A new horizon for pBR322: in vivo insertion of plasmid fragments into wide host range shuttle vectors.

Plasmid vectors based on pMB8, such as pBR322, pUC 18, pGEM5 and pBluescript (1), have become virtually indispensable tools in molecular biology. Unfortunately, pMB8 derived vectors are restricted to Entembacteriacea. Transfer of cloned DNA fragments into other bacterial species therefore necessitates alternative plasmid vectors. Insertion of the gene of interest into such (broad-host-range) plasmids may be time consuming and is often difficult to accomplish due to the lack of suitable restriction sites, expression signals and genetic markers. In this paper I report a simple procedure for the introduction of genes cloned in pBR322-type vectors into organisms which do not support the replication of these plasmids. The procedure consists of the insertion in vitro of an antibiotic resistance cassette into the plasmid of interest, followed by transposition in vivo of the relevant part of the plasmid into a shuttle vector. I anticipated that a single copy of the transposon Tni inverted repeat would be conserved in many pBR322 type plasmids, especially those which still bear the P-lactamase and the pMB8 origin of replication on a single contiguous DNA fragment. This assumption was confirmed by inspection of the published sequences of the following non-exhaustive list of cloning vectors: pBluescript SK, pBR322, pEMBL8, pGEM5, pJRD184, pKK233.8, pKOl, pMAC5-8, pSPORT-1; pUC 18. Insertion of a second copy of the 38 bp To? inverted repeat in the proper orientation into these 'pBR322 type' vectors would fulfill the minimal in cis requirements for transposition and would thus render part of their sequences transposable in the presence of Tni transposase in vivo (2). Such an insertion is facilitated by the antibiotic resistance cassettes shown in