A transposon vector for broad host range cloning

Abstract

The genetic analysis of many procaryotes is hindered by the lack of suitable broad host range cloning vectors. These vectors are often relatively large with limited cloning possibilities and frequently exhibit instability in recipient cells (1). Furthermore, the antibiotic resistance markers are not always well expressed in the recipient. We have constructed a 6.9 kb vector, based on transposon Tn5, which allows transposition of cloned DNA onto any broad host range conjugative (BHRC) plasmid and subsequent gene transfer. This flexible approach alleviates the aforementioned problems. Plasmid pJP5011 consists of pJP5010 carrying transposon Tn5-lacZ' in the ampicillin resistance (Amr/) gene. Plasmid pJP5010 is pUC19 (2) with the lacZ' fragment deleted. Tn5-lacZ' is Tn5 carrying the lacZ' fragment of pUC19. HaeU was used for deletion and insertion of the lacZ' fragment. Plasmid pJP5011 has unique Sail, Accl, Xbal, BamHl, Xmal, Smal, Kpnl, Sacl and EcoRI restriction sites available for a-complementation cloning. This was confirmed by performing clonings without insert DNA and noting the absence of white colonies on selective MacConkey (Oxoid) or X-gal media. Using pJP5011, a number of DNA fragments have been transferred to Rhodobacter sphaeroides giving the expected resultant phenotype. Routinely, a BHRC plasmid is mated into the recombinant cells followed by mating with the recipient. Growth on an appropriate contraselective antibiotic and kanamycin indicates recombinant transfer via transposition. In some cases, kanamycin resistance is not well expressed in the recipient or the recipient mutates frequently to kanamycin resistance. In such situations, transposition can first be selected by transfer to another E. coli strain followed by the use of a BHRC plasmid marker to detect BHRC plasmid: :Tn5-insert transfer to the recipient. Selection of transposition in E. coli is also advisable in situations where conjugal transfer frequency to the recipient is low due to restriction or other factors. Some advantages of this system are: a) The selection marker employed can be varied with the use of alternate BHRC plasmids. b) BHRC plasmid: :Tn-5 insert DNA constructs are very stable in a wide variety of organisms. For example, the 28 kb carotenoid gene cluster, cloned in Tn5 and expressed in a number of gram negative organisms (3), has almost never shown loss or alteration of colour due to DNA rearrangement. This experience has been repeated with other cloned pathways (unpublished data), c) Inserts in Tn5 as large as 40 kb can be transposed to BHRC plasmids (3). d) The kanamycin resistance (KanO gene of Tn5 is well expressed in many organisms (4). e) The carrier replicon (BHRC, mobilizable, suicidal, viral etc.) can be varied according to need. For instance, stable chromosomal insertions of recombinant DNA could be obtained in recipients by using mobilizable suicide vectors. For transformable recipients, pJP5011 itself could be B-y~t J|!D