Publication Date


Document Type


First Advisor

Vary, Patricia S.

Degree Name

M.S. (Master of Science)

Legacy Department

Department of Biological Sciences


Protoplasts; Plasmids; DNA; Bacillus megaterium


This thesis represents the work done to determine whether the recently developed method of polyethylene glycol induced transformation of bacterial protoplasts by plasmid DNA could be used successfully and the conditions optimized in Bacillus meqaterium QM B1551. To determine the conditions which would maximize the efficiency of this process, the growth stage of the cultures before protoplasting, the plasmid DNA concentration, the effective concentration of polyethylene glycol, the magnesium ion concentration and the post-transformation incubation time were varied. The results indicated that growing QM B1551 in hypertonic RHAF broth at 30°C with shaking at 240 rpm to an absorbance₆₆₀ of 0.6 and then exposing the cells to 200 μg/ml lysozyme in HAF buffer yielded a protoplasting'efficiency of 99% or greater. A MgCl₂ concentration of 0.02 M was necessary to maintain the integrity of the protoplast cytoplasmic membrane. A polyethylene glycol concentration of 22.5% followed by a 4-hour post-transformation incubation to allow expression of the plasmid-carried antibiotic resistance genes was found to be the most effective. Plasmids pUBllO, pBC16, pCM194, and pHV14 transformed QM B1551 protoplasts with an efficiency of 2.2 x 10² to 3.4 x 10² transformants per pg of plasmid DNA. The Gram-negative genes for ampicillin resistance of plasmid pHV14 were not expressed in QM B1551.


Bibliography: pages 75-87.


v, 87 pages




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