Research toward the development of a protocol to electrotransform Bacillus megaterium through exploration of parameters and variables that have been used to successfully electrotransform other gram-positive bacteria
Vary, Patricia S.
B.S. (Bachelor of Science)
Department of Biological Sciences
This project was proposed to develop a protocol to electrotransform Bacillus megaterium (B. megaterium) through investigation of techniques that have been used to electrotransform other species of gram-positive bacteria. Electrotransformation is a process by which exogenous DNA can be introduced into a recipient cell through the application of an electric pulse. It was decided that successful protocols be studied and applied as closely as possible to B. megaterium. Specific areas of inquiry included; finding the machine settings on the apparatus needed to achieve electric pulse times that have been found to be optimum in successful protocols, constructing a table of applied voltage vs incidence of cell death in the range of applied voltages used in other protocols, and investigating the effect of adding various substances, such as glycine and polyethelene glycol, to the growth medium or the electrotransformation buffer to assess whether damage to, or reconfiguration of the cell wall and capsule could be induced. It was found that similar, but not identical, voltages and pulse times as those utilized in the protocols studied could be achieved using the available apparatus. Surprisingly, applying similar pulse times and voltages to B. megaterium did not result in any significant or even measurable level of cell death -- a condition thought to be critical to successful electrotransformation. The most promising procedure proved to be the addition of glycine to the growth medium. Significant morphological changes were observed after growing a culture in varying concentrations of glycine to a stage that showed a 75-90% reduction in optical density when compared to a control grown without glycine. In addition, when an electric pulse was applied to these cells they appeared to have been damaged. In spite of this progress, when the procedure was followed using plasmid DNA as the donor, it yielded no transformants. There is some question as to the suitability of the plasmid that was used,(pHV 33) as an electrotransformation vector, adding complexity to the problem. In addition to the work described above, two plasmid DNA extractions using cesium chloride gradients were performed and another plasmid, the 8.1kb plasmid of B. megaterium was isolated using electroelution.
Gane, Melody K., "Research toward the development of a protocol to electrotransform Bacillus megaterium through exploration of parameters and variables that have been used to successfully electrotransform other gram-positive bacteria" (1993). Honors Capstones. 961.
Northern Illinois University
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