title: Altering the selection capabilities of common cloning vectors via restriction enzyme mediated gene disruption.
abstract: BACKGROUND
  The cloning of gene sequences forms the basis for many molecular biological studies. One important step in the cloning process is the isolation of bacterial transformants carrying vector DNA. This involves a vector-encoded selectable marker gene, which in most cases, confers resistance to an antibiotic. However, there are a number of circumstances in which a different selectable marker is required or may be preferable. Such situations can include restrictions to host strain choice, two phase cloning experiments and mutagenesis experiments, issues that result in additional unnecessary cloning steps, in which the DNA needs to be subcloned into a vector with a suitable selectable marker.
  
  
  RESULTS
  We have used restriction enzyme mediated gene disruption to modify the selectable marker gene of a given vector by cloning a different selectable marker gene into the original marker present in that vector. Cloning a new selectable marker into a pre-existing marker was found to change the selection phenotype conferred by that vector, which we were able to demonstrate using multiple commonly used vectors and multiple resistance markers. This methodology was also successfully applied not only to cloning vectors, but also to expression vectors while keeping the expression characteristics of the vector unaltered.
  
  
  CONCLUSIONS
  Changing the selectable marker of a given vector has a number of advantages and applications. This rapid and efficient method could be used for co-expression of recombinant proteins, optimisation of two phase cloning procedures, as well as multiple genetic manipulations within the same host strain without the need to remove a pre-existing selectable marker in a previously genetically modified strain.
authors.affiliation: Department of Microbiology, La Trobe University, Melbourne, VIC 3086, Australia.
authors.forename: Sam
authors.initials: S
authors.lastname: Manna
authors.forename: Ashley
authors.initials: A
authors.lastname: Harman
authors.forename: Jessica
authors.initials: J
authors.lastname: Accari
authors.forename: Christian
authors.initials: C
authors.lastname: Barth
mesh_terms: D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D017931:DNA Primers; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D005822:Genetic Vectors; D007612:Kanamycin; D016133:Polymerase Chain Reaction; D013752:Tetracycline
chemical_list: D017931:DNA Primers; D007612:Kanamycin; D004262:DNA Restriction Enzymes; D013752:Tetracycline