Open AccessThe MX 4blaster cassette: repeated and clean S accharomyces cerevisiae genome modification using the genome‐wide deletion collection
Author(s) -
Carvalho Ângela,
Pereira Filipa,
Johansson Björn
Publication year - 2013
Publication title -
fems yeast research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.991
H-Index - 92
eISSN - 1567-1364
pISSN - 1567-1356
DOI - 10.1111/1567-1364.12076
Subject(s) - expression cassette , biology , gene cassette , saccharomyces cerevisiae , genome , genetics , transformation (genetics) , computational biology , mutant , gene , terminator (solar) , plasmid , recombinant dna , ionosphere , physics , astronomy , vector (molecular biology) , integron
The kan MX 4 resistance marker is widely used for S accharomyces cerevisiae gene deletion and has been used to create a genome‐wide deletion mutant collection. Transfer of PCR ‐amplified marker loci from collection mutants is a very efficient way of introducing mutations into other S . cerevisiae strains of interest. An important limitation of this strategy is that the kan MX 4 marker is not easily removed, impairing the construction of multiple gene deletion strains. The MX 4blaster is a novel MX 4‐compatible cassette that facilitates clean or scarless kan MX 4 removal. The MX 4blaster cassette efficiently replaces the kan MX 4 cassette due to shared promoter and terminator sequences. The MX 4blaster cassette is removed by the induction of an internal double‐stranded break and transformation with a DNA fragment designed to recombine on either side of the cassette in combination with counter selection. Simple and inexpensive media are used for induction and counter selection, mitigating the use of expensive chemicals. Routinely, thirty percent of transformants successfully removed the MX 4blaster cassette. The resulting mutants contain no trace of the MX 4blaster cassette. The unique properties of the MX 4blaster cassette make it an important addition to S . cerevisiae genetic tools, especially in combination with the genome‐wide deletion collection.