Development of delta amino Levulinic acid auxotrophic of vibrio Cholerae O1 El Tor Ogawa
| dc.contributor.author | Nur Haslindawaty, Abd Rashid | |
| dc.date.accessioned | 2014-11-14T07:55:00Z | |
| dc.date.available | 2014-11-14T07:55:00Z | |
| dc.date.issued | 2005 | |
| dc.description | Master | en_US |
| dc.description.abstract | Cholera is an important diarrheal disease in developing countries. WHO estimates that cholera caused 111,575 cases with 1,894 deaths in the year 2003 worldwide. To overcome that problem, a number of cholera vaccine candidates have been developed by mutation or deletion of various genes such as thyA and gln. However, these auxotrophic strains were leaky and able to grow in the small intestine of experimental animal’s in-vivo. Objective of the study was to develop an auxotrophic vaccine strains by mutating the housekeeping gene, hemA gene in V. cholerae. The hemA gene codes for glutamyl tRNA reductase. The hemA gene plays a major rate-limiting step in delta aminolevulinic acid (ALA). The hemA gene was PCR amplified from V. cholerae O1 El Tor and cloned into pARO180 vector at EcoRI site. To mutate the hemA gene, a kanamycin cassette was inserted at the BstXI site. hemA-kan was first subcloned into conjugative suicide vector pWM91 which was then, conjugatively transferred into V. cholerae O1 El Tor and the mutant obtained was designated as VCUSM3. In order to remove the kanamycin cassette, the hemA gene was inserted with GFP gene flanking with SmiI site and subcloned onto pWM91. GFP gene was then excised and left a +1 frame shift mutation in hemA gene (hemA*/M). hemA*/M was conjugatively transferred to VCUSM3 and the mutant obtained was designated as VCUSM4. ALA auxotrophy of VCUSM3 and VCUSM4 were confirmed by their growth on ALA supplemented medium. The hemA mutants were confirmed by PCR using hemA specific primers and by DNA sequencing. Thus in this study ALA auxotrophs of V. cholerae O1 El Tor were created by mutating the hemA gene. | en_US |
| dc.identifier.uri | http://hdl.handle.net/123456789/511 | |
| dc.language.iso | en | en_US |
| dc.subject | Chemical science | en_US |
| dc.subject | Delta amino | en_US |
| dc.title | Development of delta amino Levulinic acid auxotrophic of vibrio Cholerae O1 El Tor Ogawa | en_US |
| dc.type | Thesis | en_US |
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