Leigh Lab : Markerless Mutation
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J o h n    L e i g h    L a b

University of Washington Department of Microbiology
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Email the Director :
John Leigh, Ph.D.


Markerless Mutagenesis Procedure

Currently we have two systems for producing markerless mutations in Methanococus maripaludis. Our original system, using pCRPrtNeo to make mutations in strain Mm900, takes advantage of the susceptibility to the purine analog 8-azahypoxanthine conferred by the hpt (hypoxanthine / guanine phosphoribosyltransferase) gene. More recently we developed pCRUptNeo for markerless mutagenesis of strain Mm901. This system is what we now use routinely and takes advantage of the susceptibility to the pyrimidine analog 6-azauaracil conferred by the upt (uracil phosphoribosyltransferase) gene. We also developed a method that uses pBlPrt to stably incorporate constructs into the genome, at the site of the upt gene (see markerless insertions procedure).



Markerless Mutagenesis Procedure with pCRUptNeo and Mm901 .pdf


Markerless Mutagenesis Procedure with pCRPrtNeo and Mm900 .pdf



Plasmids

pCRUPTNEOpCRPRTNEOpBLPRT
pCRUPTNEO

plasmid map
sequence text file
pCRPRTNEO

plasmid map
sequence text file
pBLPRT

plasmid map
sequence text file