Construction of new β-glucuronidase cassettes for making transcriptional fusions and their use with new methods for allele replacement

William W. Metcalf, Barry L. Wanner

Research output: Contribution to journalArticlepeer-review

Abstract

Five cassettes carrying uidA, encoding β-glucuronidase, were made for the construction of insertion mutants with transcriptional fusions to uidA. Three uidA cassettes contain antibiotic-resistance genes, for chloramphenicol (Cm), for kanamycin (Km) and neomycin (Nm), or for streptomycin (Sm) and spectinomycin (Sp). Some cause polar insertions while others provide a promoter for downstream gene expression. The expression of these uidA cassettes was compared to the expression of lacZ at the same site in phnD, a phosphate-regulated gene for phosphonate use. Several phn::uidA or phn::lacZ insertions were recombined onto the chromosome to test mutational effects and to measure gene expression in single copy. This was done using one of three methods for allele replacement. A new method involved recombination of mutations in M 13 onto the chromosome by infection of an Escherichia coli rep mutant that fails to propagate single-stranded DNA phages. Merodiploid recombinants were selected using a resistance marker carried by the M 13 phage; segregants lacking M 13 sequences were then selected as deoxycholate-resistant (DocR) ones. An improved method for recombination of mutations in pir-dependent, oriR6K vectors involved the use of plasmids containing genes for tetracycline resistance (TcR). Merodiploid recombinants were selected by conjugative transfer of such plasmids into a recipient lacking pir (encoding the n protein of the R6K plasmid); segregants lacking vector sequences were subsequently selected as Tc-sensitive ones. Both procedures are efficient and allow for recombining marked as well as unmarked mutations onto the chromosome. In addition, some insertions with an antibiotic-resistance marker were directly recombined onto the chromosome by transformation of a recD mutant with linear DNA. New E. coli mutants deleted for uidA, lacZ and phoA are also described, including ones that allow for the simultaneous use of multiple reporter genes.

Original languageEnglish (US)
Pages (from-to)17-25
Number of pages9
JournalGene
Volume129
Issue number1
DOIs
StatePublished - Jul 15 1993
Externally publishedYes

Keywords

  • Escherichia coli host
  • GUS transcriptional cassettes
  • M 13 phages
  • R6K replicons
  • lacZ fusions
  • rep mutant
  • reporter genes
  • uidA fusions

ASJC Scopus subject areas

  • Genetics

Fingerprint

Dive into the research topics of 'Construction of new β-glucuronidase cassettes for making transcriptional fusions and their use with new methods for allele replacement'. Together they form a unique fingerprint.

Cite this