Positive and negative selection using the tetA-sacB cassette: recombineering and P1 transduction in Escherichia coli

Nucleic Acids Res. 2013 Dec;41(22):e204. doi: 10.1093/nar/gkt1075. Epub 2013 Nov 6.


The two-step process of selection and counter-selection is a standard way to enable genetic modification and engineering of bacterial genomes using homologous recombination methods. The tetA and sacB genes are contained in a DNA cassette and confer a novel dual counter-selection system. Expression of tetA confers bacterial resistance to tetracycline (Tc(R)) and also causes sensitivity to the lipophillic chelator fusaric acid; sacB causes sensitivity to sucrose. These two genes are introduced as a joint DNA cassette into Escherichia coli by selection for Tc(R). A medium containing both fusaric acid and sucrose has been developed, in which, coexpression of tetA-sacB is orders of magnitude more sensitive as a counter-selection agent than either gene alone. In conjunction with the homologous recombination methods of recombineering and P1 transduction, this powerful system has been used to select changes in the bacterial genome that cannot be directly detected by other counter-selection systems.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Antiporters / genetics*
  • Bacterial Proteins / genetics*
  • Culture Media
  • Escherichia coli / genetics*
  • Escherichia coli Proteins / genetics
  • Gene Fusion
  • Genetic Engineering / methods*
  • Hexosyltransferases / genetics*
  • Recombination, Genetic*
  • Sucrose / metabolism
  • Transduction, Genetic*


  • Antiporters
  • Bacterial Proteins
  • Culture Media
  • Escherichia coli Proteins
  • sulA protein, E coli
  • tetA protein, Bacteria
  • Sucrose
  • Hexosyltransferases
  • levansucrase