1887

Abstract

Summary: An electroporation-mediated transformation method was developed and optimized for CNRZ 32. The effects of electroporation buffers, growth conditions, cell-wall-weakening agents and field strength on transformation frequency were examined. Optimal conditions yielded a frequency of 2 × 10 transformants (μg pGK12). Using this procedure, an integration plasmid was introduced into CNRZ 32 to inactivate the chromosomal X-prolyl dipeptidyl aminopeptidase gene (). The integration plasmid, designated pSUW200, consisted of pUC19, the pE194 erythromycin resistance gene and a 1.6 kb internal fragment of the gene. The erythromycin resistance transformants obtained were X-prolyl dipeptidyl aminopeptidase (X-PDAP) negative. Southern hybridization results indicated that pSUW200 had integrated into the CNRZ 32 chromosome via Campbell-type integration. After growth of the pSUW200-derived transformants for 112 generations under non-selective conditions, erythromycin-sensitive X-PDAP isolates were obtained. Southern hybridization results indicated that precise excision of pSUW200 had occurred via recombination between the 1.6 kb -derived nontandem repeats.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-139-7-1433
1993-07-01
2019-10-19
Loading full text...

Full text loading...

http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-139-7-1433
Loading
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error