WO1992006186A1 - Cloning cartridges and expression vectors in gram-negative bacteria - Google Patents

Abstract

Cloning cartridges comprising a positive regulatory gene nahR from plasmid NAH7, a promoter Pg that is regulated by nahR, a multiple cloning site, a transcription terminator, and a gene conferring tetracycline resistance. Such cartridges can be inserted into plasmids of choice to form novel expression vectors in which high level gene expression is inducible with an inexpensive non-toxic inducer at low concentrations.

Description

SPECIFICATION

TITLE: Cloning Cartridges and Expression

Vectors in Gram-Negative Bacteria

INVENTOR: Kwang-Mu Yen

BACKGROUND OF THE INVENTION

An expression plasmid vector is an indispensable tool for the study of gene expression in bacterial cells. The need to develop expression vectors is particularly acute for little- studied bacterial strains in which gene expression needs to be assessed. At least two approaches can be imagined for the introduction of an expression vector into these bacterial strains. A native plasmid, if it is known, can be converted into an expression vector or a broad host range expression vector can be introduced into these strains. The native plasmid of a poorly- studied strain is usually not very well characterized. Genetic elements essential for regulated gene expression have to be introduced from other sources in order to convert it into an expression vector. A number of broad host range vectors have been developed for Gram-negative bacteria (see Bagdasarian et al., 1983, Gene 26: 273-282; Mermod et al., 1986a, In: Sokatch, J.R and Ornston, L.N., (Eds.), The Bacteria. A Treatise On Structure and Function, Vol. 10. Academic Press, Orlando, p. 325-355; and Schmidhauser et al., 1988, Vectors: A Survey of Molecular Cloning Vectors and Their Uses (Rodriguez & Denhardt, eds.), Butterworth, Boston, pp. 287-332 for reviews). However, only a few of these vectors (Bagdasarian et al., 1983, Gene 26: 273- 282; Mermod et al., 1986b, J. Bacteriol. 162: 447-454; Furste et al., 1986, Gene 48: 119-131) allow any type of regulated gene expression.

The regulation of naphthalene catabolic genes carried by the

NAH7 plasmid has been well studied (for review, see Yen and

Serdar, 1988, CRC Crit. Rev. Microbiol., 15: 247-268). The NAH7 plasmid is a naturally-occurring plasmid in the Pseudomonas nutida (P. putida) strain G7 (ATCC 17485). It carries catabolic genes for the degradation of naphthalene to Krebs cycle intermediates

These genes are organized in two operons. The first operon encodes enzymes for the conversion of naphthalene to salicylate

(upper pathway) and the second operon encodes enzymes for the oxidation of salicylate to acetylaldehyde and pyruvate (lower pathway). Both operons are activated in the presence of the inducer salicylic acid, or some of its analogs , and the product of the regulatory gene, nahR. The nahR gene maps upstream from the lower pathway operon and next to the nahG gene which encodes the enzyme salicylate hydroxylase (Figure 1). The two genes, nahR and nahG. are transcribed in opposite directions and their promoters, P_R and P_G, share sequences. While P_G is subject to the positive regulation of NahR protein, P_R directs the synthesis of NahR protein constitutively. The nucleotide sequences of nahR, P_R and P_G have all been determined (Schell, 1986, J. Bacteriol. 166 : 9-14; You et al., 1988, J. Bacteriol. 170: 5409- 5415; and Schell and Sukordhaman, 1989, J. Bacteriol. 171: 1952- 1959). A bacterial DNA expression unit based on the nahR-p_G regulatory system has not been suggested or prepared for use in the construction of expression vectors . New vectors of broad or narrow host range that allow regulated and more efficient gene expression and are more convenient to use still need to be developed. Construction of a DNA restriction fragment carrying all of the elements essential for gene cloning, selection and expression would facilitate the development of expression vectors. Such a cloning cartridge, if it could be constructed, could then be inserted into a replicon of either broad or narrow host range to convert it into an expression vector. PCT Application PCT/GB89/00341 (Publication No. WO89/09823, 10-19-89) describes a xy1R-derived regulatory cassette from a TOL plasmid for bacterial expression vectors. This cassette contains only the xy1R gene with the binding site of its gene product Xy1R and associated promoter (P_u). The xy1R/P_u cassette is not easily transferable among replicons because there is no selective marker on the cassette. In addition, there is: (i) no transcription terminator, (ii) a paucity of cloning sites, and (iii) no engineering of the promoter to allow precise insertion of a foreign gene for optimal expression. In particular, with respect to (iii), there is no restriction site engineered to include the ATG sequence encoding the initiation codon, and there appears to be ~1.5 kb of uncharacterized DNA separating the promoter region from the cloning site (see Keil et al., J. Bacteriol. 169: 764- 770 (1987) and Harayama et al., J. Bacteriol. 171: 5048-5055 (1989)) which could likely reduce expression of a cloned gene, The regulation of the xy1R/P_u cassette requires the use of toxic chemical inducers, such as toluene. Several constructs using a xy1R/P_u cassette are mentioned, but no expression data is given to determine the usefulness of such constructs. There is, therefore, a need to develop new cloning cartridges and expression vectors that overcome the deficiencies of previously described expression units, including the cassette and vectors described in PCT/GB89/00341.

SUMMARY OF THE INVENTION

According to the present invention, a cloning cartridge and its derivative have been successfully constructed based on the NahR-regulated gene expression system. The two cartridges (also called cassettes) were tested on a derivative of the broad host range plasmid pKT231 (Bagdasarian et al. 1981, Gene 16: 237-247) for use in gene cloning and expression in different bacterial hosts. Plasmid vectors carrying one of the cartridges have allowed cloning and inducible expression of several tested genes in all of the Gram-negative bacteria tested to date. High-level protein production from a cloned gene was also demonstrated. The DNA elements assembled in the cloning cartridges provide previously unavailable convenience and efficiency in gene cloning and expression in Gram-negative bacteria. In addition, cloning cartridges according to the present invention contained on an ~3.6 kb restriction fragment allow efficient (e.g., single-step) construction of expression vectors in a wide variety of Gram- negative bacteria.

The present invention is directed to a cloning cartridge or cassette which comprises five elements essential for efficient gene cloning and expression, based on the NahR-regulated expression system. The five elements comprising a cloning cartridge according to the present invention are: a gene encoding drug-resistance, a nahR gene, a promoter P_G regulated by the nahR gene product, a multiple cloning site, and a transcription terminator. A useful drug-resistance gene is the gene encoding tetracycline-resistance (tet^r) derived from the pBR322 plasmid. In the cloning cartridge, the sequence upstream from the Hind III site in the promoter of the tet^r gene was replaced with the nahR sequence. This sequence substitution generated a new hybrid promoter for the tet^r in the cartridge and had the effect of stabilizing a plasmid carrying a cloning cartridge according to the present invention in the absence of selection. The nahR gene in the cloning cartridge is derived from the NAH7 plasmid naturally occurring in P. putida. It encodes the protein, NahR, that positively regulates the promoter P_G of the lower pathway operon for naphthalene degradation. The promoter P_G is activated in the presence of the NahR protein and an innocuous and inexpensive inducer, sodium salicylate at low concentrations (0.35 mM or lower). Within the promoter P_G of the cloning cartridge, a sequence of 3 nucleotides upstream of the ATG sequence encoding the initiation codon was altered to create an Ndel cloning site which was followed by a number of other cloning sites in the order of 5' - Hpal - Clal - Xbal - Kpnl - Sacl - Xhol - 3'. The 5' end of a characterized gene can be converted into an Ndel site without altering the coding property and cloned into this cartridge for regulated expression. A transcription terminator derived from plasmid pCFM1146 was placed immediately downstream to the multiple cloning site of P_G.

A novel five-element portable cloning cartridge according to the present invention was assembled as an ~3.6 kb EcoRI - Pstl fragment, which can be easily inserted into a variety of different replicons. Such a cloning cartridge, when inserted into a replicon of either broad or narrow host range, converts it into an expression vector. Thus, the present invention is also directed to plasmid vectors of broad or narrow host range containing the cloning cartridge. A particularly preferred embodiment is the broad host range expression vector pKMY299, constructed by replacing the EcoRI - Pstl fragment of plasmid pKMY286 with the ~3.6 kb EcoRI - Pstl cloning cartridge. In particular, plasmid pKMY299 was precisely designed for cloning and expression of genes that contain, or are engineered to contain an Ndel recognition sequence (CATATG) at the 5' end of the gene. For cloning and expression of restriction fragments carrying genes of unknown sequences, the cloning cartridge in pKMY299 was modified by the deletion of the sequence encoding the start codon AUG within the multiple cloning site. This derivative of pKMY299, designated pKMY319, thus comprises a modified cloning cartridge. The sequence deletion prevents false translational initiation in gene expression from the P_G promoter in pKMY319. The usefulness of pKMY299 and pKMY319, as examples of expression vectors containing a cloning cartridge according to the present invention, was demonstrated. Genes of various origins whose products could be easily assayed were cloned into the pKMY299 or pKMY319 expression vector. Regulated (e.g., inducible) gene expression was observed in a variety of Gram-negative host cells. Overproduction of certain gene / products was also demonstrated.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a restriction map of an ~1.7 kb Hindlll/Pstl fragment from a region of plasmid NAH7 containing the nahR gene and part of the nahG gene. P_R and P_G are promoters of nahR and nahG, respectively, with the arrows indicating direction of transcription.

Figure 2 shows the initial steps (from plasmid pKMY256 to pKMY292) in the construction of a cloning cartridge according to the present invention.

Figure 3 shows the subsequent steps (from plasmid pKMY292 to pKMY297) leading to the construction of a cloning cartridge in plasmid pKMY297.

Figure 4 shows the derivation of an expression vector pKHY319 from the pKMY299 expression vector.

Figure 5 shows the SDS-PAGE analysis of protein products from expression vectors pKMY299 and pKMY319, which comprise a cloning cartridge or modified cloning cartridge according to the present invention, in Pseudomonas putida G572. Expression of firefly luciferase under induced and uninduced conditions is shown in lanes 2,8 and 3,9, respectively. Expression of catechol 2,3 dioxygenase under induced and uninduced conditions is shown in lanes 4,6 and 5,7, respectively.

DETAILED DESCRIPTION

A cloning cartridge according to the present invention is designed to contain five elements essential for efficient gene cloning and expression based on the NahR-regulated expression system. These five elements, on a conveniently portable cartridge or cassette, comprise a drug resistance gene tet^r, nahR, P_G, a multiple cloning site, and a transcription terminator. Such a cloning cartridge can be easily inserted into a variety of vectors for efficient and regulated gene expression. A recognition site for the frequently-cutting restriction endonuclease Rsal is located three base pairs upstream from the nahG coding region (Figure 1). Initial steps in the construction of the cloning cartridge involved converting this Rsal site into a Scal restriction site convenient for inserting a multiple cloning site and placing the tet^r gene of the E. coli plasmid pBR322 (Bolivar et al., 1977, Gene 2: 95-113) next to the P_R region.

Plasmid pKMY256 has been described in co-pending and co- assigned U.S. Patent Application Ser. No. ________, filed September ____, 1990, and hereby incorporated by reference in its entirety. It is an E. coli plasmid pUC19 (Yanisch-Perron et al., 1985, Gene 33: 103-119), carrying an ~5.3 kb Pstl insert which contains the nahR gene, P_G, and ~200 base pairs (bp) of the nahG gene. Digestion of pKMY256 DNA with the enzymes Pstl and Sail and self-ligation led to the cloning of a ~400 .bp Sall-Pstl fragment containing P_R and P_G into pUC19. The resulting plasmid was designated pKMY288 (Figure 2 . In pKMY288, the promoters P_R and P_G are located on a ~200 bp Sall-Rsal fragment (Figure 2). The Rsal site of this fragment can be ligated to the Scal site of pKMY256 to regenerate the Scal site. Replacement of the ~1.4 kb Sall-Scal fragment of pKMY256 with the ~200 bp Sall-Rsal fragment of pKMY288 resulted in the deletion of the remaining nahG sequence and the conversion of the Rsal site in P_G into a Scal site (Figure 2). The newly formed plasmid was designated pKMY289 (Figure 2). In order to place the tet^r gene next to P_R, the ~470 bp Bglll-Scal fragment of pKMY289 carrying P_R and P_G was used to replace a ~4.4 kb Bplll-Scal fragment in a pBR322 plasmid carrying a copy of the transposon Tn5 (Sasakawa et al., 1982, Proc. Natl. Acad. Sci. USA 79: 7450-7454; (Figure 2). The resulting plasmid was designated pKMY291 (Figure 2). A pKMY291 derivative, designated pKMY292, was constructed by deleting the intervening sequence between the Hindlll site within nahR and the Hindlll site within the promoter of the tet^r gene (Figure 2). This step placed the tet^r gene immediately downstream from P_R.

In the following steps, a multiple cloning site was inserted at the newly-created Scal site within P_G and a transcription terminator was inserted at the end of the cloning site. In most bacterial genes, the nucleotide sequence ATG specifies the initiation codon. This trinucleotide and the sequence preceding it can be converted by site-specific mutagenesis into the recognition site of the restriction endonuclease Ndel (CATATG) without affecting the coding property of the gene. In order to accommodate genes modified in this manner, a Ndel site can be similarly created in an expression vector at the junction between the promoter and the 5' end of a gene coding region. Other restriction sites can be introduced downstream of the Ndel site for accepting the 3' end of a cloned gene. In such an expression system, the distance between the transcription start site and the gene coding region is unaltered, regardless of the gene cloned. The promoter P_R was modified to contain a Ndel restriction site at the 3' end, followed by a number of other cloning sites.

An oligonucleotide containing the following sequence was synthesized and ligated into the Scal and Pstl sites of pKMY292: Rsal Hpal XbaI

5' ACCATATGGTTAACATCGATTCTAGAGGTACC- 3' TGGTATACCAATTGTAGCTAAGATCTCCATGG- Ndel Clal Kpnl

SacI SacII PstI

GAGCTCCTCGAGCCGCGGACAGATCTCTGCA 3'

CTCGAGGAGCTCGGCGCCTGTCTAGAG 5'

Xhol Bglll

Insertion of this sequence converted the Scal site into the Rsal site naturally occurring within P_G, restored the distance between the Rsal site and the coding region, generated a Ndel cloning site at the 3' end of the P_G sequence and placed a number of other cloning sites immediately downstream of the Ndel site. The resulting plasmid was designated pKMY293 (Figure 3).

The E. coli plasmid pCFM1146 has been described in co- pending and co-assigned U.S. Patent Application Ser. No. _______ filed September ______, 1990, incorporated by reference in its entirety, carries a transcription terminator that can be easily incorporated into other systems. Other sources of transcription terminators are described in co-assigned U.S. Patent No. 4,710,473, hereby incorporated by reference. Downstream from the transcription terminator there is a restriction site for the enzyme Bglll and immediately upstream from the transcription terminator, there is a multiple cloning site including an EcoRI site, a Xhol site and a number of other restriction sites (Figure 3). The transcription terminator in pCFM1146 was placed immediately downstream of the multiple cloning site in pKMY293 in two steps. The BspMll-Pstl fragment of pKMY293 carrying the tet^r gene, P_R, P_G, and the multiple cloning site was initially cloned into the Xmal and the Pstl sites of the plasmid pUC9 (Vieira and Messing, 1982, Gene 19: 259-268) to place the EcoRI site of pUC9 downstream of the tet^r gene (Figure 3). The resulting plasmid was designated pKMY294 (Figure 3). In the next step the EcoRI- Xhol fragment of pKMY294 was cloned into the EcoRI and Xhol sites of pCEM1146 to place the transcription terminator immediately downstream of the multiple cloning site of P_G (Figure 3). The resulting plasmid was designated pKMY295 (Figure 3). The remaining steps established a unique cloning site downstream from the transcription terminator and restored the nahR gene. In pKMY295, the Bglll site downstream from the transcription terminator needed to be replaced with a restriction site unique in the cloning cartridge for the convenience of transferring the cartridge among replicons. To achieve this end, a pUC9 derivative without a Hindlll site, designated pKMY513, was initially constructed by cleaving pUC9 with the enzyme Hindlll followed by end-filling and blunt-end ligation (Figure 3). The EcoRI-Bglll fragment of pKMY295 carrying the tet^r gene, P_R, P_G, the multiple cloning site, and the transcription terminator was cloned into the EcoRI and BamHI sites of pKMY513 to eliminate the

Bglll site and incorporate a unique Pstl site next to the destroyed Bglll site (Figure 3). The resulting plasmid was designated pKKY296 (Figure 3).

In pKMY296, the part of the nahR gene downstream from the Hindlll site within nahR is still missing. To ensure correct and convenient assembly of the nahR gene, an indicator plasmid, designated pKMY512, was constructed which contained the naphthalene dioxygenase gene cluster and its NahR-regulated promoter from the plasmid NAH7. In the presence of an inducer sodium salicylate and the NahR protein, the naphthalene dioxygenase genes of pKMY512 can be turned on, which in turn catalyzes the formation of indigo dye in E. coli (Ensley et al., 1983, Science 222: 167-169). Two intermediate plasmids pN400 and pKMY239 were involved in the construction of the indicator plasmid pKKY512. The plasmid pN400 was constructed by cloning the Pvull-Bglll fragment of plasmid pE317 (Ensley et al., 1983, supra) containing the naphthalene dioxygenase genes into the Smal and BamHI sites of the plasmid pUC18 (Yanisch-Perron et al., 1985, supra). The plasmid pKMY239 was constructed by inserting the ~6.4 kb Sacl fragment of pN400 containing the naphthalene dioxygenase genes into the Sacl site of the broad host range plasmid pKMY223. Plasmid pKMY223 has been described in co- pending and co-assigned U.S. Patent Application Ser. No. ________, filed September ______, 1990, incorporated by reference in its entirety. Deletion of the BgllI-EcoRI fragment of pKMY239 carrying a portion of the nahR gene generated pKMY512. The ~1.1 kb Hindlll fragment of pKMY289 (Figure 2) carrying the portion of nahR downstream from the Hindlll site was inserted into the Hindlll site of pKMY296 to complete construction of the cloning cartridge (Figure 3). In this step the desired plasmid was selected for its ability to render E. coli cells harboring pKMY512 to produce indigo in the presence of sodium salicylate. This plasmid was designated pKMY297 (Figure 3). Thus, in pKMY297, an ~3.6 kb EcoRI-PstI fragment containing the tet^r gene, nahR, P_G, a multiple cloning site, and a transcription terminator was successfully assembled as a cloning cartridge.

To test the ~3.6 kb EcoRI-PstI fragment from pKMY297 for use as a cloning cartridge in different hosts, this fragment was inserted into a replicon derived from the broad host range plasmid RSF1010 (Scholz et al., 1989, Gene 75: 271-288). Plasmid pKT231 is a derivative of RSF1010 (Bagdasarian et al., 1981, supra) and served as a source of the RSF1010 replicon. In pKT231, a Hnal site is located ~200 bp from a Sacl site both of which occur in the polylinker of the cloning cartridge. These two sites were removed from pKT231 by digestion of the plasmid DNA with Hpal and Sacl followed by treatment with the Klenow fragment of E. coli DNA polymerase I to generate blunt ends and self-ligation. The resulting plasmid was designated pKMY286. The broad host range expression vector pKMΥ299 was constructed by replacing the EcoRI-Pstl fragment in pKMY286 with the 3.6 kb EcoRI-Pstl cloning cartridge (Figure 4).

In pKMY299 , the expected nucleotide sequences at the junction between P_G and the multiple cloning site and at the Pstl junction between the cloning cartridge and the RSF1010 replicon were completely confirmed by DNA sequence analysis. Sequences of 79 bp in P_G, upstream of the Ndel site and of 158 bp downstream of this site were determined. The sequence data demonstrated that, as expected, the 5' end of the synthetic polylinker was ligated at the Rsal site close to the 3' end of P_G and that the polylinker sequence downstream of the Xhol site was replaced by pCFM1146 sequences (Figure 3). Downstream from P_G, seven unique restriction sites including a Ndel site followed by Hpal, Clal, Xbal. KpnI. SacI. and Xhol sites, can be used for precise insertion of genes with the 5' end lying within a Ndel recognition sequence. A sequence of 337 bp including 54 bp of the cloning cartridge upstream of the PstI recognition sequence and 277 bp of the RSF1010 replicon downstream of this sequence, was determined. This sequence indicated that the Pstl end of the cloning cartridge was ligated, as expected, at the corresponding Pstl site in RSF1010 DNA starting at nucleotide 7768 (Scholz et al., 1989, supra).

Similar sequence analysis of 303 bp of the RSF1010 replicon upstream of the EcoRI recognition sequence and 36 base pairs of the cloning cartridge downstream of this sequence, also demonstrated, as expected, that the EcoRI end of the cloning cartridge was ligated at the corresponding EcoRI site in RSF1010 DNA starting at nucleotide 8676 (Scholz et al., 1989, supra). However, the same analysis revealed that the sequence from nucleotides 1 to 1653 in RSF1010 DNA (Scholz et al., 1989, supra) was completely deleted in pKMY299. The deleted region contains the entire strA gene and most of the strB gene determining streptomycin resistance (Scholz et al., 1989, supra). Restriction patterns of pKMY286 and pKT231 DNA suggested that this deletion occurred in the plasmid pKT231 used, as described herein. Further analysis of pKMY286 and pKMY299 DNA with various restriction enzymes did not detect other aberrations in pKMY299. Plasmid pKMY299 can be considered, therefore, as an RSF1010 plasmid with nucleotides 1 to 1653 deleted and nucleotides 7768 to 8676 replaced with a 3.6 kb cloning cartridge.

Plasmid pKMY299 was designed for precise cloning and regulated expression of genes that contain, or are engineered to contain, an Ndel recognition sequence at the 5' end. For cloning and expression of restriction fragments carrying genes of unknown sequences, the cloning cartridge in pKMY299 was modified. The sequence ATG within the Ndel recognition sequence in pKMY299 was removed to prevent false translational initiation in gene expression from P_G. This was achieved by digestion of pKMY299 DNA with Ndel and Hpal followed by treatment with Mung Bean nuclease to remove the overhang, and self-ligation of the remaining plasmid DNA. The resulting plasmid was designated pKMY319 (Figure 4). Sequence analysis confirmed the expected location of the five remaining cloning sites in pKMY319 in the order of Cla I , Xbal, Kpnl. Sacl, and Xhol sites. The same analysis revealed that in addition to removal of the AT overhang generated by Ndel digestion as expected, the Mung Bean nuclease unexpectedly removed another eight bp. The expected sequence and the actually observed sequence around the ligation site in pKMY19 are shown as follows:

* * * * *

expected T C A C G A G T A C C A A A C A T C G A T

Clal

* * * *

observed T C A C G A C A T C G A T

ClaI

The asterisks mark bases complementary to the 3' end of the Pseudomonas aeruginosa 16S RNA and define a coding region for the putative ribosome-binding site (Shine and Delgarno, 1975, Nature 254: 34-38). One of the important bases encoding ribosome- binding site was removed in pKMY319. A disruption of this ribosome binding site might prove advantageous in the use of pKMY319 for the expression of cloned fragments containing genes of unknown sequence. A Shine-Delgarno sequence, which is normally located only a few bp upstream from a gene, is usually cloned on a fragment along with the gene. A second ribosome binding site encoded by the expression vector might act to reduce expression (Schauder and McCarthy, 1989, Gene 78 : 59-72).

To evaluate the use of pKMY299 and pKMY319 as expression vectors, genes of various origins whose products could be easily assayed were cloned into the plasmids and their expression tested. An intronless luciferase gene constructed from Photinus pyralis (firefly) cDNA and genomic clones (de Wet et al., 1987, Mol. Cell. Biol. 7 : 725-737), was reconstructed to contain a Ndel site at the 5' end. The reconstructed luciferase gene was cloned into pKMY299 at the Ndel and Asp718 (Kpnl) sites and the resulting plasmid, pKMY520, was introduced into E. coli and P. putida host cells. Expression of the luciferase gene was analyzed in the presence or absence of sodium salicylate as an inducer in both recombinant host cells. Luciferase activity was determined by measuring the light produced in a reaction catalyzed by this enzyme. Production of luciferase protein in P. putida was also analyzed by SDS polyacrylamide gel electrophoresis (SDS-PAGE) of a crude extract. Luciferase production in uninduced recombinan P. putida cells carrying pKMY520 was barely visible on an SDS gel (Figure 5). However, the high sensitivity of the luciferase assay (de Wet et al., 1987, supra) allowed detection of relatively high enzyme activity from uninduced recombinant P. putida or E. coli host cells carrying pKMY520 (Table II). The same assay detected a ~90 fold induction of the luciferase activity in recombinant P. putida host cells (Table II) and an ~80 fold induction of the same activity in recombinant E. coli host cells. The amount of luciferase produced in induced P. putida cells carrying pKMY520 represented ~3.7% of the total soluble proteins (Figure 5). These results demonstrated regulated expression of a eukaryotic gene from pKMY299 in two different Gram-negative hosts.

To test the use of pKMY319 as an expression vector, restriction fragments carrying the toluene monooxygenase (TMO) gene cluster tmoABCDEF from Pseudomonas mendocina KR1 or the catechol 2, 3-dioxygenase gene from the plasmid NAH7 (see Yen and Serdar, 1988 supra, for review) were individually cloned into pKMY319 to generate plasmids ρKMY342 and pKMY517, respectively. The tmoABCDEF gene cluster is described and claimed in co- pending and co-assigned U.S. Patent Application Ser. No. _________, filed September _____, 1990 , incorporated by reference in its entirety. Recombinant plasmid pKMY342 carrying the TMO gene cluster was introduced into a number of Gram-negative bacterial species and plasmid pKMY517 carrying the catechol 2,3- dioxygenase gene was introduced into P. putida. Expression of these genes was measured under induced and uninduced conditions. Significantly higher specific activities of both toluene monooxygenase and catechol 2, 3-dioxygenase were observed from induced cultures than from uninduced cultures of all bacterial strains tested (Tables I and III). These results demonstrated the wide use of pKMY319 as an expression vector in obtaining regulated gene expression in Gram-negative bacteria. Comparing to the level of catechol 2, 3-dioxygenase produced from NAH7, a 25-fold overproduction of this enzyme from pKMY517 was observed under the experimental conditions (Table I). Production of catechol 2, 3-dioxygenase protein in P. putida harboring the plasmid pKMY517 was analyzed by SDS-PAGE of a crude cell extract. The amount of catechol 2, 3-dioxygenase produced represented ~10% of the total soluble proteins, as detected by densitometer analysis of the gel (Figure 5). These results demonstrated the usefulness of pKMY319 in the overproduction of gene product(s).

The stability of the cloning cartridges was tested in view of reports that a plasmid carrying the tet^r gene, used as an element of the cloning cartridges described herein, was not stably inherited in P. putida or E. coli in the absence of selection (Bagdasarian et al., 1982, supra: Kolot et al., 1989, Gene 75: 335-339). It has been suggested that a short sequence within the promoter of the tet^r gene forms a "hot spot" for recombination (James and Kolodner, 1983, in Mechanisms of DNA Replication and Recombination. Cozzarelli, (ed.), pp. 761-772, Liss, New York), and might be responsible for destabilization of this plasmid carrying the tet^r gene (Kolot et al., 1989, supra). This sequence contains a Hindlll site and disruption of the Hindlll recognition sequence or the sequence in its vicinity stabilized the plasmid carrying the tet^r gene (Kolot et al., 1989, supra). In the construction of a cloning cartridge according to the present invention, the sequence upstream from the Hindlll site in the promoter of the tet^r gene was replaced with the nahR sequence (Figure 3). This sequence substitution generated a new hybrid promoter for the tet^r gene and stabilized the plasmid carrying either of the cloning cartridges according to the present invention. The hybrid promoter in the cloning cartridges described herein, allowed the use of the tet^r gene as a selection marker in all of the bacterial strains tested (Tables 2, 3 and 4). To test the stability of a plasmid carrying a cloning cartridge according to the present invention, P. putida KT2440 and P. putida G572 host cells (Table 1) harboring pKMY299 or pKMY319 were grown in L-broth in the absence of tetracycline for over 50 generations. Each of the cultures was streaked on L-agar plates for single colony formation and 100 colonies from each culture were tested on L-agar plates supplemented with tetracycline (50 mg/ml) for tetracycline resistance. All of the colonies tested were tetracycline resistant. This result suggested that use of the tet^r gene in the form carried by the cloning cartridges according to the present invention did not lead to the elimination of either cartridge or elimination of a plasmid carrying either of the cartridges, in the absence of selection.

The invention is now illustrated by the following Examples, with reference to the accompanying drawings.

EXAMPLE 1

Construction of Intermediate Plasmid PKMY289

A. Preparation of Plasmid pKMY256

The construction of plasmid pKMY256 (formerly designated pKY256) has been described in co-pending and co-assigned U.S. patent application Ser. No. 177,631, which is hereby incorporated by reference. The starting material for the construction of pKMY256 was plasmid pKY217 described by Yen and Gunsalus, 1985, J. Bacteriol. 162: 1008-13. Plasmid pKMY256 was constructed according to the following series of steps. In the first step, an ~4.3 kb Hindlll fragment from plasmid pKMY217 containing the nahR and nahG genes was cloned into the Hindlll site of the pKT240 plasmid described by Bagdasarian et al., 1983, Gene 26- 273-82. The resulting plasmid from this first step was designated pKMY219. In the second step, an ~7 kb BamHI - Sacl fragment from pKMY219 containing the nahR and nahG genes was cloned into the BamHI and Sacl sites of the pKT231 plasmid described by Bagdasarian et al., 1981, Gene 16: 237-47. The resulting plasmid was designated pKMY223. In the next step, an ~6 kb Pstl fragment from pKMY223 containing the nahR gene, ~200 base pairs of the nahG gene and the gene conferring kanamycin resistance, was cloned into the Pstl site of the pUC19 plasmid described by Yanisch-Perron et al., 1985, supra. The resulting ~8.0 kb plasmid was designated pKMY256. (Figure 2). The orientation of the ~6 kb Pstl fragment in pKMY256 placed the multi-cloning site of pUC19 from the Sall to the EcoRI site immediately downstream of the Pstl site in the nahG gene. Plasmid pKMY256 was then used to construct intermediate plasmid pKMY289 as follows.

B. Preparation of Plasmid pKMY288

Plasmid pKMY256 DNA was digested with Pstl and SaIl, resulting in 4 PstI-Sail fragments of ~4.9 kb, ~2.7 kb, ~420 bp, and ~10 bp. The digestion mixture was self-ligated and used to transform E. coli JM109 cells. The transformed cells were plated on L-agar with ampicillin (250 μg/ml), IPTG (isopropyl-β-D- thiogalactopyranoside) as inducer and X-gal (5-bromo-4-chloro-3- indolyl-β-D-galactoside)as substrate for the lacZ gene product. The desired construct was screened by picking colorless colonies followed by miniprep analysis. It contained the ~420 bp Pstl- Sall fragment carrying P_R and P_G. The resulting ~3.1 kb plasmid was designated pKMY288 (Figure 3), and comprises pUC19 (~2.7 kb), ~60 bp of the nahR gene, P_R, P_G and ~200 bp of the nahG gene (originally derived from pKY217 as described above). Plasmid pKMY288 is thus the equivalent of subcloning the ~420 bp Pstl- Sall fragment of pKMY256 into plasmid pUC19.

C. Preparation of Plasmid pKMY289

Plasmid pKMY288 DNA (Section B above) was digested with Sall and Rsal. Plasmid pKMY256 DNA (Section A above) was digested with Sall. Scal (compatible with Rsal) and Xbal (to prevent reformation of original plasmid pKMY256 during subsequent ligation). The digested pKMY288 and pKMY256 DNAs were mixed, ligated, and used to transform E. coli JM109. Transformants were selected by plating on L-agar with kanamycin (50 μg/ml). The resulting ~6.8 kb plasmid was designated pKMY289 (Figure 2), and comprises the ~6.6 kb Sall-Scal fragment of pKMY256 and the ~200 bp Sall-Rsal (after ligation, Rsal is converted to Scal) fragment of pKKY288. This ~200 bp fragment comprises ~60 bp of the nahR gene, P_R, P_G (minus the 5 bp sequence ACAGC before the ATG of the nahG gene). None of the nahG gene remains. In the construction of pKMY289, the RsaI site immediately upstream of the nahG gene has been converted to a Scal site, which allows certain manipulations in the following construction steps. EXAMPLE 2

Construction of Intermediate Plasmid PKMY293 A. Preparation of Plasmid pKMY291

Plasmid pKMY289 DNA (Example 1) was digested with Bglll, Scal and Pstl (to prevent reformation of original plasmid pKMY289 during subsequent ligation). Plasmid pBR322::Tn5 (Sasakawa et al., 1982, Proc. Natl. Acad. Sci. USA 79: 7450-7454) DNA was digested with Bglll and Scal. The digested pKMY289 and pBR322::Tn5 DNAs were mixed, ligated and used to transform E. coli JM109 cells. Transformants were selected by plating on L- agar with tetracycline (10 μg/ml). The resulting ~6.1 kb plasmid was designated pKMY291 (Figure 2) and comprises the ~5.6 kb Bglll-Scal fragment of pBR322::Tn5 containing DNA regions essential for replication and the ~470 bp Bglll-Scal fragment of pKMY289 containing ~330 bp of the nahR gene, P_R and P_G (minus the 5 bp sequence ACAGC before the ATG of the nahG gene). In addition to positioning the nahR region with respect to the tet^r gene for further manipultion, this step placed a desirable restriction site (Pstl) downstream of the Scal site for unidirectional insertion of a polylinker at the Scal site.

B. Preparation of Plasmid pKMY292

Plasmid pKMY291 DNA (Section A above) was digested with

Hindlll and Xhol. The digestion mixture was ligated, digested again with Xhol (to prevent the reformation of pKMY291), then used to transform E. coli JM109 cells. Transformants were selected by plating on L-agar with tetracycline (10 μg/ml). The desired ~4.2 kb plasmid was designated pKMY292 (Figure 2). Plasmid pKMY292 resulted from the deletion of an ~1.9 kb Hindlll fragment, including the desired deletion of the Pstl site. In addition, the deletion eliminated the nucleotide sequences upstream of the tet^r promoter and brought the tet^r gene of pBR322::Tn5 as close as possible to the nahR gene sequence, and in a desired orientation.

C. Preparation of Plasmid pKMY293

Plasmid pKMY292 DNA (Section B above) was digested with Scal and PstI. An ~240 bp Scal-PstI fragment of pKMY292 was deleted and replaced with a polylinker of the following sequence: 5 ' -ACCATATGGTTAACATCGATTCTAGAGGTACCGAGCTCCTCGAGCCGCGGACAGATCTCTGCA 3 ' -TGGTATACCAATTGTAGCTAAGATCTCCATGGCTCGAGGAGCTCGGCGCCTGTCTAGAG This double-stranded polylinker contains multiple restriction sites in the order of 5 ' -Rsal-Ndel-Hpal-Clal-Xbal-KpnI-SacI-Xhol- SacII-BglII-PstI-3'.

The single-stranded DNA fragments used in the construction of the polylinker of the above-described sequence were chemically synthesized by using an ABS 380B DNA synthesizer (Applied Biosystems, Inc., 850 Lincoln Centre Drive, Foster City, CA 94404). Many DNA synthesizing instruments are known in the art and can be used to make the fragments. In addition, the fragments can also be conventionally prepared in substantial accordance with the procedures of Itakura et al., 1977, Science 128: 1056 and Crea et al., 1978, Proc. Natl. Acad. Sci. US 25: 5765. The synthesized single strands were annealed to form the double-stranded polylinker as follows. Four single strands were synthesized for annealing and designated 140-27 (31 mer), 140- 28 (35 mer), 140-29 (32 mer) and 140-30 (24 mer), having the following sequences, respectively:

140-27 5'-ACC ATA TGG TTA ACA TCG ATT CTA GAG GTA C-3' 140-28 5'-CTC GGT ACC TCT AGA ATC GAT GTT AAC CAT ATG GT-3' 140-29 5'-CGA GCT CCT CGA GCC GCG GAC AGA TCT CTG CA-3' 140-30 5 '-GAG ATC TGT CCG CGG CTC GAG GAG-3'

The 5' ends of strands 140-28 and 140-29 were kinased (marked with an asterisk below) prior to annealing according to conventional methods, for example, Yansura, et al., 1977, Biochem. 16:1772-1780. The scheme for annealing was:

5' 140-27 3' 5'_* 140-29 3'

3' 140-28 _*5' 3' 140-30 5' The Scal and Pstl digested pKMY292 DNA was ligated with the above-described 5' -Rsal-Pstl-3' polylinker, and the ligation mixture was used to transformE. coli HB101 cells. Transformants were selected on L-agar plates with tetracycline (10 μg/ml). The desired ~4.1 kb plasmid, designated pKMY293 (Figure 3), was identified by miniprep analysis of Xhol and Asp718 digested DNA. The pKMY292 DNA remains uncut by Xhol and Asp718 digestion. Plasmid pKMY293 comprises the synthetic polylinker, P_G, P_R, ~270 bp of the nahR gene, and the tet^r gene immediately downstream. The synthetic polylinker replaced the deleted 5 bp sequence ACAGC at the Scal site of plasmid pKMY292 with the sequence ACCAT to generate the Ndel site in the polylinker. Thus, the insertion of the synthetic polylinker as described: (i) converted the Scal site into the Rsal site naturally occurring within P_G; (ii) restored the distance between the Rsal site and the coding region; (iii) generated an Ndel cloning site at the 3' end of the P_G sequence; and (iv) placed a number of other cloning sites immediately downstream of the Ndel site.

EXAMPLE 3

Construction of Intermediate Plasmid pKMY295 A. Preparation of Plasmid pKMY294

Plasmid pKMY293 DNA (Example 2) was digested with Pstl and BSPMII. In addition, plasmid pUC9 DNA (Vieira and Messing, 1982, Gene 19: 259-268) was digested with Pstl and Xmal. The digested pKMY293 and pUC9 DNAs were mixed, ligated and used to transform E. coli JM109 cells. Transformants were selected by plating on L-agar with tetracycline (10 μg/ml) and ampicillin (500 μg/ml). The desired ~4.8 kb plasmid, designated pKMY294 (Figure 3), was identified by miniprep analysis of Xhol and EcoRI digested DNA. Plasmid pKMY294 comprises the ~2.0 kb Pstl-BspMII fragment of pKMY293 inserted by ligation into the Pstl and Xmal sites of pUC9. The ligation eliminated the Xmal and BspMII sites and placed an EcoRI site downstream of the tet^r gene.

B. Preparation of Plasmid pKMY295

Plasmid pKMY294 DNA (Section A above) was digested with Xhol and EcoRI. In addition, plasmid pCFM1146 DNA was similarly digested with Xhol and EcoRI. The construction of pCFM1146 is described in co-pending and co-assigned U.S. Patent Application Ser. No. 177,631, which has been incorporated by reference. The XhoI-EcoRI-digested pKMY294 and pCFM1146 DNAs were mixed, ligated and used to transform E. coli FM5 cells. E. coli FM5 cells were derived from a strain of E. coli K-12 and contained an integrated λ phage repression gene, CI₈₅₇ (Burnette et al., 1988, Bio/Technology 6: 699-706). Transformants were selected by plating on L-agar with tetracycline (10 μg/ml) and kanamycin (50 μg/ml). The resulting ~6.8 kb plasmid was designated pKMY295 (Figure 3). Miniprep analysis of Xhol-EcoRI digested pKMY295 confirmed that an ~2.0 kb fragment of pKMY294 had been successfully inserted in pCEMH46 with the concomitant deletion of a small segment of the pCFM1146 cloning site comprising the EcoRI-Hpal-KpnI-Ncol-Hindlll-Xhol sites. In pKMY295, the transcription terminator in pCFM1146 is located immediately downstream of the multiple cloning sites of P_G.

EXAMPLE 4

Construction of Intermediate Plasmid PKMY297

A. Preparation of Plasmid pKMY296

The starting materials for the construction of plasmid pKMY297 were plasmid pKMY296 DNA (Example 3) and plasmid pKMY513 DNA. Plasmid pKMY513 is essentially the pUC9 plasmid (Vieira and Messing, 1982, supra) in which the Hindlll site has been deleted as follows. Hindlll-digested pUC9 DNA was treated with the Klenow fragment of DNA polymerase I to fill in the sticky ends created by the Hindlll digestion. The Klenow-treated DNA was ligated, treated with Hind III, and used to transform E. coli JM109 cells. Transformants were selected on L-agar plates with ampicillin (250 μg/ml). Miniprep analysis of selected transformants confirmed that the DNA was resistant to Hindlll digestion. The Hindlll resistant plasmid DNA was designated pKMY513.

The pKMY513 DNA thus obtained was digested with EcoRI and BamHI. Plasmid pKMY295 DNA (Example 3) was digested with EcoRI and Bglll. The digested DNAs were mixed, ligated, and the ligation mixture used to transform E. coli JM109 cells. Transformants were selected on L-agar plates containing tetracycline (10 μg/ml) and ampicillin (500 μg/ml). The desired ~5.1 kb plasmid, designated pKMY296 (Figure 10), contained the ~2.4 kb EcoRI-Bglll fragment of pKMY295 ligated with the ~2.7 kb EcoRI -BamHI fragment of pKMY513. The ligation eliminated the Bglll and BamHI sites and placed a unique Pst I site downstream of the transcription terminator.

B. Preparation of Plasmid pKMY297

Plasmid pKMY296 DNA (Section A above) was digested with Hindlll. Similarly, plasmid pKMY289 DNA (Example 1) was digested with Hindlll. The digested pKMY296 and pKMY289 DNAs were mixed, ligated and used to transform E. coli HB101 cells containing plasmid pKMY512. Plasmid pKMY512 was used, in order to specifically select only those transformants that contained a plasmid having the ~1.1 kb Hindlll fragment of pKMY289 inserted into the unique Hindlll site of pKMY296 to recreate a complete nahR gene. The construction of pKMY512 and its use in the selection of the desired transformant are described as follows.

The starting material for the construction of pKMY512 was plasmid pN400 DNA. Plasmid pN400 was itself constructed by cloning the ~7.5 kb PvuII-Bglll fragment of plasmid pE317 (Ensley et al., 1983, Science 222: 167-169), which contains the naphthalene dioxygenase genes from plasmid NAH7 , into the Smal and BamHI sites of plasmid pUC18 (Yanisch-Perron et al., 1985, supra). Plasmid pN400 DNA was digested with Sacl and an ~6.4 kb Sacl fragment containing the naphthalene dioxygenase genes was inserted by ligation in the same orientation as the nahG gene into SacI-digested pKMY223 DNA to yield intermediate plasmid pKMY239. The desired plasmid pKMY512 was obtained by deletion of an ~6.1 kb Bglll-EcoRI fragment from pKMY239 to delete a portion of the nahR gene and its activity. Plasmid pKMY512 thus contains all the structural naphthalene dioxygenase genes, but no functional nahR gene. The nahR gene product positively controls the expression of the naphthalene dioxygenase structural genes, and these structural gene products are able to catalyze the production of indigo in E. coli as shown by Ensley et al., 1983, suora. Without the nahR gene product, no expression of naphthalene dioxygenase activity and no indigo production is possible in transformants with pKMY512 alone. When a transformant contains plasmid pKMY512, and a derivative of plasmid pKMY296 in which the ~1.1 kb Hindlll fragment of pKMY289 has been inserted in the correct orientation so as to yield a functional nahR gene, such a transformant contains two complementing plasmids and will be able to produce indigo in the presence of an inducer of the naphthalene dioxygenase genes.

Using this complementation system, transformants were initially selected on L-agar plates with 500 μg/ml ampicillin (pKMY296 selection marker), 50 μg/ml kanamycin (pKMY512 selection marker), and 1.0 mM sodium salicylate as inducer of the naphthalene dioxygenase genes. Blue colonies, due to indigo production, were selected for further analysis. Miniprep DNA from the blue colonies was used to transform E. coli HB101 cells, and these secondary transformants were selected on L-agar plates with 500 μg/ml ampicillin only. Miniprep analysis of Hindlll digested DNA from these secondary transformants confirmed that the ~1.1 kb Hindlll fragment of pKMY289 had been successfully cloned into the Hindlll site of pKMY296, thus generating the desired ~6.2 kb plasmid designated pKMY297 (Figure 3).

With the successful construction of plasmid pKMY297, the ~3.6 kb EcoRI-Pstl cloning cartridge (or cloning cassette) containing the 5 desired elements was completed. The cartridge contains a regulatory gene, a promoter regulated by the regulatory gene, a multiple cloning site (polylinker), a transcription terminator, and a gene encoding antibiotic resistance.

EXAMPLE 5

Construction of Plasmid PKMY299

To test the ~3.6 kb EcoR l-Pst I fragment from pKMY297 for use as a cloning cartridge or cassette in different hosts, this fragment was inserted into a replicon derived from a broad host range plasmid RSF1010 (Scholz et al., 1989, supra). The broad host range expression vector pKMY299 was constructed as follows. A. Preparation of Intermediate Plasmid pKMY286

Plasmid pKT231 DNA (Example 1) was digested with Sacl and Hpal. The Hpal site is between the EcoRI and Sacl sites of pKT231, ~200 bp from the Sacl site. The SacI- and Hpal-digested pKMY231 DNA was treated with the Klenow fragment of DNA polymerase I to create blunt ends. The Klenow-treated DNA was then ligated and used to transform E. coli HB101 cells. Transformants were selected on L-agar plates with 50 μg/ml kanamycin. The resulting ~10.3 kb plasmid was designated pKMY286. In pKMY286, the Hpal and Sacl sites from pKT231 were deleted, so that these two sites within the polylinker would be available as cloning sites.

B. Preparation of Plasmid pKMY299

Plasmid pKMY286 DNA (Section A above) was digested with EcoRI and PstI. Plasmid pKMY297 DNA (Example .4) was also digested with EcoRI and Pstl. and, in addition, with Scal (to prevent regeneration of plasmid pKMY297). The digested pKMY286 and pKMY297 DNAs were mixed, ligated and used to transform E. coli HB101 cells. Transformants were selected by plating on L- agar with tetracycline (10 μg/ml). Tetracycline-resistant colonies were picked and tested on L-agar with tetracycline (10 μg/ml) and ampicillin (500 μg/ml). Tetracycline-resistant and ampicillin-sensitive colonies were picked and examined by miniprep analysis for the ligation of the ~3.6 kb EcoRI-Pstl cassette of plasmid pKMY297 with the ~6.1 kb EcoRI-Pstl fragment of plasmid pKMY286. The desired ~9.7 kb plasmid containing this cassette was designated pKMY299 (Figure 4). Plasmid pKMY299 in E. coli HB101 cells has been deposited with the American Type Culture Collection as strain EcY5103 on September 25, 1990 and given accession number A.T.C.C. 68427.

C. Selected Sequence Analysis of Plasmid pKMY299 DNA

In pKMY299 the expected nucleotide sequences at the junction between P_G and the multiple cloning site and at the Pstl junction between the cloning cartridge and the RSF1010 replicon were completely confirmed by DNA sequence analysis. Sequences of 79 base pairs in P_G, upstream of the Ndel site and of 158 base pairs downstream of this site were determined. The sequence data demonstrated that, as expected, the 5' end of the synthetic polylinker was ligated at the Rsal site close to the 3' end of P_G and that the polylinker sequence downstream of the Xhol site was replaced by pCFM1146 sequences (Figure 3). Downstream from P_G, seven unique restriction sites including a Ndel site followed by Hpal, Clal, Xbal, Kpnl, Sacl, and Xhol sites can be used for precise insertion of genes with the 5' end lying within a Ndel recognition sequence. A sequence of 337 bp, including 54 bp of the cloning cartridge upstream of the Pstl recognition sequence and 277 bp of the RSF1010 replicon downstream of this sequence, was determined. This sequence indicated that the Pstl end of the cloning cartridge was ligated, as expected, at the corresponding Pstl site in RSF1010 DNA starting at nucleotide 7768 (Scholz et al., 1989, supra).

Similar sequence analysis of 303 bp of the RSF1010 replicon upstream of the EcoRI recognition sequence and 36 bp of the cloning cartridge downstream of this sequence, also demonstrated, as expected, that the EcoRI end of the cloning cartridge was ligated at the corresponding EcoRI site in RSF1010 DNA starting at nucleotide 8676 (Scholz et al., 1989, supra). However, the same analysis revealed that the sequence from nucleotides 1 to 1653 in RSF1010 DNA (Scholz et al., 1989, supra) was completely deleted in pKMY299. The deleted region contains the entire strA gene and most of the strB gene determining streptomycin resistance (Scholz et al., 1989, supra). Restriction patterns of pKMY286 and pKT231 DNA suggested that this deletion occurred in the plasmid pKT231 used, as described herein. Further analysis of pKMY286 and pKMY299 DNA with various restriction enzymes did not detect other aberrations in pKMY299. Plasmid pKMY299 can be considered, therefore, as an RSF1010 plasmid with nucleotides 1 to 1653 deleted and nucleotides 7768 to 8676 replaced with a 3.6 kb cloning cartridge.

EXAMPLE 6

Construction of Plasmid PKMY319

Plasmid pKMY299 containing the ~3.6 kb EcoRI-Pstl cloning cartridge, described in Example 5 above, was designed for the cloning and expression of genes containing the Ndel recognition sequence at the 5' end. For the cloning and expression of restriction fragments carrying genes of unknown sequences, the cloning cartridge in pKMY299 was modified to remove the sequence ATG within the Ndel recognition sequence in pKMY299 to prevent false translational initiation in gene expression from P_G. Specifically, the ATG sequence within the Ndel recognition sequence of the polylinker from plasmid pKMY299 was removed as follows. Plasmid pKMY299 DNA (Example 5) was digested with Ndel and Hpal, then treated with Mung Bean nuclease (New England Biolabs, 32 Tozer Road, Beverly, MA 01915) according to the manufacturer's instructions, to remove the overhang. The nuclease-treated DNA was then ligated and used to transform E. coli HB101 cells. Transformants were selected on L-agar plates with tetracycline (10 μg/ml). The desired plasmid, in which the Ndel and Hpal sites were effectively deleted was designated pKMY319 (Figure 4). Plasmid pKMY319 in E. coli HB101 cells has been deposited with the American Type Culture Collection as strain EcY5110 on September 25, 1990 and given accession number A.T.C.C. 68426. When the sequence around the ligation site in pKMY319 (i.e., ligation after Ndel. Hpal and Mung Bean nuclease treatment of pKMY299 DNA) was analyzed, it was found that in addition to removing the single-stranded AT overhang created by Ndel. Mung Bean nuclease had removed 8 bp of sequence, including at least one of the nucleotides thought to be important for encoding the ribosomal binding site 5' to the AUG translation start codon. (The sequence of this ribosomal binding site is reviewed in Yen and Serdar, 1988, CRC Crit. Rev. Microbiol. 15: 247-267 (see Figure 4 at p. 255). The expected sequence and the actually observed sequence around the ligation site in pKMY319 is shown as follows:

* * * * *

expected T C A C G A G T A C C A A A C A T C G A T

Clal

* * * *

observed T C A C G A C A T C G A T

Clal

A disruption of this ribosomal binding site may prove advantageous in the use of pKMY319 for the expression of cloned fragments containing genes of unknown sequences. Such a fragment often contains sequence(s) encoding the ribosomal binding site(s) for the gene(s) it carries. In such cases, a second ribosome binding site might act to effectively reduce expression (Schauder and McCarthy, 1989, Gene 78 : 59-72).

EXAMPLE 7

Construction of a pKMY319-derived Expression System

for the Catechol 2,3-Dioxygenase Gene of Plasmid NAH7

A. Preparation of Intermediate Plasmid pKMY514

Plasmid pKY67 is the NAH7 plasmid containing a Tn5 insertion in the nahG gene (Yen and Gunsalas, 1982, Proc. Natl.

Acad. Sci. 79 : 874-878). Plasmid pKY67 DNA and plasmid pUC19 DNA

(Yanisch-Perron et al., 1985, supra) were digested with Xmal.

The digested pKY67 and pUC19 DNAs were mixed, ligated and used to transform E. coli HB101 cells. Transformants were selected by plating on L-agar with kanamycin (50 μg/ml) and ampicillin

(500 μg/ml). The desired ~5.4 kb plasmid, designated pKMY514, was pUC19 carrying an ~2.7 kb Xmal insert containing the Tn5 gene encoding kanamycin resistance and the NAH7 gene nahH encoding catechol 2,3-dioxygenase. B. Preparation of Intermediate Plasmid ρKMY515

Plasmid pKMY514 DNA (Section A above) was digested with

Ncol and Xhol. A Ncol site and an Xhol site have been mapped upstream and downstream of the nahH gene respectively (Ghosal et al., 1987, Gene 33: 19-28). Plasmid pCFM1146 DNA (Example 3) was also treated with Ncol and Xhol. The digested pKMY514 and pCFM1146 DNAs were mixed, ligated and used to transform E. coli

FM5 cells. Transformants were selected by plating on L-agar with kanamycin (50 μg/ml) at 28°C. At 28°C, the temperature inducible λ promoter derived from pCFM1146 is off and transcription of the nahH gene is not induced. This master plate is kept under conditions whereby the nahH gene is not induced. Replica plates were made from the master plate. The replica plates were then incubated at 42°C, so as to turn on the temperature inducible λ promoter and induce the production of the nahH gene product, catechol 2,3-dioxygenase. To detect those colonies producing catechol 2, 3-dioxygenase, the replica plates were sprayed with

0.5 M catechol. The catechol is converted to a yellow-colored product, 2-hydroxymuconic semialdehyde, to yield yellow colonies. The colonies on the master plate corresponding to the yellow colonies on the replica plate were picked, and grown at 28°C.

The desired ~6.2 kb plasmid designated pKMY515 was thereby obtained, comprising an ~1.5 kb Ncol-Xhol fragment from pKMY514.

This fragment contains the nahH gene inserted into the Ncol and Xhol sites of the ~4.7 kb pCFMH46. The Xbal site in pKMY514 which was derived from pCIM1146 is thus available for cloning the nahH gene in pKMY319 in the last step in the construction of the plasmid pKMY517 as described in Section C below.

C. Construction of pKMY517 for the Expression of NAH7 Catechol 2,3-Dioxygenase Gene

Plasmid pKMY515 DNA (Section B above) was digested with

Xbal and Xhol. Similarly, plasmid pKMY319 DNA (Example 6) was digested with Xbal and Xhol. The digested pKMY515 and pKMY319

DNAs were mixed, ligated, and used to transform E. coli HB101 cells. Transformants were selected on L-agar plates containing tetracycline (10 μg/ml). To detect those colonies producing catechol 2, 3-dioxygenase, the plates were sprayed with catechol, as described in Section B above, and yellow colonies were selected. The desired ~11.2 kb plasmid for the expression of the NAH7 catechol 2, 3-dioxygenase gene was obtained and designated pKMY517. It comprised an ~1.5 kb Xbal-Xhol fragment from pKMY515 containing the nahH gene inserted into the Xbal and Xhol sites of pKMY319. These results suggested that inducible promoter in pKMY517 that was derived from pKMY319 was slightly leaky so that when a very sensitive assay with catechol was used, even small amounts of catechol 2, 3-dioxygenase were easily detected in the pKMY517-transformed cells in the absence of induction. When the cells are induced, large amounts of catechol 2, 3-dioxygenase may be produced, as described in Example 9 below.

EXAMPLE 8

Construction of a pKMY299-derived Expression System for the Luciferase Gene of Firefly Photinus Ovralis

A. Preparation of pLu2

A double-stranded synthetic oligonucleotide was prepared with the following sequence:

Ndel

5' - TATGGAAGACGCCAAAAACATAAAGAAAGGCCCGGCGCCATTCTATCCT-3 '

3 ' - ACCTTCTGCGGTTTTTGTATTTCTTTCCGGGCCGCGGTAAGATAGGAGATC-5 '

Xbal

Each strand was synthesized using an Applied Biosystems Model 380B nucleic acid synthesizer. The two strands were annealed by conventional methods, for example, Yansura et al., 1977, supra. The two termini of the annealed oligonucleotide are compatible with termini generated by cleavage with Ndel and Xbal. This double-stranded oligonucleotide was mixed with pUC19 DNA (Yanisch- Perron et al., supra) that had been digested with Ndel and Xbal. then ligated and used to transform E. coli JM83 cells (Vieira and Messing, 1982, Gene 19: 259-268). Transformants were selected by plating on L-agar with ampicillin (100 μg/ml), IPTG (1 mM) and β-gal (2 mg/ml) . The desired transformants, in which the ~230 bp Ndel-Xbal fragment of pUC19 containing the lacZ gene was replaced by the synthetic oligonucleotide during the ligation, were ampicillin resistant and colorless. Miniprep analysis of the DNA from such transformant colonies confirmed the presence of an ~2.5 kb plasmid with expected Ndel and Xbal sites. One such isolate was designated pAD6. Plasmid pAD6 thus contains a coding sequence for the 5' end of the firefly luciferase gene beginning with the sequence ATG encoding the start codon (within the synthetically derived Ndel site) and ending with the Xbal site at codon 16 (nucleotide 100) (see Figure 1 of deWet et al., 1987, Mol. Cell. Biol. 7:725-737).

In order to construct an intact firefly luciferase gene, plasmid pAD6 DNA was digested with Ndel and Xbal. releasing the synthetic oligonucleotide insert. The small insert fragment was isolated from a 10% polyacrylamide gel according to conventional methods. Plasmid pJD201 (deWet et al., 1987, supra) comprises a full-length, intronless Photinus pyralis (firefly) luciferase gene constructed by a genomic DNA-cDNA fusion cloned into plasmid pUC19. Plasmid pJD201 DNA was digested with Xbal and Kpnl. The ~1.7 kb fragment containing the majority of the firefly luciferase gene coding sequence (i.e. from the Xbal cleavage site at codon 16 (nucleotide 101) to the Kpnl site in the polylinker of pUC19) was isolated from a 0.7% agarose gel according to conventional methods. The two purified fragments (Ndel-Xbal and Xbal-Kpnl) encoding the complete firefly luciferase gene were mixed with plasmid pCFM1156 DNA that had been digested with Ndel (at the ATG of pCFM1156) and Kpnl. Plasmid pCFM1156 is identical to plasmid pCFM4722 described by Burnette et al., 1988, Bio/Technology 6: 699-796, and contains an inducible P_L promoter, a ribosome binding site, a cloning cluster, E. coli origin of replication, a transcription terminator, genes regulating plasmid copy number, and a kanamycin-resistance gene. The 3- fragment mixture was ligated and used to transform E. coli FM5 cells. Transformants were selected on L-agar plates containing 50 μg/ml kanamycin. The desired ~6.4 kb plasmid was designated pLu2. Miniprep analysis of restriction endonuclease digested pLu2 DNA confirmed that an ~1.7 kb Ndel-Xbal-Kpnl insert had been successfully cloned into pCFM1156. The Ndel-Hpal-MluI-EcoRI- Ncol-Kpnl linker was thus deleted from pCFM1156. Since there was a possibility that multiple copies of the small fragment purified from pAD6 for the ligation could have been inserted, plasmid pLu2 DNA was subjected to DNA sequence analysis. The sequence analysis showed that a single copy of the small fragment of pAD6 had been inserted and confirmed the desired coding sequence of luciferase.

B. Preparation of Plasmid pKMY520

Plasmid pLu2 DNA (Section A above) was digested with Ndel and Asp718 (Boehringer Cat. No. 814253). ASP718 recognizes the same 6 bp sequence as KpnI. but cuts at a different site, as follows:

Similarly, plasmid pKMY299 DNA (Example 5) was digested with Ndel and Asp718. The designated pLu2 and pKMY299 DNAs were mixed, ligated and used to transform E. coli HB101 cells. Transformants were selected on L-agar plates with tetracycline (10 μg/ml). Colonies were picked, screened for luciferase activity, and tested by miniprep analysis as follows. Each colony picked was grown ~15 hours in 5 ml of L-broth with 10 μg/ml tetracycline. Luciferase activity of each was assayed in accordance with the procedure of Example 10. Those with detectable luciferase activity were checked by miniprep analysis, using Hindlll and Asp718 to digest the miniprep DNA. The desired plasmid contained three fragments: an ~1.1 kb Hindlll fragment, an ~2.2 kb Hindlll- ASP718 fragment and an ~8.1 kb (vector) Hindlll-ASP718 fragment. It had a size of ~11.4 kb and was designated pKMY520. Plasmid pKMY520 thus contains an ~1.7 kb Ndel-Asρ718 fragment derived from pLu2 comprising the firefly luciferase gene inserted into the Ndel and KpnI sites of pKMY299.

EXAMPLE 9

Catechol 2,3-Dioxygenase Assay

Cells were grown in 50 ml of PAS medium (Chakrabarty, et al., 1973, Proc. Natl. Acad. Sci. USA 70:: 1137-1140) containing 0.4% glutamate or 50 ml of L-broth in the presence or absence of 0.35 mM sodium salicylate (as inducer) for ~13-14 hrs. at 30°C. The cells were harvested by centrifugation, the pellet washed with 20 ml of 100 mM sodium phosphate buffer at pH 8.3. The cells were resuspended in 5 ml of the same buffer with 10% (v/v) acetone for enzyme stabilization. The resuspended cells were sonicated using a cell disrupter manufactured by Heat Systems - Ultrasonics, Inc. (Plainview, New York; available as model No. W-375) and giving 5 pulses of 10 seconds/pulse with 1 minute between each pulse. After sonication, the suspension was centrifuged for 30 minutes at 15,000 rpm in a Beckman Instruments, Inc. (Somerset, NJ 08875) J2-21 centrifuge with a JA20 rotor to yield a crude extract for assay and for SDS-PAGE analysis. The pellet was discarded and the supernatant (crude extract) was used in the assay for catechol 2, 3-dioxygenase activity essentially according to Sala-Trepat and Evans, 1971, Eur. J. Biochem. 20: 400-413, as follows. The total volume for the assay was 1 ml. One to 10 μl of crude extract (or an appropriate dilute of extract) was mixed with 100 μl of 3.3 mM catechol (substrate) and then diluted up to 1 ml with assay buffer (100 mM sodium phosphate, pH 8.3 with 10% (v/v) acetone). Formation of the yellow product, 2-hydroxymuconic semialdehyde (extinction coefficient ε = 33.4 mM^-1 cm^-1) was measured at O.D.₃₇₅ using a Beckman DU-70 spectrophotometer. An aliquot of the crude extract was also used for the determination of protein concentration by the method of Bradford, 1976, Anal. Biochem. 72: 248 using the Bio-Rad Protein Assay Kit obtained from Bio-Rad Laboratories, Richmond, CA 94804. The calculated specific activity (μmole/min/mg) is reported in Table I below. Strain PpG1901 (Yen and Gunsalas, 1982, Proc. Natl. Acad. Sci. 79: 874- 878; Yen and Gunsalas, 1985, J. Bacteriol. 162: 1008-1013) is Pseudomonas putida G1343 containing the wildtype NAH7 plasmid. Strain PpY1006 is Pseudomas utida G572 (Shaham et al., 1973, J. Bacteriol. 116: 944-949) containing plasmid pKMY517 (Example 7). Table I shows that when cells from these 2 strains were grown in the presence of an inducer of the P_G promoter (e.g., 0.35 mM sodium salicylate), significant amounts of exizymatically active catechol 2, 3-dioxygenase were expressed. The plasmid pKMY517 contains the catechol 2,3-dioxygenase gene derived from the NAH7 plasmid. Even uninduced pKMY517-containing cells exhibit detectable levels of catechol 2,3-dioxygenase activity, indicating that the inducible promoter is somewhat "leaky" (i.e., a small amount of enzyme is made even without an inducer of the nahH gene). Nonetheless, the results shown in Table I clearly demonstrate that when cells containing pKMY517 were grown in the presence of inducer, the highest levels of catechol 2,3- dioxygenase activity were observed.

TABLE I

Expression of the Catechol 2, 3-dioxygenase Gene of the Plasmid NAH7 from NAH7 and the Plasmid

Vector pKMY319 in Pseudomonas putida

Specific Activity of catechol

These results demonstrated the usefulness of pKMY319 as an expression vector in obtaining regulated gene expression. Comparing the level of catechol 2,3-dioxygenese produced from NAH7 with that from pKMY517, a 25-fold overproduction was observed under the experimental conditions (Table I). Production of catechol 2, 3-dioxygenase relative to other proteins produced in P. putida harboring plasmid pKMY517 was analyzed on SDS- polyacrylamide gels (Figure 5). SDS-PAGE was performed essentially according to Laemmali 1970, Nature 227: 680-685. Protein samples (crude extracts prepared as described above) were heated at 65°C for 15 minutes in a loading buffer containing 2% SDS, 5% 2-mercaptoethanol, 10% glycerol, 0.02% bromophenol blue and 62.5 mM Tris-Cl (pH 6.8) before they were loaded on the gel. The gel was stained with Coomassie blue and scanned with a laser densitometer (Ultrascan XL, Pharmacia LKB Biotechnology, Inc., Piscataway, NJ 08854) to determine relative protein productions. As shown in Figure 5: lanes 1 and 10 are molecular weight standards (hen egg white lysozyme, 14,400; soybean trypsin inhibitor, 21,500; bovine carbonic ahhydrase, 31,000; hen egg white ovalbumin, 42,699; bovine serum albumin, 66,200; and rabbit muscle phosphorylase b, 97,400): lanes 4 and 5 are PpY1006 in PAS, induced and uninduced, respectively: and lanes 8 and 9 are PpY1006 in L-broth, induced and uninduced, respectively. Similar percentages of catechol 2,3-dioxygenase were produced whether the cultures were grown in PAS medium or L-broth. The amount of catechol 2,3-dioxygenase produced represented ~10% of the total soluble cell proteins (Figure 5, lanes 4 and 8). These results demonstrated the usefulness of the pKMY319 expression vector for the overproduction of gene product(s).

EXAMPLE 10

Luciferase Assay

Strain PpY1009 cells were grown in 5.0 ml of L-broth with 10 μg/ml tetracycline at 30°C to a density of O.D.₅₅₀

4.5 in the presence or absence of 0.35 mM sodium salicylate as an inducer. Strain PpY1009 is Pseudomas putida G572 (Shaham et al., supra) containing plasmid pKMY520. An aliquot of cell suspension (3-10 μl) was mixed with water and 30 μl of assay buffer (0.2 M HEPES, pH 7.7, 50 mM MgSO₄) in a total volume of 100 μl in a cuvette. The cuvette was placed in a luminometer, for example, a LUMAC BIOCOUNTER M 2500 (LUMAC B.V. , P. O. Box 31101, 6370 AC Landgraaf, The Netherlands). The light-emitting reaction was initiated by injection of 100 μl of 1 mM luciferin (Sigma Chemical, St. Louis, MO 63178) in 5 mM citrate buffer, pH 5.5, and 100 μl of 100 mM rATP in water. [The BIOCOUNTER M 2500, a very sensitive photon counter controlled by a microprocessor, was operated for these analyses in the "A2 mode"]. The light produced was displayed in relative light units (RLU) by the luminometer. [According to the Biocounter manufacturer's instructions, the photomultiplier is calibrated such that 200 pg ATP in 100 μl LUMIT-PM gives 7,200 RLU]. The data are reported as specific activity of luciferase (RLU/μg protein). Protein concentration was measured as described in Example 9, using the method of Bradford, 1976, supra and the Bio-Rad Protein Assay Kit. Cells were resuspended in 0.1 N NaOH and incubated in a boiling water bath for 20 minutes before protein determination.

TABLE II

Expression of the Luciferase Gene of the Firefly Photinus pyralis from the Expression Vector pKMY299

in Pseudomonas putida and Escherichia coli

aPlasmid pKMY520 is pKMY299 carrying an insert containing firefly luciferase gene.

bCells grown in L-broth as described in this Example 10.

cCells not carrying the luciferase gene gave a background value of less than 30 RLU per μg protein.

Production of luciferase protein in P. putida was also analyzed by SDS-PAGE, according to the method described in Example 9 for the analysis of catechol 2, 3-dioxygenase. Luciferase production in uninduced P. putida cells harboring pKMY520 was barely visible on SDS gels when the cells were grown in PAS medium (Figure 5, lane 3) or in L-broth (Figure 5, lane 7). However, the high sensitivity of the luciferase assay allowed detection of relatively high enzyme activity from uninduced P. putida or E. coli cells harboring pKMY520, as shown in Table II above. This assay also allowed detection of an ~90-fold induction of activity in P. putida and an ~80-fold induction in E.coli (Table II). The SDS-PAGE analysis revealed that the amount of luciferase produced in induced P. putida harboring pKMY520 represented ~3.7% of the total soluble proteins whether the cells were grown in PAS medium (Figure 5, lane 2) or in L-broth (Figure 5, lane 6). These results demonstrated regulated expression of a eukaryotic gene from pKMY299 in two different Gram-negative host cells.

EXAMPLE 11

Construction of and Assay for pKMY319-Derived Expression System for Toluene Monooxygenase (TMO) Genes

The tmoABCDEF gene cluster from Pseudomonas mendocina KR- 1 has been cloned and sequenced as described in co-pending and co-assigned U.S. Patent Application Ser. No. _____________, filed

September _____, 1990, incorporated by reference herein. To further test the use of pKMY319 as a regulated expression vector in Gram-negative bacteria, restriction fragments carrying the TMO gene cluster from P. mendocina KR1 were cloned into pKMY319. Specifically, pKMY342 was constructed by cloning the ~4.7 kb Xbal-SacI fragment of pKMY341 carrying the TMO gene cluster into pKMY319. Plasmid pKMY341 was constructed by cloning the ~4.7 kb Xbal-BamHI fragment of pKMY336 carrying the TMO gene cluster into the E. coli vector pT7-5. The construction of pKMY336 has been described in detail in the above-referenced co-pending and co- assigned application. Analysis of pKMY342 DNA with restriction enzymes demonstrated that two copies of the Xbal-SacI fragment joined by a SacI-KpnI-Xbal linker derived from the multiple cloning site in pKMY319 had been cloned into pKMY319. The pKMY342 recombinant plasmid carrying the TMO gene cluster was then introduced into a number of Gram-negative bacterial species as shown in Table III below. Expression of the TMO genes was measured under induced and uninduced conditions. The toluene monooxygenase assay has been described in co-pending and co- assigned U.S. Patent Application Ser. No. _____ filed September _____, 1990, previously incorporated by reference. As shown in

Table III, significantly higher specific activities of toluene monooxygenase were observed from induced cultures as compared with uninduced cultures of all bacterial strains tested. These results demonstrated the wide use of pKMY319 as an expression vector in obtaining regulated gene expression in Gram-negative bacteria.

TABLE III

Expression of the Toluene Monooxygenase Gene Cluster

ABCDEF of Pseudomonas mendocina KR1 from the Plasmid Vector pKMY319 in Gram-Negative Bacteria

Specific Activity of Toluene Monooxygenase^b

Bacterial Strain* (nmole min^-1mg^-1)

Aeromonas hydrophila Y21, uninduced 0.3

Aeromonas hydrophila Y21, induced 17.0

Enterobacter cloacae Y81, uninduced 0.9

Enterobacter cloacae Y81, induced 23.0

Escherichia coli Y5250, uninduced 0.9

Escherichia coli Y5250, induced 19.0

Klebsiella pneumoniae Y61, uninduced 1.3

Klebsiella pneumoniae Y61, induced 15.0

Pseudomonas putida Y2511, uninduced 0.8

Pseudomonas putida Y2511, induced 27.0

Pseudomonas mendocina Y4075 , uninduced 1.2

Pseudomonas mendocina Y4075 , induced 19.0 ^aAll bacterial strains contain the recombinant plasmid pKMY342 , which is pKMY319 carrying an insert containing the toluene monooxygenase gene cluster from P. mendocina KR1. The parent strains into which pKMY342 was introduced are all natural isolates.

bThe specific activities in toluene-induced and uninduced P. mendocina KR1 cells were 30 and 0.5 nmole min^-1mg^-1, respectively. EXAMPLE 12

Tailoring 5' End of Known Genes for Insertion into

Plasmid pKMY299 Expression System

Since the Ndel recognition site ends with the sequence ATG, altering the 5' end of any gene beginning with the sequence ATG to generate an Ndel site does not lead to a change in the coding property of the gene. This is an ideal manner of generating a suitable 5' end restriction site for precise cloning of a gene for expression and requires the alteration of no more than 3 nucleotides. The 3 nucleotides that are 5' to the sequence ATG encoding the start codon of a gene may be specifically converted by site-directed mutagenesis to CAT, thus creating an Ndel recognition site for cloning into the pKMY299 broad host range expression system.

Site-directed mutagenesis is either accomplished according to conventional methods (for example, see Chapter 15 of Sambrook et al., 1989, Molecular Cloning - A Laboratory Manual (Second Edition), Cold Spring Harbor Laboratory Press, N.Y.; Chapter 8 of Current Protocols in Molecular Biology. Ausubel et al., eds., 1989, Greene Publishing Associates and Wiley-Interscience) or accomplished with the following steps: (i) locate a restriction site just inside the coding region; and (ii) attach a synthetic oligonucleotide linker at this restriction site that restores the reading frame of this gene and contains an Ndel site at the 5' end. Examples of generating an Ndel site at the 5' end of a gene using the latter method can be found in Burnette et al., 1988, supra. or in Example 8 above. Such modified genes are useful for cloning into the pKMY299 broad host range expression system according to the present invention.

Claims

WHAT IS CLAIMED IS:

1. A cloning cartridge comprising a positive regulatory gene nahR from plasmid NAH7, a promoter P_G that is regulated by nahR, a multiple cloning site, a transcription terminator and a modified gene conferring tetracycline resistance.

2. A cloning cartridge according to Claim 1, wherein the promoter P_G comprises a sequence of 3 nucleotides upstream of an ATG sequence encoding an initiation codon that has been altered to create an Ndel cloning site.

3. A cloning cartridge according to Claim 2, further comprising a cloned characterized gene, wherein the 5' end of the gene has been converted into an Ndel site without altering coding of the gene.

4. A cloning cartridge according to Claim 3, wherein the cloned characterized gene is luciferase.

5. A cloning cartridge according to Claim 2, wherein the ATG sequence has been deleted.

6. A cloning cartridge according to Claim 5, further comprising a cloned restriction fragment containing a gene or a gene cluster.

7. A cloning cartridge according to Claim 6 , wherein the cloned restriction fragment comprises a gene encoding catechol 2,3-dioxygenase.

8. A cloning cartridge according to Claim 6 , wherein the cloned restriction fragment comprises genes encoding toluene monooxygenase.

9. A bacterial expression vector comprising the cloning cartridge of Claim 1.

10. A bacterial expression vector comprising the cloning cartridge of Claim 2.

11. A bacterial expression vector comprising the cloning cartridge of Claim 3.

12. A bacterial expression vector comprising the cloning cartridge of Claim 5.

13. A bacterial expression vector comprising the cloning cartridge of Claim 6.

14. A bacterial expression system comprising pKMY299.

15. A bacterial expression system comprising pKMY319.

16. A bacterial DNA expression unit comprising the cloning cartridge of Claim 1, operably linked to a heterologous gene.

17. A bacterial DNA expression unit comprising the cloning cartridge of Claim 2, operably linked to a heterologous gene.

18. A bacterial DNA expression unit comprising the cloning cartridge of Claim 5, operably linked to a heterologous gene.

19. A host cell transformed with the bacterial expression vector of Claim 9.

20. A host cell transformed with the bacterial expression vector of Claim 10.

21. A host cell transformed with the bacterial expression vector of Claim 11.

22. A host cell transformed with the bacterial expression vector of Claim 12.

23. A host cell transformed with the bacterial expression vector of Claim 13.

24. A host cell transformed with the bacterial expression system of Claim 14.

25. A host cell transformed with the bacterial expression system of Claim 15.

26. A host cell transformed with the bacterial expression unit of Claim 16.

27. A host cell transformed with the bacterial expression unit of Claim 17.

28. A host cell transformed with the bacterial expression unit of Claim 18.

29. A process for inducibly expressing a protein in a bacterial host cell comprising the steps of:

(a) transforming the host cell with the bacterial DNA expression unit of Claim 16;

(b) growing the transformed host cell in an appropriate medium; and

(c) inducing the transformed host cell to express the protein by adding an inducer.

30. A process according to Claim 29, wherein the host cell is a gram-negative bacterial cell.

31. A process according to Claim 30, wherein the inducer is sodium salicylate or an analog of sodium salicylate.