title: A high-throughput, restriction-free cloning and screening strategy based on ccdB-gene replacement.
abstract: BACKGROUND
  In high-throughput demanding fields, such as biotechnology and structural biology, molecular cloning is an essential tool in obtaining high yields of recombinant protein. Here, we address recently developed restriction-free methods in cloning, and present a more cost-efficient protocol that has been optimized to improve both cloning and clone screening.
  
  
  RESULTS
  In our case study, three homologous β-lactamase genes were successfully cloned using these restriction-free protocols. To clone the genes, we chose a gene replacement strategy, where the recombinant genes contained overhangs that targeted a region of the expression vector including a cytotoxin-encoding ccdB-gene.
  
  
  CONCLUSIONS
  We provide further evidence that gene replacement can be applied with high-throughput cloning protocols. Targeting a replacement of the ccdB-gene was found to be very successful for counterselection using these protocols. This eliminated the need for treatment with the restriction enzyme DpnI that has so far been the preferred clone selection approach. We thus present an optimized cloning protocol using a restriction-free ccdB-gene replacement strategy, which allows for parallel cloning at a high-throughput level.
authors.forename: Bjarte Aarmo
authors.initials: BA
authors.lastname: Lund
authors.forename: Hanna-Kirsti Schrøder
authors.initials: HK
authors.lastname: Leiros
authors.affiliation: NorStruct, Department of Chemistry, Faculty of Science and Technology, UiT - The Arctic University of Norway, N-9037 Tromsø, Norway. gro.bjerga@uni.no.
authors.forename: Gro Elin Kjæreng
authors.initials: GE
authors.lastname: Bjerga
mesh_terms: D003001:Cloning, Molecular; D003603:Cytotoxins / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D011995:Recombination, Genetic; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism
chemical_list: D003603:Cytotoxins; D011994:Recombinant Proteins; D001618:beta-Lactamases; C108449:oxacillinase