Related Experiment Videos
Electrotransformation in Salmonella typhimurium LT2.
J Binotto1, P R MacLachlan, K E Sanderson
1Department of Biological Sciences, University of Calgary, Alta., Canada.
Canadian Journal of Microbiology
|June 1, 1991
Summary
Electroporation offers high transformation efficiency in Salmonella typhimurium LT2, unaffected by lipopolysaccharide (LPS) variations. This method bypasses cell envelope property limitations seen in calcium chloride shock transformations.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Bacterial transformation is crucial for genetic studies.
- Calcium chloride shock is a common transformation method but is sensitive to cell envelope properties.
- Electroporation offers an alternative transformation technique.
Purpose of the Study:
- To evaluate the efficiency of electroporation for transforming Salmonella typhimurium LT2.
- To compare the influence of lipopolysaccharide (LPS) composition on electroporation versus calcium chloride shock transformation.
- To investigate the effect of the reciprocal restriction barrier between Escherichia coli K-12 and S. typhimurium LT2 on electroporation efficiency.
Main Methods:
- Electroporation of Salmonella typhimurium LT2 using pBR322 DNA.
- Comparison of transformation efficiencies across strains with varying LPS structures (Rc, Rd2, smooth, rough, Re).
- Assessment of transformation efficiency using calcium chloride shock methods for comparison.
- Evaluation of the impact of host-restriction mutants on electroporation.
Main Results:
- Electroporation yielded high transformation efficiencies (10(8)-10(9) electrotransformants/microgram DNA).
- LPS composition had minimal impact on electroporation efficiency, unlike calcium chloride shock methods.
- Calcium chloride shock efficiencies varied significantly with LPS type (10(6) to 10(2) transformants/microgram DNA).
- The restriction barrier between E. coli K-12 and S. typhimurium LT2 reduced electroporation efficiency by approximately 100-fold.
- Host-restriction mutants diminished or eliminated this barrier.
Conclusions:
- Electroporation is a highly efficient transformation method for Salmonella typhimurium LT2.
- Cell envelope properties, specifically LPS composition, are less critical for electroporation success compared to calcium chloride shock.
- The reciprocal restriction barrier can be overcome using host-restriction mutants to improve interspecies transformation.