Bacillus licheniformis PWD-1 is a keratin-degrading, spore-forming bacterium isolated from a poultry waste digester. A mutant of B. licheniformis PWD-1, named B. licheniformis WBG-2, was developed that is deficient in sporulation. The mutation was created using the Splicing by Overlap-Extension PCR method (Gene SOE?ing) to create a 256bp deletion in the spoIIAC gene, which encodes an essential sporulation-specific sigma factor. In-vivo gene replacement was accomplished with the use of a temperature-sensitive plasmid that is able to integrate and excise from the B. licheniformis chromosome. PCR and DNA sequencing were used to confirm the deletion, while heat-treatment assays and electron microscopy verified the absence of spores. The mutant, while completely asporogenic, is able to expresses normal levels of keratinase as compared with B. licheniformis PWD-1.
| Identifer | oai:union.ndltd.org:NCSU/oai:NCSU:etd-12172003-143418 |
| Date | 19 December 2003 |
| Creators | Greenhut, William Bradley |
| Contributors | Jason Shih, Dr. Walter Dobrogosz, Hosni M. Hassan |
| Publisher | NCSU |
| Source Sets | North Carolina State University |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://www.lib.ncsu.edu/theses/available/etd-12172003-143418/ |
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