The conversion of raw forage crops such as grass and cereals into silage is brought about by a fermentation carried out by the indigenous lactic acid bacteria. The most important of the lactic acid bacteria in terms of silage fermentations are members of the general <i>Lactobacillus</i> and <i>Pediococcus</i>. Due to recent advances in the genetics of the lactic acid bacteria, there is now significant potential to modify strains by genetic manipulation and hence create a new range of strains with additional desirable characteristics such as cellulase production which can be used as silage additives. The strains used in this study were all natural silage isolates and were identified as either <i>Lactobacillus plantarum</i> or <i>Pediococcus pentosaceus</i> using a range of characteristics including homo/heterofermentative activity, Gram staining, the API 50CH system and growth temperature. Six strains of <i>L. plantarum</i> and twelve strains of <i>P. pentosaceus</i> were isolated. All were screened for the presence of native plasmids, with a range of sizes from two kilobases (kb) to eleven kb identified within the <i>L. plantarum</i> strains and a more limited size range identified within the <i>P. pentosaceus</i> strains. None of the ioslates were naturally transformable so electroporation was used as a means to facilitate the uptake of exogenous DNA. The transformation protocol was optimised by examining the effects of electroporation buffer, growth conditions of the bacteria, DNA concentration and various electrical parameters. The optimised procedure used exponential phase cells, 0.5M sucrose as the electroporation solution, one μg plasmid DNA, electrical parameters of 2.5 kilovolts, 100 ohms resistance and 25 microFarads capacitance followed by an expression period of two hours.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:661339 |
| Date | January 1993 |
| Creators | Rodger, Margaret L. |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/11900 |
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