A novel xylB-based positive selection vector.
Expression of a plasmid-borne Escherichia coli xylulokinase gene (xylB) under the control of the lac promoter yields constitutively high levels of xylulokinase activity. When a plasmid containing this lac-xylB fusion (pLEK100) is transformed into a xylB- mutant the Xyl+ phenotype is restored on xylose-containing media. When the same transformants are plated on xylitol medium, growth inhibition is observed. Positive selection is achieved by cloning DNA into the unique restriction sites of pLEK100, to disrupt xylB expression, transforming E. coli, and then plating transformants on xylitol medium. With this protocol only transformants with insert containing plasmids will be obtained. This results in a considerable reduction in the time and effort needed to construct genomic libraries or perform routine DNA cloning experiments. Three unique sites are available which are suitable for positive selection of DNA fragments, via the disruption of translation (BglII) or transcription (HindIII, SalI, and BglII) of the xylB gene.
Stevis
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Laboratory of Renewable Resources Engineering, Purdue University, West Lafayette, Indiana 47907.
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D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D005822:Genetic Vectors; D010770:Phosphotransferases / Q000235:genetics*; D017853:Phosphotransferases (Alcohol Group Acceptor); D010957:Plasmids; D015183:Restriction Mapping
D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C032700:xylulokinase