Allelic exchange in Escherichia coli using the Bacillus subtilis sacB gene and a temperature‐sensitive pSC101 replicon

Abstract

To facilitate efficient allelic exchange of genetic information into a wild-type strain background, we improved upon and merged approaches using a temperature-sensitive plasmid and a counter-selectable marker in the chromosome. We first constructed intermediate strains of Escherichia coli K12 in which we replaced wild-type chromosomal sequences, at either the fimB-A or lacZ-A loci, with a newly constituted DNA cassette. The cassette consists of the sacB gene from Bacillus subtilis and the neomycin (kanamycin) resistance gene of Tn5, but, unlike another similar cassette, it lacks IS1 sequences. We found that sucrose sensitivity was highly dependent on incubation temperature and sodium chloride concentration. The DNA to be exchanged into the chromosome was first cloned into derivatives of plasmid pMAK705, a temperature-sensitive pSC101 replicon. The exchanges were carried out in two steps, first selecting for plasmid integration by standard techniques. In the second step, we grew the plasmid integrates under non-selective conditions at 42 degrees C, and then in the presence of sucrose at 30 degrees C, allowing positive selection for both plasmid excision and curing. Despite marked locus-specific strain differences in sucrose sensitivity and in the growth retardation due to the integrated plasmids, the protocol permitted highly efficient exchange of cloned DNA into either the fim or lac chromosomal loci. This procedure should allow the exchange of any DNA segment, in addition to the original or mutant allelic DNA, into any non-essential parts of the E. coli chromosome.

Reciprocal exchange of minor components of type 1 and F1C fimbriae results in hybrid organelles with changed receptor specificities

“…Meanwhile, when plasmid pPKL65, encoding the FimH adhesin of type 1 fimbriae, was introduced into such hosts, a D-mannose binding phenotype resulted ( Table 2). The ability to agglutinate guinea pig erythrocytes was comparable to that of hosts expressing wild-type type 1 fimbriae, indicating acceptance of the FimH adhesin by the FlC system. Electron microscopy showed the bacteria to be profusely fimbriated (Fig.…”

Integrated regulatory responses of fimB to N -acetylneuraminic (sialic) acid and GlcNAc in Escherichia coli K-12

“…Allelic exchange of WT sequences was carried out as reported (34,35). Intermediate strains containing deletions of the yjhA-fimB intergenic region, nanR or pdhR, replaced by a sacB-kan r cassette, were transformed with derivatives of the temperature-sensitive plasmid, pMAK705 (35).…”

Robust functional gene validation by adenoviral vectors: one-step Escherichia coli-Derived Recombinant Adenoviral Genome construction

“…(a) Plasmid pJJ301 was constructed in a pIG5 23 plasmid background by removal of all adenoviral-(5 0 ITR, C, pIX and IVa2 coding regions), and nls-LacZ coding sequences, followed by insertion of desired elements, by standard molecular biology techniques. 29 This plasmid contains an 'upstream region' (UR) (a PCR fragment amplified from nt 57 627 to 57 951 of pAdDE1-CMV-nlsLacZ (pXL2822 10 ), which confers 325 bp of sequence homology with pAdDE1-CMV-nlsLacZ), modified pIX and IVa2 coding regions (a complete Syngen #2 fragment 23 ), nts 4355-5191 of Ad5 IVa2, 20 which confer 848 bp of sequence homology with pAdDE1-CMV-nlsLacZ, the sacB gene of Bacillus subtilus, which is lethal to E. coli in the presence of sucrose 30,31 (and was taken from pXL2756 10 ), an R6Kg conditional bacterial origin of replication, 21,22 and a transposon incorporating a Kanamycin resistance cassette. 32,33 (b) pAdDE1-CMV-nlsLacZ, which has been described (as pXL2822) previously, 10 consists of an E1E3-deleted recombinant Ad5 genome expressing LacZ with an N-terminally fused nuclear localisation signal (under control of a CMVie promoter) inserted into an E. coli incompatibility group P (incP)-derived replicon containing a vegetative origin of replication (oriV).…”