Journal of General Microbiology (1988), 134, 605-609. Printed in Great Britain 605 Construction of a Shuttle Vector for Inducible Gene Expression in Escherichia coli and Bacillus subtilis By HEINRICH LEONHARDT AND JUAN C. ALONSO* Max-Planck-lnstitut jiir Molekulare Genetik, Ihnestr. 73, D- 1000 Berlin 33, FRG (Received 4 August 1987; revised 13 November 1987) The construction of a shuttle vector for inducible gene expression allowing fast and easy cloning in Escherichia coli and subsequent transformation of Bacillus subtilis is presented. The expression is based on the regulation of the tac promoter by the Lac repressor which was assayed with the xylE gene from Pseudomonas putida as a marker gene. The laclq gene, transcribed by the strong spo promoter, allowed full repression of the weak tac promoter. INTRODUCTION A number of proteins produced by genetic engineering techniques are detrimental to cell growth. These problems can be overcome by the use of either chemically inducible or thermoinducible expression systems. Yansura & Henner (1983) adapted the lac operator- repressor system to achieve IPTG-induci ble expression of interferon in Bacillus subtilis. Another approach is to use a promoter-repressor system from phage 4105. The repressor itself may be temperature-sensitive (Osburne et al., 1985) or its gene can be placed on a plasmid temperature- sensitive for replication (Dhaese et al., 1984); in other words, the repression can be relieved by incubation at a non-permissive temperature. In these thermoinducible systems the expression of cloned genes can be repressed only about 30-fold while a chemically inducible system is repressed no more than 100-fold. Utilizing the well-characterized lac repressor-operator system, which operates in B. subtilis (Yansura & Henner, 1983), we have developed an expression system regulated at the level of transcription. The use of the weak tac promoter (Moran et al., 1982; Peschke et al., 1985) should allow a high repression and, therefore, avoid interference with cell growth. METHODS Bacteria. Bacillus subtilis strain PSLl (Ostroff & Pene, 1983) and Escherichia coli strain HBlOl (Boyer & Roulland-Dussoix, 1969) were used. Ty broth for B. subtilis (Rottlander & Trautner, 1970) and L broth for E. coli (Luria & Burrous, 1957) were used throughout both as liquid and solid medium. The media were supplemented either with ampicillin (50 pg ml-I), neomycin (5 pg ml-I) or phleomycin (0.1 pg ml-I). Plasmids. pKK223-3 (Brosius & Holy, 1984), placIq (Bagdasarian et al., 1983), pTG402 (Zukowski et al., 1983) and PUB 1 I0 (Lacey & Chopra, 1974) were used. Genes encoded by PUB 1 10 are kan and ble (conferring NmRand PmR, respectively) and repU, the replication initiator protein (unpublished results). Recombinant DNA procedures. Restriction endonucleases and DNA modification enzymes were purchased from Boehringer and used as described by the manufacturer. Transjormation. E. coli cells were transformed as described by Cohen et al. (1 972). Transformation of competent B. subtilis cells was done as described previously (Rottlander & Trautner, 1970). Catechol2,3-oxygenase assay. This was done essentially as described by Zukowski et al. (1983). Cell cultures were grown to an OD,,, of 0.2 units; if required, IPTG was added and cells were further incubated for 120 min. All values reported are the mean of four independent experiments with SE values within 15%. Abbreviation : IPTG, isopropyl P-D-thiogalactopyranoside. 0001-4342 0 1988 SGM