Raheel Nazakat (2014) Cloning and expression of Phy2GENE in Escherichia coli. Masters thesis, Universiti Malaysia Sabah.
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Abstract
One particular bacterium that demonstrates high phytase activity is Mitsuokella Jalaludinii. However, the anaerobic nature of the bacterium prohibits its massproduction. Cloning and expression of the phytase gene into aerobes microorganism is essential in overcoming this problem. Thus, this study aims in isolating the Phy2 gene of Mitsuokella Jalaludinii followed by cloning and expression in Escherichia colt: The Phy2 gene was isolated from the genomic DNA of Mitsuokella Jalaludinii through polymerase chain reaction using PhyF and PhyR primers. PCR product of 1127 base pairs containing the probable promoter sites, Phy2 coding region and putative terminator site was then inserted into pJETl.2/blunt cloning vector in the direction downstream of the T7 promoter. It was used to transform chemically-competent Escherichia coli strain DHSa. Growth of transformants was seen on LB agar containing 50 µg/ml ampicillin. The recombinant clone was then confirmed. by Bgl II restriction digestion. Two fragments were seen; one representing the plasmid (2974 base pairs) while the other one represents the Phy2 gene construct (1127 base pairs). The orientation of the insert was confirmed through PCR by using pJETl.2 forward sequencing primer and PhyR reverse primer. Screening of phytase activity among the recombinant clones was carried out qualitatively by growing the clones on LB agar plate supplemented with ampicillin and 2% sodium phytate. Clear zone was seen in the area where recombinant Escherichia coli DHSa carrying the Phy2 construct grew. Meanwhile, no halo zone was seen for those clones carrying control PCR product as insert. This revealed that only those clones with Phy2 gene construct exhibit phytase activity as the enzyme was expressed from the multi-copied vector. It can be concluded that the transcription of Phy2 gene initializes from the gene's own promoter because Escherichia coli DHSa lacks the gene that encodes for T7 RNA polymerase. However, the identified candidate promoter sites were only tentative. Prior to its industrial application, further purification and characterization of the crude phytase will be required to determine its functionality and efficacy in reducing phytic acid content in animals' feed.
| Item Type: | Thesis (Masters) |
|---|---|
| Keyword: | Mitsuokella jalaludinii, Phytase, Gen Phy2, Kloning gen, Ekspresi protein |
| Subjects: | Q Science > QR Microbiology > QR1-502 Microbiology > QR75-99.5 Bacteria |
| Department: | INSTITUTE > Biotechnology Research Institute (BRI) |
| Depositing User: | DG MASNIAH AHMAD - |
| Date Deposited: | 18 Jun 2025 10:09 |
| Last Modified: | 19 Jun 2025 15:42 |
| URI: | https://eprints.ums.edu.my/id/eprint/44186 |
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