Previously, triparental mating was used for the introduction of plasmids into plant-pathogenic bacteria. Triparental mating requires a helper strain, carrying the genes that code for conjugation and DNA transfer, and a donor strain, carrying the plasmid to be introduced into the new bacterial strain. At least five to seven days are required in order to determine if the plasmid was successfully introduced into the new bacterial strain and confirm that there is no carryover of the helper or donor strain. Electroporation does not require a helper or donor strain.
This helps avoid possible contamination with other strains. In addition, introduction of the plasmid can be verified in the recipient strain within two days, making electroporation a faster and more efficient method of transformation. Electroporation is being used for transformation of both Gram-positive and Gram-negative species of plant-pathogenic and plant-associated bacteria. The electroporation conditions can differ not only for bacterial species but also strains within a species. Consequently, it is important to define the optimal parameters of electroporation to ensure that each bacterial strain of interest is transformed efficiently.
These parameters include the time constant and the field strength applied to the sample 8. The time constant is dependent on the total resistance of the sample and the capacitance of the pulse circuit for the electroporation device. The field strength is dependent on the initial voltage delivered by the electroporator and the distance between the electrodes of the cuvette 8. These parameters are discussed in detail by Lurquin 8. This laboratory exercise is designed to be adapted to the instructor's available equipment as well as the goals of the instructor. It is divided into three sections with separate protocols, and the instructor can choose which sections will be most effective in their respective courses.
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Electrotransformation of Bacteria by Plasmid DNA
Bacterial strains with relevance in the food industry and biotechnology, in medical and veterinary fields, agroindustry and environmental sciences are covered. Visit Seller's Storefront.
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Payment Methods accepted by seller. AbeBooks Bookseller Since: August 14, Stock Image. Published by Springer, Transformants were cultivated for 25 generations in medium without erythromycin and subsequently plated on solidified medium with and without erythromycin. Structural stability of the plasmid was also examined by cultivation in selective medium for 25 generations, plating on solid medium and analysis of plasmid DNA from a number of colonies. Sequencing revealed no deletions indicating that pTRKH3 was also structurally stably maintained.
Treatment of L. The described host-vector systems are reproducible and highly efficient. The vector has a medium copy number 30—40 in E.
Tetracycline resistance is only expressed in E. Plasmid DNA was extracted using the alkaline lysis procedure [ 21 ]. Extracted DNA was purified by cesium chloride-ethidium bromide density centrifugation [ 21 ]. DNA concentration was determined spectrophotometrically at nm. The nucleotide sequence of pTRKH3, linearized by Ava I was determined by the primer walking strategy, starting from both ends.
To cover the complete 7. Sequencing was accomplished by using an Applied Biosystems model A automatic sequencer according to procedures provided by the supplier and fluorescent-dye-labeled dideoxyribonucleotides. Electroporation was done according to a modification of the method of Van der Lelie [ 9 ], proposed by Dornan and Collins [ 10 ]. Overnight cultures of L. Cells were harvested by centrifugation at 10, g for 10 min when the optical density at nm was between 0. Cell number was calculated according to McIntyre and Harlander [ 5 , 6 ].
The cells were washed sequentially with the following ice-cold solutions by alternate centrifugation and resuspension: bidistilled water 2. Electroporation was performed by a single pulse at 2. Appropriate controls confirmed the absence of transformants if either the electric pulse or the plasmid DNA was omitted. Transformation was confirmed by selection in erythromycin containing medium and plasmid analysis. Pretreatment of L.
Electroporation and marker exchange of Pseudomonas syringae pv. syringae.
Following treatment, the cells were pelleted, resuspended in 1. Genetics and biotechnology of lactic acid bacteria. Genetics of lactic acid bacteria. Harlander SK: Transformation of Streptococcus lactis by electroporation. Streptococcal genetics. Appl Environ Microbiol. Biochim Biophys Acta.
Competent cell preparation
Nucleic Acids Research. Lett Appl Microbiol. Electrotransformation of bacteria. Edited by: Eynard N, Teissie J. Eynard N, Teissie J: General principles of bacteria electrotransformation: key steps. Brown TA: Essential molecular biology.
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Wu S, Letchworth GJ: High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol. Pichia protocols. Edited by: Higgins D, Cregg J.
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Suga M, Hatakeyama T: High efficiency transformation of Schizosaccharomyces pombe pretreated with thiol compounds by electroporation. Download references. This work was financed within the framework of the Cell Factory Programme of the Commission of the European Communities. Correspondence to Maria Papagianni.