title: A BioBrick™-Compatible Vector for Allelic Replacement Using the XylE Gene as Selection Marker.
abstract: BACKGROUND
  Circular plasmid-mediated homologous recombination is commonly used for marker-less allelic replacement, exploiting the endogenous recombination machinery of the host. Common limitations of existing methods include high false positive rates due to mutations in counter-selection genes, and limited applicability to specific strains or growth media. Finally, solutions compatible with physical standards, such as the BioBrick™, are not currently available, although they proved to be successful in the design of other replicative or integrative plasmids.
  
  
  RESULTS
  We illustrate pBBknock, a novel BioBrick™-compatible vector for allelic replacement in Escherichia coli. It includes a temperature-sensitive replication origin and enables marker-less genome engineering via two homologous recombination events. Chloramphenicol resistance allows positive selection of clones after the first event, whereas a colorimetric assay based on the xylE gene provides a simple way to screen clones in which the second recombination event occurs. Here we successfully use pBBknock to delete the lactate dehydrogenase gene in E. coli W, a popular host used in metabolic engineering.
  
  
  CONCLUSIONS
  Compared with other plasmid-based solutions, pBBknock has a broader application range, not being limited to specific strains or media. We expect that pBBknock will represent a versatile solution both for practitioners, also among the iGEM competition teams, and for research laboratories that use BioBrick™-based assembly procedures.
authors.affiliation: Department of Electrical, Computer and Biomedical Engineering, Laboratory of Bioinformatics, Mathematical Modelling and Synthetic Biology, University of Pavia, 27100 Pavia, Italy ; Centre for Health Technologies, University of Pavia, 27100 Pavia, Italy.
authors.forename: Michela
authors.initials: M
authors.lastname: Casanova
authors.affiliation: Department of Electrical, Computer and Biomedical Engineering, Laboratory of Bioinformatics, Mathematical Modelling and Synthetic Biology, University of Pavia, 27100 Pavia, Italy ; Centre for Health Technologies, University of Pavia, 27100 Pavia, Italy.
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Pasotti
authors.affiliation: Department of Electrical, Computer and Biomedical Engineering, Laboratory of Bioinformatics, Mathematical Modelling and Synthetic Biology, University of Pavia, 27100 Pavia, Italy ; Centre for Health Technologies, University of Pavia, 27100 Pavia, Italy.
authors.forename: Susanna
authors.initials: S
authors.lastname: Zucca
authors.affiliation: Department of Electrical, Computer and Biomedical Engineering, Laboratory of Bioinformatics, Mathematical Modelling and Synthetic Biology, University of Pavia, 27100 Pavia, Italy ; Centre for Health Technologies, University of Pavia, 27100 Pavia, Italy.
authors.forename: Nicolò
authors.initials: N
authors.lastname: Politi
authors.affiliation: Department of Electrical, Computer and Biomedical Engineering, Laboratory of Bioinformatics, Mathematical Modelling and Synthetic Biology, University of Pavia, 27100 Pavia, Italy ; Centre for Health Technologies, University of Pavia, 27100 Pavia, Italy.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Massaiu
authors.affiliation: Department of Biology and Biotechnology, University of Pavia, 27100 Pavia, Italy ; Centre for Health Technologies, University of Pavia, 27100 Pavia, Italy.
authors.forename: Cinzia
authors.initials: C
authors.lastname: Calvio
authors.affiliation: Centre for Health Technologies, University of Pavia, 27100 Pavia, Italy.
authors.forename: Maria Gabriella
authors.initials: MG
authors.lastname: Cusella De Angelis
authors.affiliation: Department of Electrical, Computer and Biomedical Engineering, Laboratory of Bioinformatics, Mathematical Modelling and Synthetic Biology, University of Pavia, 27100 Pavia, Italy ; Centre for Health Technologies, University of Pavia, 27100 Pavia, Italy.
authors.forename: Paolo
authors.identifier: 0000-0002-8931-4676
authors.initials: P
authors.lastname: Magni
keywords: Allelic replacement; BioBrick; Knockout; Standard vector; XylE