The pSVJ21 shuttle vector provides a cloning system that can be used both with E. coli and high G+C Gram-positive psychrophilic (cold-loving) bacteria. In combination with the efficient electroporation protocol, this vector can be used for low temperature expression of cold-active enzymes in high G+C Gram-positive hosts, and for genetic studies of Gram-positive microorganisms.
The pSVJ21 shuttle vector was constructed by ligating p54 and pUC18 and inserting a 1.3 bp CAT cassette conferring resistance to chloramphenicol (cm). The resulting 6002 bp construct contains genes for ampicillin and chloramphenicol resistance and replicons for both Gram-positive and Gram-negative bacteria. The segregational stability of the vector in three Gram-positive recipients under non-selective conditions was higher at 5C than at 25C, making them suitable candidates for protein expression at low temperature.
From the laboratory of Jean Brenchley, PhD, Penn State University.
Part of The Investigator's Annexe program.
|Antibiotic Resistance:||Ampicillin (amp), Chloramphenicol (cm)|
|Buffer:||Liquid (10mM Tris pH 8)|
|Grow in E. coli at 37 C:||Yes|
|Selectable markers:||Amp and cm in E. coli, cm in Gram-positives|
|Vector Backbone and Size:||pSVJ21, 6002 bp|
|High or low copy:||high copy in E. coli, low copy in Gram-positives|
|Storage:||4C or -20C|
Transformation Protocol: As a starting point, transform the plasmid in E. coli using either competent cells or electroporation and selection on LB agar with ampicillin (100 µg/ml or chloramphenicol (5-10 µg/ml), or both. Usually 40-50 ng is used per transformation. Cultures of transformants can be kept as stocks with glycerol and used to isolate new plasmid DNA when necessary. Details on the transformation procedure in Gram-positive recipients are provided in Miteva V., et al. Extremophiles, 12, 441-449 (2008).
NOTE: The plasmid has some single restriction sites from the pUC18 multicloning site, such as Hind III and Sal I, which do not disrupt the two replicons and can be used to linearize the plasmid an and insert DNA fragment.
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