A New Horizon for Pbr322: in Vivo Insertion of Plasmid Fragments Into Wide Host Range Shuttle Vectors Menno Kok

1995 Oxford University Press Nucleic Acids Research, 1995, Vol. 23, No. 24 5085-5086 A new horizon for pBR322: in vivo insertion of plasmid fragments into wide host range shuttle vectors Menno Kok Departement de Genetique et Microbiologie, Universit6 de Geneve, 1 Rue Michel Servet, 1211 Geneve, Switzerland Received August 3, 1995; Revised and Accepted November 17, 1995 Plasmid vectors based on pMB8, such as pBR322, pUC18, pGEM5 Figure 1, which encode either kanamycin or tetracycline resistance and pBluescript (1), have become virtually indispensable tools in and bear a copy of the 38 bp inverted repeat at their extremities. molecular biology. Unfortunately, pMB8 derived vectors are A typical experiment is illusuated in Figure 2. The Escherichia restricted to Enterobacteriacea. Transfer of cloned DNA fragments coli thyA gene present in the pBR322 derivative pBTAH1.2 (3) was into other bacterial species therefore necessitates alternative plasmid rendered transposable by the insertion of a 1673 bp kanamycin vectors. Insertion ofthe gene ofinterest into such (broad-host-range) cassette, obtained from plasmid pGMK833 by BanHI digestion. plasmids may be time consuming and is often difficult to accomplish Next, the thyA gene, flanked by the ampicillin and kanamycin due to the lack of suitable restriction sites, expression signals and resistance markers, was inserted into broad host range plasmid R751 genetic markers. In this paper I report a simple procedure for the by transposition. This was done by transforming E.coli MS967, introduction of genes cloned in pBR322-type vectors into organisms canying plasmids pSC259, encoding Tn3 transposase, and the which do not support the replication of these plasmids. The shuttle plasmid R751 (2), with pBTAH1.2::Kml plasmid DNA. As procedure consists ofthe insertion in vitro of an antibiotic resistance controls, pBTAH1.2::Km2 DNA, in which the kanamycin resistance cassette into the plasmid ofinterest, followed by transposition in vivo cassette had been inserted in the inverse orientation, and unmodified of the relevant part of the plasmid into a shuttle vector. pBTAH1.2 were also introduced into MS967. Transposition of the I anticipated that a single copy of the transposon Tn3 inverted thyA gene into conjugative plasmid R751 was detected by mating repeat would be conserved in many pBR322 type plasmids, especially those which still bear the flactamase and the pMB8 106 MS967(pBTAHI.2::Kml) with a >10-fold excess of the E.coli origin of replication on a single contiguous DNA fragment. This MC4100 thyA mutant Exconjugants were obtained at a frequency assumption was confirmed by inspection ofthe published sequences of -0.04% per donor per generation. I have tested 300 R751::thyA of the following non-exhaustive list of cloning vectors: pBluescript exconjugants, all of which were found to be resistant to ampicillin, SK, pBR322, pEMBL8, pGEM5, pJRD184, pKK233.8, pKOl, kanamycin and trimethoprim and to complement the thymidylate pMAC5-8, pSPORT-1; pUC18. Insertion of a second copy ofthe 38 synthase mutation of MC4100(thyA), suggesting that the plasmid bp Tn3 inverted repeat in the proper orientation into these 'pBR322 coded thyA fragment was copied faithfully into the shutfle vector type' vectors would fulfill the minimal in cis requirements for R751. The integrity ofthe transposable fiagments was confirmed by transposition and would thus render part of their sequences restriction mapping of four R751::thyA recombinants (results not transposable in the presence of Tn3 transposase in vivo (2). Such an shown). No recombinants were obtained from MS967 transformed insertion is facilitated by the antibiotic resistance cassettes shown in with either of the two control plasmids. Table 1. pBR322 type plasmids as source of transposable sequences Vector Insertion site in the Transposable markersa Fidelity of vector/inserted cassette transposition (%)b pBluescript SacI/Tet Tet, Ap 100 pGEM7 BamHI/Km Km, Ap 100 pUC18 BamHI/Km Km, Ap 100 pBTAHI.2 BamHI/Km Km, Ap, thyA 100 pKK232.8 PvuII/Km Km, Ap 100 pJRD184 Xho/Km Km, Tc, Ap 100 pSPORT1 BssHIlITet Tet, lacI, lacZa, Ap 100 aMarkers encoded by the DNA fragment flanked by the two Tn3 inverted repeats. bTransposition was evaluated by transforming MS967 with plasmid DNA into which a Tn3 cassette had been inserted, transferring R75 1 recombinants to a plasmid free Ecoli strain, and selecting for the marker encoded by the inserted cassette (kanamycin or tetracycline resistance). The fidelity of transposition is defined as the percentage of exconjugants which bear all the transposable markers. In all cases >50 exconjugants were tested. 5086 Nucleic Acids Research, 1995, Vol. 23, No. 24 EcoRI HindIl m } J Tetracyclhne tsette (1636 bp) ~0] Ap / i1i I IRTn3 r pBTAHI.2 HindlIl ,i iia z z-1 BamHI (tet) :,i i .It,A .1, Kanamycin cassette (1952 bp) --, IK~III 14M-1~~~~~I1' 11 4 Q A~~k 4P1I Hinduil 3amHI Figure 1. Insertion of Tn3 cassettes into pBR322 type plasmids. The kanamycin- and tetracycline-cassettes may be obtained from plasmids pGMK833 or pGMK836, respectively, by digestion with Nsil (as shown here) or by using the restriction sites shown above the DNA bar, such as MluI, SmaI or Sacl. The restriction sites indicated below the DNA barcut close to or within the antibiotic marker, and cannot be used to prepare the cassette. IR = Tn3 inverted repeat; Pla = ,B-lactamase; Tet = Tetracycline resistance gene; Km = Kanamycin resistance gene; cos = Phage lambda cos site; ori = origin of replication derived from pMB8; kb = kilobasepairs. IRTn3 I have tested a number of other plasmid vectors using the protocol outlined above. In all cases the predicted plasmid sequences were rendered transposable by the insertion of the Figre 2. Cassette insertion and transposition. Plasmid pBTAH1.2 bears the kanamycin or the tetracycline cassette, and were stabily inserted E.coli thyA fragment inserted into the unique Hindm site ofpBR322. A BamHI kanamycin fragment was inserted into the unique BamHI site of this plasmid into broad host range vector R751 (Table 1). This confirmed that to yield pBTAHl.2::Kml and pBTAHI.2::Km2. Ecoli MS967 bearing in these plasmid vectors the 38 bp transposon inverted repeat plasmid pSC259 encoding Tn3 transposase and conjugative plasmid R751, downstream of the P-lactamase gene was functionally intact. In encoding trimethoprim resistance, was transformed with the thyA plasmids to these experiments, I took advantage of conjugation of plasmid ampicillin resistance. In this strain, pBTAHl.2::Km1 can act as a substrate for Tn3 transposase, whereas pBTAHl.2Km2 cannot, due to the relative orienta- R75 1 to identify transposition recombinants, and to transfer the tion of the Tn3 inverted repeats. Transposition events were identified by DNA fragment of choice into alternative bacterial host back- conjugation of the shuttle vector to Ecoli MC4100 (thyA). grounds. Recombinants of plasmid R751 were readily transfer- able from one E.coli strain to another and may be transferred to a wide variety of Gram-negative bacteria (2). The procedure is straight forward, reliable, and quick: cassette insertion, trans- ACKNOWLEDGEMENTS formation of E.coli MS967, and conjugation can be completed The author wishes to thank Dr P. Ahrweiler for providing plasmid within 2 days. pBTAHI.2. Part of the research was supported by the Swiss If, alternatively, a non-conjugative shuttle vector would be used National Science Foundation. as a target for transposon insertion, extraction of plasmid DNA from the transposition proficient E.coli strain (carrying pSC259 and the shuttle vector of choice) followed by transformation of REFERENCES plasmid free E.coli, selecting the appropriate markers, may yield 1 Sambrook J., Fritsch E.F. and Maniatis T. (1989) In Molecular Cloning: A appreciable amounts of transposition recombinants, as the Laboratory Manual, Second edition. Cold Spring Harbor Laboratory observed transposition frequencies are high (2). Although I have Press, Cold Spring Harbor, New York. 2 Kok M., Rekik M., Witholt B. and Harayama S. (1994) J. Bacteriol. 176, focused here on the application of the system to cloning in 6566-6571. Gram-negative bacteria, Tn3 mediated gene transfer is potentially 3 Bell-Pedersen D., Galloway Salvo J.L. and Belfort M. (1991) J. Bacteriol. applicable to all organisms for which shuttle vectors are available. 173, 1193-1200..

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