Peng
Format: Thesis
Language:English
English
Published: 2011
Subjects:
QK504-(638) Cryptogams
Online Access:https://eprints.ums.edu.my/id/eprint/38592/1/ABSTRACT.pdf
https://eprints.ums.edu.my/id/eprint/38592/2/FULLTEXT.pdf
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spelling my-ums-ep.385922024-05-02T08:43:10Z Construction of a gene knockout cassette for leucosporidium antarcticum 2011 Koh, Joseph Soon Peng QK504-(638) Cryptogams Leucosporidium .antarcticum, a psychrophilic yeast was isolated from Antarctica. It grew optimally at 12°C, and adapted well to the cold temperature in Antarctic. The whole genome of the L. antarcticum has been sequenced using the 454 system. It is ideal to conduct systematic knockout of genes to determine its gene function especially .genes that are responsible for the adaption to the cold. Hence, there is a need to construct a knock-out system for L. antarcticum. The gene knockout constructs were built .by fusing ,an .antibiotic (Genetidn-E418) resistance .gene, KanMX used for gene knock out in Saccharomyces cerevisiae to a series of L. antarcticum gene's promoters. The gene promoters used belonged to the ACT, SNF and Cydophilic genes of L. antarcticum. Gene fusion was conducted using the. polymerase chain reaction (PCR). One set of primer was designed to amplify the genes' promoter region with an overhang that was homologous to the second set of primer used to amplify the KanMX.gene. The promoter that was fused to the KanMX gene was cloned onto a CloneJet Vector (Fermentas) and subsequently used as the template to prepare the gene knock-out DNA fragment for L. antarcticum. The significances of the study involved the verification of the putative promoters obtained from L. antarcticum in the expression of the kanMX gene, secondly, the constructed gene knockout cassette can be used to knockout the targeted gene in order to reveal the function of the gene, and lastly, to demonstrate that gene fusion can be established seamlessly using PCR. Preliminary transformation of L. antarcticum using knockout cassette constructed was tested by using electroporation and' heat-shock transformation systems applied in S. cerevisiae. The transformation of L. antarcticum was not successful. L. antarcticum's colonies were found after electroporation transformation but did not grow onto .the medium containing geneticin (G418). No colony was found after heat-shock transformation. 2011 Thesis https://eprints.ums.edu.my/id/eprint/38592/ https://eprints.ums.edu.my/id/eprint/38592/1/ABSTRACT.pdf text en public https://eprints.ums.edu.my/id/eprint/38592/2/FULLTEXT.pdf text en validuser masters Universiti Malaysia Sabah Institut Penyelidikan Bioteknologi
institution Universiti Malaysia Sabah
collection UMS Institutional Repository
language English
English
topic QK504-(638) Cryptogams
spellingShingle QK504-(638) Cryptogams
Koh, Joseph Soon Peng
Construction of a gene knockout cassette for leucosporidium antarcticum
description Leucosporidium .antarcticum, a psychrophilic yeast was isolated from Antarctica. It grew optimally at 12°C, and adapted well to the cold temperature in Antarctic. The whole genome of the L. antarcticum has been sequenced using the 454 system. It is ideal to conduct systematic knockout of genes to determine its gene function especially .genes that are responsible for the adaption to the cold. Hence, there is a need to construct a knock-out system for L. antarcticum. The gene knockout constructs were built .by fusing ,an .antibiotic (Genetidn-E418) resistance .gene, KanMX used for gene knock out in Saccharomyces cerevisiae to a series of L. antarcticum gene's promoters. The gene promoters used belonged to the ACT, SNF and Cydophilic genes of L. antarcticum. Gene fusion was conducted using the. polymerase chain reaction (PCR). One set of primer was designed to amplify the genes' promoter region with an overhang that was homologous to the second set of primer used to amplify the KanMX.gene. The promoter that was fused to the KanMX gene was cloned onto a CloneJet Vector (Fermentas) and subsequently used as the template to prepare the gene knock-out DNA fragment for L. antarcticum. The significances of the study involved the verification of the putative promoters obtained from L. antarcticum in the expression of the kanMX gene, secondly, the constructed gene knockout cassette can be used to knockout the targeted gene in order to reveal the function of the gene, and lastly, to demonstrate that gene fusion can be established seamlessly using PCR. Preliminary transformation of L. antarcticum using knockout cassette constructed was tested by using electroporation and' heat-shock transformation systems applied in S. cerevisiae. The transformation of L. antarcticum was not successful. L. antarcticum's colonies were found after electroporation transformation but did not grow onto .the medium containing geneticin (G418). No colony was found after heat-shock transformation.
format Thesis
qualification_level Master's degree
author Koh, Joseph Soon Peng
author_facet Koh, Joseph Soon Peng
author_sort Koh, Joseph Soon Peng
title Construction of a gene knockout cassette for leucosporidium antarcticum
title_short Construction of a gene knockout cassette for leucosporidium antarcticum
title_full Construction of a gene knockout cassette for leucosporidium antarcticum
title_fullStr Construction of a gene knockout cassette for leucosporidium antarcticum
title_full_unstemmed Construction of a gene knockout cassette for leucosporidium antarcticum
title_sort construction of a gene knockout cassette for leucosporidium antarcticum
granting_institution Universiti Malaysia Sabah
granting_department Institut Penyelidikan Bioteknologi
publishDate 2011
url https://eprints.ums.edu.my/id/eprint/38592/1/ABSTRACT.pdf
https://eprints.ums.edu.my/id/eprint/38592/2/FULLTEXT.pdf
_version_ 1804890330313523200

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