A general system for generating unlabelled gene replacements in bacterial chromosomes

K. Leenhouts, G. Buist, A. Bolhuis, A. Ten Berge, J. Kiel, I. Mierau, Magdalena Dabrowska, G. Venema, J. Kok

Research output: Contribution to journalArticlepeer-review

262 Citations (SciVal)


A general system is described that facilitates gene replacements such that the recombinant strains are not labelled with antibiotic resistance genes. The method is based on the conditional replication of derivatives of the lactococcal plasmid pWV01, which lacks the repA gene encoding the replication initiation protein. Replacement vectors can be constructed in and isolated from gram-positive and gram-negative helper strains that provide RepA in trans. Cointegrate formation of the integration vectors with the chromosome of the target strain is selected by antibiotic resistance. Resolution of the cointegrate structure is identified in the second step of the procedure by the loss of the lacZ reporter gene present in the delivery vector. The second recombination event results either in gene replacement or in restoration of the original copy of the gene. As no antibiotic resistance marker is present in the genome of the mutant the system can be used to introduce multiple mutations in one strain. A feasibility study was performed using Lactococcus lactis and Bacillus subtilis as model organisms. The results indicate that the method should be applicable to any non-essential gene in numerous bacterial species.

Original languageEnglish
Pages (from-to)217-224
Number of pages8
JournalMolecular and General Genetics
Issue number1-2
Publication statusPublished - 1 Jan 1996


  • Conditional replication
  • Gene replacement
  • lacZ reporter
  • Mutagenesis

ASJC Scopus subject areas

  • Genetics


Dive into the research topics of 'A general system for generating unlabelled gene replacements in bacterial chromosomes'. Together they form a unique fingerprint.

Cite this