Greenhut, William Bradley ; Jason Shih, Committee Chair,Dr. Walter Dobrogosz, Committee Member,Hosni M. Hassan, Committee Member,Greenhut, William Bradley ; Jason Shih ; Committee Chair ; Dr. Walter Dobrogosz ; Committee Member ; Hosni M. Hassan ; Committee Member
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.
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Development of An Asporogenic Mutant of Bacillus Licheniformis PWD-1.