Electrotransformation of Streptococcus agalactiae with plasmid DNA

A protocol for efficient electrotransformation of Streptococcus agalactiae (group B streptococcus) Lancefield's strain O90R (NTCT 9993) (an unencapsulated derivative of type Ia strain O90) was developed. The Escherichia coli-Streptococcus shuttle vector pDP28 (7.8 kb) carrying the ermB gene for resistance to erythromycin was used as donor DNA. Frozen 'electrocompetent' cells were prepared by repeated washes in 10% glycerol. A 50-microliters aliquot containing about 5 x 10(9) colony forming units of bacteria was subjected to the electric pulse. Optimal conditions for electrotransformation were determined using different media, harvesting cells at different points of the growth curve, and using different field strengths. The dose-response curve for transformation of S. agalactiae with pDP28 showed one-hit kinetics as donor DNA varied between 0.01 and 3 micrograms. The efficiency of electrotransformation for this range of amounts of donor DNA was 1.2 x 10(4) cfu micrograms-1. The transformation frequencies obtained with this electroporation protocol are high enough to allow both subcloning and shotgun cloning of streptococcal DNA in S. agalactiae

Electrotransformation of Streptococcus agalactiae with plasmid DNA.

A protocol for efficient electrotransformation of Streptococcus agalactiae (group B streptococcus) Lancefield's strain O90R (NTCT 9993) (an unencapsulated derivative of type Ia strain O90) was developed. The Escherichia coli-Streptococcus shuttle vector pDP28 (7.8 kb) carrying the ermB gene for resistance to erythromycin was used as donor DNA. Frozen 'electrocompetent' cells were prepared by repeated washes in 10% glycerol. A 50-microliters aliquot containing about 5 x 10(9) colony forming units of bacteria was subjected to the electric pulse. Optimal conditions for electrotransformation were determined using different media, harvesting cells at different points of the growth curve, and using different field strengths. The dose-response curve for transformation of S. agalactiae with pDP28 showed one-hit kinetics as donor DNA varied between 0.01 and 3 micrograms. The efficiency of electrotransformation for this range of amounts of donor DNA was 1.2 x 10(4) cfu micrograms-1. The transformation frequencies obtained with this electroporation protocol are high enough to allow both subcloning and shotgun cloning of streptococcal DNA in S. agalactiae

Total reflection X-ray fluorescence used to distinguish mechanically separated from non-mechanically separated meat

This paper presents the successful application of total reflection X-ray fluorescence spectroscopy (TXRF) for the identification of products obtained from mechanically separated meat (MSM), a very important issue for food quality and safety. According to the European Food Safety Authority, one of the most important parameters to distinguish MSM is the content of Ca. Consequently, the development of reliable and cost-effective analytical tools is very important to monitor the chemical composition of these foods. In this study, we have developed an analytical method for elemental analysis of meat samples based on total reflection X-ray fluorescence. A simple sample preparation by suspending the grinded meat sample in a solution of diluted Triton X-100 and polyvinyl alcohol in water showed to be the best for this kind of samples. Fresh chicken meat, chicken meat with different percentage of MSM, pure MSM and meat products prepared with MSM were analyzed. The content of K, Ca, Fe, Cu and Zn was determined. Results show that Ca, but also K and Fe are significant markers to distinguish MSM from fresh meat. A limit of 40% MSM for differentiation was achieved by applying principal component analysis. The method accuracy was evaluated comparing the obtained results with those obtained after acidic digestion and ICP-MS analysis