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Team:Alberta/References/Publications/Simple and highly efficient BAC recombineering using galK selection
From 2009.igem.org
Soren Warming, Nina Costantino, Donald L. Court, Nancy A. Jenkins and Neal G. Copeland
Nucleic Acids Research, 2005, Vol. 33, No. 4 e36
Abstract: Recombineering allows DNA cloned in Escherichia
coli to be modified via lambda Red-mediated
homologous recombination, obviating the need for
restriction enzymes and DNA ligases to modify DNA.
Here, we describe the construction of three new
recombineering strains (SW102, SW105 and SW106)
that allow bacterial artificial chromosomes (BACs) to
be modified using galK positive/negative selection.
This two-step selection procedure allows DNA to be
modified without introducing an unwanted selectable
marker at the modification site. All three strains contain
an otherwise complete galactose operon, except
for a precise deletion of the galK gene, and a defective
temperature-sensitive lambda prophage that makes
recombineering possible. SW105 and SW106 cells
in addition carry L-arabinose-inducible Cre or Flp
genes, respectively. The galK function can be selected
both for and against. This feature greatly reduces
the background seen in other negative-selection
schemes, and galK selection is considerably more
efficient than other related selection methods
published. We also show how galK selection can be
used to rapidly introduce point mutations, deletions
and loxP sites into BAC DNA and thus facilitate
functional studies of SNP and/or disease-causing
point mutations, the identification of long-range regulatory
elements and the construction of conditional
targeting vectors.
Link: [http://nar.oxfordjournals.org/cgi/content/abstract/33/4/e36 Nucleic Acids Research]
