EP1104472A1 - Gene switch - Google Patents

Abstract

The present invention relates inter alia, to a method of initiating transcription of a target gene in a eukaryotic cell comprising: (a) providing a eukaryotic cell which is capable of producing a response protein; and (b) inserting into the genome of said cell a polynucleotide defining an inducible promoter sequence operably linked to and capable when induced of initiating transcription of said target gene; and (c) applying to said cell a chemical inducer capable of binding to said response protein whereby said chemical inducer binds to said response protein to form an inducing complex which binds to and induces said inducible promoter thereby initiating transcription of said target gene.

Description

GENE SWITCH

The present invention relates inter alia, to the induction of gene expression in a eukaryote by the application of a chemical inducer to the eukaryote and to materials and methods for achieving induction. Such systems are referred to as "gene switches".

In particular, the present invention relates to a method of controlling expression of a target gene in a plant, animal or yeast.

Bacterial cells have the ability to respond to the surrounding environment. The response to different environmental cues is essential for survival of bacteria. It is apparent that individual bacteria in a population are also able to sense the density and state of the local bacterial population (the "quorum") of which they are members. That is, an individual bacterium can detect the presence of like bacteria in the surrounding environment. Quorum sensing allows bacteria to synchronise growth, development which when a minimal population level is reached initiates a concerted response from the population.

In the case of Photobacterium fischeri, N-(3-oxohexanoyl)-L-homoserine lactone or autoinducer regulates bioluminescence in a cell density-dependent manner. There are two main genes in the lux operon of P. fischeri involved in the signal production and signal detection. Luxl is the gene involved in the biosynthesis of the homoserine lactone but the mechanism by which this takes place is unclear. It has been proposed that S-adenosylmethionine and coenzymeA or the acyl carrier protein adduct of 3-oxohexanoic acid are substrates for the luxl gene in P. fischeri.

In bacteria, the autoinducer regulates expression of the luxl gene and thus creates a positive autoregulation of autoinducer synthesis. LuxR, the response regulator or autoinducer receptor, is a protein involved in responding to the presence of N-(3-oxo)hexanoyl-L-homoserine lactone (OHHL) in P. fischeri. At the C- terminal end LuxR contains a DΝA binding domain and a transcriptional activator. LuxR C-terminal end shows amino acid homology to transcriptional activators known as the two component environmental-sensing systems such as UhpA, FixJ and ΝarL. LuxR is thought to interact with DΝA as a homodimer to a palindrome within the luxl operator sequence termed lux box. The Ν-terminal end of the protein is called the - 9 .

receptor module as it has no similarity to the two-component environmental sensing systems.

Quorum sensing systems are found in other bacteria and may be activated by different homoserine lactones. Such is the case in P. aeruginosa PAO1, Lasl directs the synthesis of the autoinducer N-(3-oxododecanoyl)-L-homoserine lactone

(OdDHL), which activates the positive transcriptional activator, LasR (Winson et al., 1995). Moreover, in the same P.auruginosa, PAO1, a second signalling pathway termed vsm containing vsmR and vsml genes was isolated. The vsml gene product is involved in production of N-butanoyl-L-homoserine lactone (BHL) and N-hexanoyl- L-homoserine lactone (HHL). These compounds are present in the spent culture supematants of P. aeruginosa and when either BHL or HHL to PAΝ067, a pleiotropic P. aeruginosa mutant unable to synthesize either of these autoinducers, restored elastase, chitinase, and cyanide production (Winson et al., 1995). Other evidence suggesting the presence of different homoserine inducers in one species has recently been observed in Vibrio anguillarum (Milton et al., 1997) and P. aeroginosa (Pesci et al, 1997). Furthermore, Table 1, shows examples of characterised systems where different compounds are known to be inducers of different receptor molecules in different bacteria.

Table 1.

I w I

I

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The present invention therefore seeks to provide inter alia, methods and materials for the induction of gene expression in eukaryotes by the application of chemical inducers to the eukaryote.

According to the present invention there is provided a method of initiating transcription of a target gene in a eukaryotic cell comprising:

(a) providing a eukaryotic cell which is capable of producing a response protein;

(b) inserting into the genome of said cell a polynucleotide defining an inducible promoter sequence operably linked to and capable when induced of initiating transcription of said target gene; and

(c) applying to said cell a chemical inducer capable of binding to said response protein whereby said chemical inducer binds to said response protein to form an inducing complex which binds to and induces said inducible promoter thereby initiating transcription of said target gene. The eukaryotic cell may already contain the mechanisms to produce the said response protein or may alternatively be provided with them by inserting into the said cell, a polynucleotide which provides for the production of the response protein using techniques well known within the art. The inducible promoter for use in the above mentioned method may comprise the nucleotide sequence depicted as SEQ ID No. 2 and the response protein may comprise the amino acid sequence depicted in SEQ ID No. 16. Alternatively the inducible promoter may comprise a functional variant such as a polynucleotide which is the complement of one which binds to SEQ ID No. 2 at a temperature of between 60°C and 65°C in 0.3 strength citrate buffered saline containing 0.1% SDS followed by rinsing at the same temperature with 0.3 strength citrate buffered saline containing 0.1 % SDS wherein the said polynucleotide is still capable of acting as an inducible promoter upon binding with the said inducing complex. In particular the response protein for use in the method of the present invention may be encoded by the polynucleotide comprises the sequence depicted in SEQ ID No. 5. Alternatively the polynucleotide encoding the said response protein may comprise the complement of one which binds to SEQ ID No. 5 at a temperature of between 60°C and 65°C in 0.3 strength citrate buffered saline containing 0.1% SDS followed by rinsing at the same temperature with 0.3 strength citrate buffered saline containing 0.1% SDS wherein the said polynucleotide still encodes a protein which is capable of forming an inducing complex with the said chemical inducer. It is particularly preferred that the polynucleotide defining the inducible promoter according to the present invention contains the region depicted as SEQ ID No. 21. The method of present invention is particularly applicable to initiating transcription in cells of plants, more particularly in the cells of: melons, mangoes, soybean, cotton, tobacco, sugarbeet, oilseed rape, canola, flax, sunflower, potato, tomato, alfalfa, lettuce, maize, wheat, sorghum, rye, bananas, barley, oat, turf grass, forage grass, sugar cane, pea, field bean, rice, pine, poplar, apple, peaches, grape, strawberries, carrot, lettuce, cabbage, onion, citrus or nut plants. In particular the method of the present invention may be used to initiate transcription in a variety of tissues, including roots, leaves, stems and reproductive tissues. The chemical inducer which may be used in the above mentioned method is N-(3-oxo)hexanoyl-L-homoserine lactone or a functional equivalent thereof and may be applied to the plant cell as part of an agriculturally acceptable formulation. Alternatively the chemical inducer may be produced within the plant by inserting into the genome of the plant a polynucleotide encoding a protein which provides for the production of the chemical inducer within the plant. This polynucleotide may, for example, be under the transcriptional control of a constitutive promoter, a gene switch (such as the alcA/R, Heliothis ecdysone and GST-27 gene switch), wound inducible, tissue or temporal promoters. The advantage of producing the chemical inducer in planta is that there is no need to spray or exogenously treat plants to induce gene expression.

Further alternative inducible promoters, response proteins and chemical inducers which may be used in the method of the present invention are referenced in Table 1. For example, the promoter sequence or a part thereof may obtainable from the vanl gene of Vibiro anguillarum and the response protein encoded by the vanR gene may be used with the chemical N-(3-oxo-decanoyl)-L-homoserine lactone (ODHL) or a functional variant thereof.

The polynucleotide encoding the said response protein in the method referred to above may be bounded by a promoter and a terminator sequence and in particular the promoter may be inducible such as the Ale A/ R switch system, the GST switch system and the ecdysone switch. Alternatively the promoter may be constitutive such as cauliflower mosaic virus 35S/19S, maize Ubiquitin and Arabidopsis Ubiquitin 3 or may be developmentally regulated specific promoter such as those controlling expression of gene required during seed formation, germination such as cysteine proteinases (as specified in our International Publication No WO 97/35983 ) and malate synthase.

The target gene in the method referred to above may be any gene of interest, for example, β-glucuronidase; Bacillus thuringenesis toxin; barnase or barstar.

In a further aspect of the present invention there is provided a method of providing plants containing an inducible target gene comprising: (a) inserting into a plant cell which cell provides for production of a response protein a polynucleotide defining an inducible promoter sequence operably liked to said target gene; and (b) regenerating morphologically normal fertile plants thereof; and (c) applying to the population of regenerants a chemical inducer or a functional variant thereof capable of binding to said response protein, whereby said chemical inducer binds to said response protein to form an inducing complex which binds to and induces said inducible promoter thereby initiating transcription of said target gene; and (d) selecting those plants which are expressing the said target gene. The inducible promoter sequence referred to in the preceding paragraph may comprise the nucleotide sequence depicted as SEQ ID No. 2 or a functional variant thereof and the response protein comprises the amino acid sequence depicted as SEQ ID No 16 or a functional variant thereof and the said chemical inducer is N-(3-oxo)hexanoyl-L- homoserine lactone or a functional equivalent thereof. In particular the inducible promoter sequence may contain the nucleotide sequence depicted as SEQ ID No. 10 or a functional variant thereof and the response protein may comprises the amino acid sequence depicted as SEQ ID No 17 or a functional variant thereof and the said chemical inducer may be N-(3-oxo)dodecanoyl-L-homoserine lactone or a functional equivalent thereof. Alternatively, the said inducible promoter sequence may contain the nucleotide sequence depicted as SEQ ID No. 12 or a functional variant thereof and the response protein may comprise the amino acid sequence depicted as SEQ ID No 18 or a functional variant thereof and the said chemical inducer may be N-(3- oxo)octanoyl-L-homoserine lactone or a functional equivalent thereof. The present invention also provides plants produced according to the method of the preceding paragraph which plants may be selected from the group consisting of: melons, mangoes, soybean, cotton, tobacco, sugarbeet, oilseed rape, canola, flax, sunflower, potato, tomato, alfalfa, lettuce, maize, wheat, sorghum, rye, bananas, barley, oat, turf grass, forage grass, sugar cane, pea, field bean, rice, pine, poplar, apple, peaches, grape, strawberries, carrot, lettuce, cabbage, onion, citrus or nut plants.

The method employed for transformation of plant cells is not especially germane to the present invention and any method suitable for the target plant may be employed. For example, transgenic plants are obtained by regeneration from the transformed cells. Numerous transformation procedures are known from the literature including agroinfection using Agrobacterium tumefaciens or its Ti plasmid, electroporation, micro injection or plants cells and protoplasts, microprojectile transformation. All of these methods are well known within the art. The present invention may also be applied to any plant for which transformation techniques are, or become, available.

In a further aspect of the present invention there is provided a DNA construct which comprises a first polynucleotide region which comprises an inducible promoter sequence operably liked to and capable when induced of initiating transcription of a target gene and a second polynucleotide region which provides for the production of a response protein wherein upon contact of the said response protein with a chemical inducer the said response protein binds the chemical inducer to produce an inducer complex which binds the said inducible promoter sequence thereby initiating transcription of the said target gene. The said first polynucleotide of the DNA construct may comprise the nucleotide sequence depicted as SEQ ID No. 2 or a functional variant thereof and the second polynucleotide region may comprise a polynucleotide which encodes the amino acid sequence depicted as SEQ ID No. 16 or a functional variant thereof. Alternatively the said first polynucleotide may comprise the nucleotide sequence depicted as SEQ ID No. 10 or a functional variant thereof and the second polynucleotide region may comprise a polynucleotide which encodes the amino acid sequence depicted as SEQ ID No. 17 or a functional variant thereof. Alternatively, the said first polynucleotide may comprise the nucleotide sequence depicted as SEQ ID No. 12 or a functional variant thereof and the second polynucleotide region may comprise a polynucleotide which encodes the amino acid sequence depicted as SEQ ID No. 18 or a functional variant thereof. The said second polynucleotide region referred to above may comprise a promoter operably linked to the polynucleotide encoding the said response protein. The said promoter may be inducible and constitutive or developmentally controlled.

The present invention still further provides the use of a DNA construct referred to above in the production of a plant containing a target gene of which the expression may be controlled by the application of a chemical inducer to the said plant. In a further aspect of the present invention there is provided a method of screening compounds in a bioassay comprising applying an amount of a chemical to a plant which plant contains a polynucleotide comprising an inducible promoter operably linked to a region encoding a reporter protein and which plant is also capable of producing a response protein and testing the said plant for the production of the said reporter protein. The chemical could be applied to the plant and the activity of the reporter gene monitored for e.g. improved activity, mobility or stability or to assess if the chemical had an inhibitory effect on the response protein which resulted in a decrease in activity of the reporter gene.

In a further aspect of the present invention there is provided a method of selectively controlling pests in a field which field comprises crop plants and pests wherein the plants are those obtained according to the methods referred to above and the said target gene encodes a target protein which is capable of controlling the said pests said method comprising applying to the plants an amount of a chemical inducer which is sufficient to bind to the said response protein to produce the said inducing complex which is capable of initiating transcription of the target gene which provides for the production of the target protein in an amount which is sufficient to control the said pests.

In a further aspect of the present invention there is provided a method of providing a plant which contains a target gene which is inducibly controlled comprising: (a) inserting into a first plant cell a polynucleotide comprising a first inducible promoter operably linked to a target gene and regenerating a first morphologically normal fertile plant therefrom;

(b) inserting into a second plant cell a polynucleotide comprising a promoter operably linked to a region encoding a response protein which is capable of binding to a chemical inducer to produce an inducing complex which is then capable of binding the said inducible promoter to allow for the initiation of transcription of the said target gene and regenerating a second morphologically normal fertile plant therefrom;

(c) cross pollinating said first plant with the said second plant or said second plant with said first plant and harvesting the seed therefrom;

(d) growing said seed and applying to the resultant plants an amount of said chemical inducer which provides an inducing complex capable of binding the said inducible promoter to allow for the initiation of transcription of the said target gene.

The term "functional variant" with respect to a polynucleotide encoding the inducible promoter of the present invention includes variant sequences which are the complement of a sequence which hybridises to the inducible promoter sequence at a temperature of between 60°C and 65°C in 0.3 strength citrate buffered saline containing 0.1 %> SDS followed by rinsing at the same temperature with 0.3 strength citrate buffered saline containing 0.1 %> SDS and which are still capable of acting as an inducible promoter.

The term "functional variant" with respect to the response protein includes variant proteins obtained by conservative substitutions within the amino acid sequence which substitutions do not significantly adversely affect the ability of the response protein to bind the chemical inducer. In particular substitutions may be made between the following amino acid groups viz.

(a) Alanine, Serine, Glycine and Threonine

(b) Glutamic acid and Aspartic acid

(c) Arginine and Lysine (d) Isoleucine, Leucine, Naline and Methionine (e) Phenylalanine, Tyrosine and Tryptophan The term "transgenic" in relation to the present invention does not include a wild type regulator promoter in its natural environment in combination with its associated functional gene in its natural environment.

The term "target gene" with reference to the present invention means any gene of interest. A target gene can be any gene that is either foreign or natural to the eukaryote in question.

The term "construct" - which is synonymous with terms such as "cassette", "hybrid" and "conjugate" - includes a target gene directly or indirectly attached to the regulator promoter, such as to form a cassette. An example of an indirect attachment is the provision of a suitable spacer group such as an intron sequence intermediate the promoter and the target gene. The same is true for the term "fused" in relation to the present invention which includes direct or indirect attachment. Such constructs also include plasmids and phage which are suitable for transforming a cell of interest. The term "expression system" means that the system defined above can be expressed in an appropriate organism, tissue, cell or medium. The system may comprise one or more constructs and may also comprise additional components that ensure to increase expression of the target gene by use of the regulator promoter.

One possible use of the inducible promoters of the present invention is in the control of male sterility. The anther is the site of male reproductive processes in flowering plants. It is composed of several tissues and cell types and is responsible for producing pollen grains that contain the sperm cells. The tapetum is a specialised tissue which plays a critical role in pollen formation. It surrounds the pollen sac early in pollen development, degenerates during the latter stages of development and is not present in an organised form in the mature anther. The tapetum produces a number of compounds which aid pollen development or are incorporated into the pollen outer wall and it has been demonstrated that many of the natural male sterility mutations have impaired tapetum differentiation or function.

Tapetal tissue is therefore critical to the formation of functional pollen grains. A number of genes have been identified and cloned that are specifically expressed in tapetal tissue. They include Osg6B, Osg4B (Tsuchiya et al. 1994, Yokoi, S et al. 1997), pEl, p T72 (WO9213957), p CA55 corn (WO92/13956) , TA29, TA13,(Seurinck et al. 1990), RST2 corn ( WO9713401), MS 14, 18, 10 and A6, A9 from Brassica napus (Hird et al. 1993). Anther specific clones have been isolated from a number of species Bp4A and C (Albani et al. 1990), chs petunia (Koes et al. 1989), rice (Xu et al. 1993, Zou et al. 1994), amongst others. In higher plants the female reproductive organ is represented by the pistil, composed of the ovary, style and stigma. The gynoecium has been shown to contain up to 10,000 different mRNAs not present in other organs (Kamalay and Goldberg 1980). These include regulatory genes responsible for controlling pistil development as well as "downstream" ones encoding proteins associated with differentiated cell types in the pistil. Genes governing self-incompatibility and their homologues are one class of gene with pistil predominant expression patterns (Nasrallah et al. 1993). Other cloned genes which are applicable as target genes in the present invention include β glucanase (Ori et al. 1990), pectate lyase ( Budelier et al. 1990) and chitinase (Lotan et al. 1989) which are expressed in the transmitting tissue and a proteinase inhibitor (Atkinson et al. 1993) which are expressed in the style. Others are pathogenesis related or are homologues of genes involved in the cleavage of glycosidic bonds. These enzymes may facilitate pollen tube growth by digesting proteins in the tissue through which the pollen tube grows. A number of female sterile mutants have been identified in Arabidopsis. For example, sinl (short integument) (Robinson-Beers et al. 1992) and bell (bell) (Robinson-Beers et al. 1992) affect ovule development. In the short integument a mutation blocks megasporogenesis at the tetrad stage

(Elliot,R.C, et al. 1996, Klucher,K.M, 1996). A lethal ovule 2 mutation has been observed but not cloned in maize (Nelson et al. 1952). Pistil specific basic endochitinases have been cloned from a number of species (Ficker et al. 1997, Dzelzkalns et al. 1993, Harikrishna et α/. 1996, Wemmer et al. 1994) and extensin- like genes have been shown to be expressed in the styles of Nicotiana alata (Chen C- G, et al. 1992). The following are ovule specific clones ZmOV23,13, (Greco R., et al. unpublished), OsOsMAB3A (Kang H.G., et al. 1995), ZmZmM2 (Theissen G., et al. 1995) and stigma specific stigl (Goldman, M.H et al. 1994) , STG08, STG4B12 (EP- 412006-A). Mariani et al. used the promoter from the STIG1 gene to drive expression of barnase in the stigmatic secretory zone.

In summary then, the present invention therefore provides a gene switch which is operably linked to a foreign gene or a series of foreign genes whereby expression of said foreign gene or said series of foreign genes may be controlled by application of an effective exogenous inducer. The gene switch of the present invention, therefore, when linked to an exogenous or foreign gene and introduced into a eukaryote by transformation, provides a means for the external regulation of expression of that foreign gene.

It is possible to use one, two or more of these inducible promoter regions according to the present invention to activate different processes in plant cells, thereby obtaining a plant which will have multiple inducible cassettes all controlled by for example, different homoserine lactones. Also, a plant may contain, for example, an inducible promoter according to the present invention in conjunction with other switch type mechanisms examples of which include inducible promoters include the Ale A/ R switch system described in International Publication No. WO. 93/21334, the GST switch system described in International Publication Nos WO 90/08826 and WO 93/031294 and the ecdysone switch described in our International Publication No. WO 96/37609.

The methods and products of the present invention may also be used to control expression of foreign proteins in eukaryotes such as yeast and mammalian cells. Many heterologous proteins for different applications may be produced by expression in such eukaryotic cells. The present invention is advantageous in that it provides control over the expression of foreign genes in such cells. It also provides a further advantage, particularly in yeast and mammalian cells, where accumulation of large quantities of a heterologous protein can damage the cells, or where the heterologous protein is damaging such that expression for short periods of time is required in order to maintain the viability of the cells. The inducible system of the present invention also has applicability in gene therapy as it allows the timing of the therapeutic gene to be controlled. The present invention is therefore not only advantageous in transformed mammalian cells but also to mammals per se. Furthermore, the present invention may be used to switch on genes which produce potentially damaging or lethal proteins. Such a system may be employed in the treatment of cancer in which cells are transformed with genes which express proteins which are lethal to the cancer. The timing of the action on such proteins on the cancer cells may be controlled using the switch of the present invention. Various preferred features and embodiments of the present invention will now be described by way of non-limiting examples with reference to the accompanying

Figures of which:-

Figure 1 shows a schematic representation of the general structure of the response regulator protein of the bacterial quorum sensing system.

Figure 2 shows a schematic representation of the homoserine lactone gene switch system. Figure 3 is a plasmid map of the reporter gene construct, p221.91ux6.

Figure 4 is a plasmid map of p221.91ux2.

Figure 5 Plasmid map of p221.91ux3.

Figure 6 Plasmid map of p221.91uxC.

Figure 7 Plasmid map of p221.9Lpro. Figure 8 Depicts the expression vector containing the LuxR gene in the pDH51LuxR plasmid.

Figure 9 Shows the expression cassette containing the enhanced N-terminal fusion protein of SV40-NLS-Gal4-LuxR in the plasmid pDH51SVLuxR.

Figure 10 Shows the expression cassette containing the enhanced C-terminal fusion of LuxR-SV40-NLS-Gal4 in the plasmidDH5 lLuxRSV.

Figure 1 1 is the plasmid map of p221.91asbox. Containing one copy of the LasR Box.

Figure 12 is a plasmid map of the transient expression construct containing LasR, pSinLASR.

Figure 13 expression plasmid pFunLuxR for expression in monocotyledon protoplasts.

Figure 14 expression plasmid pFunLasR for expression in monocot protoplasts.

Figure 15 expression plasmid pFunTraR for expression in monocot protoplasts.

Figure 16 reporter plasmid p221.9traboxl containing tra box sequence 1 described by

Zhu and Winans, (1999) Figure 17 reporter plasmid p221.9trabox2 containing tra box of sequence 2 described by Zhu and Winans, (1999). Figure 18 is a plasmid map of binary vectors containing the effector and reporter cassettes, pAHLl.

Figure 19. is a plasmid map of binary vectors containing the effector and reporter cassettes, pAHL2. Figure 20. is a plasmid map of binary vectors containing the effector and reporter cassettes, pAHL3.

Figure 21 is a plasmid map of binary vector containing luxl gene, pBDHELI.

Figure 22 is a plasmid map of pSB401 containing the Lux operon.

Figure 23 shows the root of a transgenic tobacco plant that was grown in tissue culture under aseptic conditions was placed on an LB agar plate and overlain with top agar containing E. coli with the plasmid pSB401 (Figure 22). This strain expresses the lux operon and will bioluminesce in response to OHHL and HHL. Bioluminescence in the region around the root was clearly seen with the naked eye.

Figure 24. Shows a leaf of a transgenic tobacco plant that was grown in tissue culture under aseptic conditions. The leaf was excised from the plant and placed on an LB agar plate and overlaid with top agar containing Chromobacterium violaceum indicator strain. The violet colour detected represent the induction of the indicator stain gene showing the extrusion of OHHL from the plan tissue into the bacterial medium. Sequences

SEQ ID No. 1 is the Lux/ box promoter region.

SEQ ID No. 2 is the Luxl promoter region.

SEQ ID No. 3 & 4 are the LuxRBavnhl fragment.

SEQ ID No. 5 & 6 are the LuxR sequence/protein sequence. SEQ ID No. 7 & 8 are the NVLuxR fusion flanked by BamHI and Pstl sites/protein sequence.

SEQ ID No. 9 & 10 are the LuxRNV sequence/protein sequence.

SEQ ID No. 11 is a TraRl fragment.

SEQ ID No. 12 is the TraR2 fragment. SEQ ID No. 13 is the LasBoxl region.

SEQ ID No. 14 is the LasBox2 region.

SEQ ID No. 15, 16, 17 and 18 are the TraBoxl,2,3,4 regions respectively. SEQ ID No. 19 is the LuxR response protein sequence. SEQ ID No. 20 is the LasR response protein sequence. SEQ ID No. 21 is the TrasR response protein sequence.

SEQ ID No. 22 & 23 are the open reading frames of LasR and TraR respectively. SEQ ID No.24 is the Lux Box promoter region.

Example 1. Transient expression of LuxR and reporter plasmid in Tobacco mesophyll protoplasts.

Preparation of Reporter gene expression cassette. Six copies of the 20bp palindromic lux box sequence will be fused upstream of the -60CaMV minimal promoter which in turn is fused to the reporter gene GUS. The palindrome of sequence 5' GATCACCTGTACGATCGTACAGGT 3' (Sequence ID. 1) was self annealed and introduced into a BamHI pBluescript vector. Sequence determination of a number of recombinants lead to the identification of a plasmid with 6 copies of the palindrome. The identified plasmid was digested with H dIII and Sail to release the in 6 copies of the palindrome and introduced into a Hindlϊl/Sall p221.9 vector. p221.9 plasmid contains a -60CaMV minimal promoter fused to GUS downstream of the Hinάlll and Sail unique cloning sites. A recombinant plasmid was identified and named p221.91ux6 (Figure 3). The same oligonucleotide was used to generate p221.9LuxR2 (Figure 4), p221.9LuxR3 (Figure 5) and p221.9LuxC (Figure 6). p221.91pro was generated using the bacterial promoter containing the Lux box sequences (Figure 7 Sequence ID 2).

Preparation of LuxR expression vector. The lux receptor was altered at both ends of the coding sequence. At the 5 'end a plant Kozac consensus sequence was placed with an Ncol site at the ATG start of the coding sequence. Upstream from the Kozac consensus sequence a BamHI unique site was introduced using PCR. The sense oligonucleotide was luxrbamhl 5' CCCGGATCCTAACAATGGGTATGAAAGACATAAATG 3' (Sequence ID.3 )and the antisense primer was luxrbamh2 5'

CGAACTCGAGTCATGATTTTAAAGTATGGGCAA-TCAATTG 3' (Sequence ID.4 ). The PCR reaction was carried out using Tag polymerase (2.5 U) in a reaction buffer containing lOOng of template DNA, 100 ng of each oligonucleotide, 20 mM TRIS-HC1 pH 8.4, 50 mM KC1, 10 mM MgCl₂, 50 mM dNTPs and using hot start conditions followed by 15 cycles of denaturing (94°C for 1 minute), annealing (66°C for 1 min) and synthesis (72°C for 3 min). The fragment was purified and digested using BamHVXhol and was introduced into pDH51 BamHl/Sall vector to give pDH511uxR (Xhol and Sa l restriction enzymes produce compatible ends) (Figure 8). The sequence of the insert was determined and compared to the published LuxR sequence (Sequence ID. 5) (Devine et al, 1988).

Tobacco mesophyll transformation.

Tobacco shoot cultures cv. Samsun, were maintained on solidified MS medium + 3% sucrose in a controlled environment room (16 hour day / 8 hour night at 25°C, 55%o R.H), were used as the source material for protoplasts. Leaves were sliced parallel to the mid-rib, discarding large veins and the slices were placed in CPW13M (13%) mannitol, pH5.6, 860mmol/kg) for 1 hour to pre-plasmolyse the cells. This solution was replaced with enzyme mixture (0.2%> cellulase R10, 0.05%> macerozyme R10 in CPW9M (CPW13M but 9% mannitol), pH5.6, 600mmol/kg) and incubated in the dark at 25°C overnight (16 hours). The enzyme mixture was passed through a 75μm sieve and the filtrate was centrifuged at 600rpm for 3.5 minutes, discarding the supernatant. The pellet was resuspended in 0.6M sucrose solution and centrifuged at 600rpm for 10 minutes. The protoplasts were removed and diluted with CPW9M (pH5.6, 560mmol/kg) and pelleted by centrifuging at 600rpm for 3.5 minutes. The protoplasts were resuspended in CPW9M, counted, diluted to 2xl0⁶/ml in MaMg medium (Negrutui et al., 1987) and aliquoied at 4xl0⁵ protoplasts per treatment. 20μg each of effector and reporter plasmid DNA (lmg/ml) were added followed by 200μl PEG solution (Negrutui et al., 1987). The protoplasts were incubated at room temperature for 10 minutes before addition of 5ml MSP19M medium (MS medium, 3%> sucrose, 9% mannitol, 2mg/l NAA, 0.5mg/l BAP, pH5.6, 700mmol/kg) in the presence or absence of ligand (N-(-3-oxohexanoyl)-L-homoserine lactone (OHHL)). The protoplasts were cultured in their tubes lying horizontally at 25°C and they were harvested for the GUS assay after 24 hours. Transicnt Expression in Nicotiana piimbaginifoUa suspension cells derived protoplasts

Protoplasts isolation

Protoplasts were isolated from Nicotiana plumb aginifolia suspension cells. Suspension cells were sub-cultured once a week in Np suspension medium in 250 ml Erlenmeyer flasks that were shaken at 100 RPM , at 25°C with 16h 8h light/dark regime. Protoplasts were isolated 2 or 3 days after subculture. Two or 2.5 g fresh weight of cells were incubated with 20 ml filter-sterilised enzyme solution and shaken at 40 rpm at 25°C. The enzyme solution comprised 1% cellulysin (Calbiochem 219466), 1 % Macerozyme RIO™ (Yakult, Tokyo), and 1% Driselase (Sigma™ D- 9515) dissolved Artificial Sea Water Mannitol. After 3 h cell digestion in enzyme solution, they were washed through 100-, 50-um diameter sieves with W5 solution before being collected by centrifugation at 80 x g for 4 min. This method is also used for the isolation of wheat protoplasts.

Protoplasts transformation.

Protoplasts were resuspended in W5 medium at densities ranging from 0.1 to 0.2 x 10⁶ /ml and sedimented for 4 min at 80 g. They were then taken up in 0.2 to 0. 5 ml of: 0.4 M mannitol, 15 mM MgCl₂, 0.1 % MES, 2 % glucose, pH 5.6 containing 15 μg plasmid DNA. Five minutes later poly-ethylene glycol (PEG) 4000 (Fluka™) at 40%) (wlv) in 0.4 M mannitol and 0. 1 M Ca(N0₃).4H₂0 pH 9.0 was added to give final PEG concentrations of 20%. After half an hour 1 ml 0.2 M CaCl₂.2H₂0 were added, and the protoplasts centrifuged for 4 min at 40 g. The protoplasts were then cultured in transformation buffer at 0.1-0.2 x 10⁶ protoplasts per ml during 48h at 25°C in the dark. This method is also applicable to the transformation of wheat protoplasts.

GUS Assays

Transient transformed tobacco protoplasts were harvested by centrifugation at 3000 rpm. by collecting them by centrifugation at 3000 rpm. The supernatant was discarded and the protoplasts resuspended in GUS extraction buffer (Jefferson et al., 1987) for preparation of β-glucoronide extracts. The tubes were vortexed for 1 minute and then spun at 13000rpm for 2 minutes. The supernatant was collected and placed in a fresh eppendorf tube. 20μl of the extract were used in the GUS assays. Fluorometric assays for GUS activity were performed with the substrate 4- methylumbelliferyl-D-glucuronide (Sigma™) using a Perkin-Elmer™ LS-35 fluorometer (Jefferson et al., 1987). Protein concentration of tissue homogenates were determined by the Bio-Rad™ protein assay (Bradford, 1976).

Example 2. Transient expression of enhanced LuxR.

The transient expression experiments were carried out as described above. The same reporter plasmid was used but different effector constructs were produced. In order to address whether the presence of a strong activator such as VP 16 would enhance transcriptional efficacy of the receptor, it was introduced in both the N- and C- terminal ends of LuxR. The VP16 was also fused to the nuclear localisation signal (NLS) from SV40. Both the NLS of SV40 and VP16 were obtained from the yeast two hybrid plasmid pPC86 as a fused fragment which meant that it could be fused as one on to LuxR. The N-terminal fusion was constructed by isolating and introducing into pBluescript a ClallNoil fragment of pPC86 containing the SV40 NLS and Gal4 activation domain. The resulting pBluescriptSV40V was digested with Hindlll and filled in followed by Bglϊl digestion. The fragment was introduced into SmallBamHl pDH51 luxR vector (BamHI and Bglϊl enzymes produce compatible ends) to produce the N-terminal fusion in LuxR and yield plasmid pDH51NVLuxR (Figure 10). The methionine start site is provided by the SV40 fragment.

The C-terminal end fusion was carried out by placing at the Real site in LuxR the blunted fragment of NLS/VP16. pDH51LuxR was digested with Real, blunt ended and ligated to the ClallNoil SV40-Gal4 blunt ended fragment. The fusion resulted in plasmid pDH5 ILuxRNV which contains four linking amino acids, C A K L, between the end of LuxR and the start of the nuclear locating sequence.

The plasmids were tested in tobacco mesophyll protoplasts as described in Example 1, where p221.91ux6 was introduced with or without either of the expression vectors containing the enhanced luxR. GUS assays were carried out as described above. Example 3. Transient expression of LuxR, LasR and TraR in monocot protoplasts.

Expression plasmid construction.

The vector of choice for expression in monocot transient system was pFun which contains ubiquitin promoter region and the Nos terminator. In between the two there is multiple unique cloning sites. LuxR was transferred into pFun as a BamHI fragment. pLasR was transferred as a BamHI/Kpnl fragment. TraR was isolated from Agrobacterium tumefaciens by PCR using oligonucleotides TraRl 5' AATTGGTACCCACC ATGCAGC ACTGGCTGGACAAGTTGACC 3 ' (Sequence ID. 11) and TraR2 5' AATTGGATCCCAGATCAGCTTTCTTCTGCTTGGCGAGG 3' (Sequence ID 12). The purified fragment was restriction enzyme digested with BamHI and introduced into pFun BamHI vector. On each case the expression vectors were named as follows: pFunLuxR (Figure 13), pFunLasR (Figure 14) and pFunTra (Figure 15).

Reporter plasmid construction

Reporter plasmid used with the expression vector encoding for LuxR is the same as that used in the transient experiments carried out in Tobacco (Examples 1 and 2). In the case of reporter vectors for both the expression of LasR and TraR the parental plasmid p221.9 was used. In both cases oligonucleotides encoding the

LasBox and the two TraBoxes were synthetically made. The LasBox oligonucleotides are as follows LasBox 1 5' TCGACACCTGCGAGTTCTCCGAGGTG 3' (Sequence ID 13) and LasBox2 is 5' TCGACACCTCGGAGAACTCGCAGG TG 3' (Sequence ID 14). The TraBoxes as described by Zhu and Winans, (1999) were used and the oligos are as follows, TraBoxl 5 ' TCGACTACACGTCTAGACGTGTAGG 3'

(sequence ID 15), TraBox2 5' TCGACCTACACGTCTAGACGTGTAG 3 '(Sequence ID 16) (first pair), TraBox3 5' TCGACTACACGTCTAGACGTGTAAG 3' (Sequence ID 17) and trabox4 5' TCGACTTACACGTCTAGACGTGTAG 3' (second pair) (Sequence ID 18). Equimolar amounts of each pair oligo in the pair were mixed denatured and allow to cool down slowing to form double stranded DNA which had Sail cohesive ends. The double stranded DNA was then ligated into a Sail digested p221.9 vector. Recombinants were screened by colony hybridisation and sequenced to ascertain number of elements incorporated into the vector. The vectors were named as follows p221.9Traboxl (Figure 16), p2219Trabox2 (Figure 17) and p221.9Lasbox (Figure 11).

Example 4. Stable expression of LuxR in Tobacco plants Binary vector construction

The reporter cassette was isolated from p221.91ux6 by digesting with EeoRI and H dIII to yield a 2.0kb fragment. The fragment was purified and introduced into pBin 19 Eco RI/H/πdlll vector to produce pBinrepAΗL. The three different variants of the LuxR receptor, that is LuxR and the two enhanced versions were restriction enzyme digested with EcoRI (site flanking both sides of the effector cassette) and introduced into a dephosphorylated EcoRI pBinl9repAΗL vector to produce either pAHLl (LuxR) (Figure 18), pAHL2 (SVLuxR) (Figure 19) or pAHL3 (LuxRSV) (Figure 20).

Plant transformation

The plant transformation construct pAHL 1 (Figure 18), pAHL2 (Figure 19) and pAHL3 (Figure 20), containing LuxR or chimeric LuxR and a reporter gene cassette, were transformed into Agrobacterium tumefaciens LBA4404 using the freeze/thaw method described by Holsters et al. (1978). Tobacco (Nicotiana tabacum cv Samsun) transformants were produced by the leaf disc method (Horsch et al., 1988). Shoots were regenerated on medium containing 1 OOmg/1 kanamycin. After rooting, plantlets were transferred to the glasshouse and grown under 16 h light 8 h dark conditions

PCR analysis

Analysis of transgenic tobacco plants by PCR was carried out using leaf sample extracted in 300μl of extraction buffer. The DNA was precipitated with isopropanol at 4°C for 10 minutes and then centrifuged. The pellet was dried and resuspended in lOOμl of TΕ (lOmM Tris HCl pH 8.0, ImM ΕDTA). 2.5 μl were placed in a 500 μl eppendorf tube and a master mix containing buffer, dNTPs and oligonucleotides was added. The Taq polymerase (Gibco-BRL™) was added after samples were denatured for 3 minutes.

Chemical treatments

OHHL was dissolved in methanol (Sigma) and the stock maintained at -20°C. The compound was diluted in growth media used to germinate seedlings. Uninduced seedlings were treated with equivalent amount of methanol. Seeds were germinated in MS media supplemented with 0.8%> (w/v) agar. Seedlings were collected 2 days post-germination (two cotyledon stage).

GUS Assay

In seedling induction experiments 10 two day old seedlings were collected and flash frozen in liquid nitrogen. The seedlings were homogenised in 300 μl of GUS extraction buffer and centrifuged for 5 minutes at 13000rpm. The supernatant was used for both GUS and Bio-Rad™ protein assays.

Example 5 Stable constitutive expression of yenl gene in tobacco plants

Binary vector construction. pBDHELI (Figure 21) was constructed by fusing the alfalfa mosaic virus (AMV) translation enhancer sequence from pBi526 (Datla et al., Plant Science 94,

139-140 (1993)) to the yenl coding sequence from Yersinia ente ocolitica. AMV -Luxl gene fusion was directional cloned into pDH51 (Pietrzak et /.,1986) vector to produce pDHELI. A 1.8kb fragment of pDHELI was cloned into pBinl9 (Bevan, 1994) to give pBDHELI (Figure 21). The plasmid was introduced into Agrobacterium as described above. A tobacco plant population of was produce and screened as detailed above. A high expressor of yenl gene was selected by their ability to synthesised OHHL. The assay consists of placing a leaf of the transgenic plant on an agar plate overnight. The leaf was then removed and the evil mutant of Chromobacterium violaceum spread over the plate. Violacein (a purple pigment) production by the bacterium can be seen when OHHL had diffused out of the leaf and into the agar (Figure 24). Example 6. LuxR is activated by compounds produced by yenl plants.

The root of a transgenic tobacco plant containing t zyenl expression cassette, that was grown in tissue culture under aseptic conditions, was placed on an LB agar plate and overlain with top agar containing E. coli with the plasmid pSB401 (Figure 22). This strain expresses the lux operon (i.e. luxR, lux ABCDE) and will bioluminesce in response to OHHL and HHL. Figure 24 shows that acyl-homoserine lactones produced by yenl introduction into tobacco plants are capable of activating LuxR harboured in E .coli and activate reporter gene expression. These data suggest that introduction of LuxR expression and reporter cassettes into plants harbouring the yenl gene will result in activation of reporter gene expression.

Example 7. Cross of LuxR tobacco high expressor plant with yenl tobacco plant.

Plants are produced by crossing the yenl expressing plant with the AHL switch plants. The seed of the progeny was collected and assayed for GUS activity as described above. GUS activity was assayed for in all tissues and different ages as the expectation was that GUS protein would be present in all tissues expressing the yenl gene, yenl Is under control of the 35S CaMV promoter as is the LuxR protein in the AHL switch. Initially, progeny seedlings were grown in 1/2MS and were collected 2 days post-germination or when cotyledons were fully extended. Other modifications of the present invention will be apparent to those skilled in the art without departing from the scope of the invention.

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Claims

1. A method of initiating transcription of a target gene in a eukaryotic cell comprising: (a) providing a eukaryotic cell which is capable of producing a response protein;

(b) inserting into the genome of said cell a polynucleotide defining an inducible promoter sequence operably linked to and capable when induced of initiating transcription of said target gene; and (c) applying to said cell a chemical inducer capable of binding to said response protein whereby said chemical inducer binds to said response protein to form an inducing complex which binds to and induces said inducible promoter thereby initiating transcription of said target gene.

2. A method according to claim 1 wherein a polynucleotide is inserted into the genome of said eukaryotic cell which polynucleotide provides for the production of the said response protein.

3. A method according to claim 1 or 2 wherein the inducible promoter comprises the nucleotide sequence depicted as SEQ ID No. 2 and the response protein comprises the amino acid sequence depicted in SEQ ID No. 16.

4. A method according to claim 3 wherein the said inducible promoter comprises a polynucleotide which is the complement of one which binds to SEQ ID No. 2 at a temperature of between 60┬░C and 65┬░C in 0.3 strength citrate buffered saline containing 0.1 %> SDS followed by rinsing at the same temperature with 0.3 strength citrate buffered saline containing 0.1 %> SDS wherein the said polynucleotide is still capable of acting as an inducible promoter upon binding with the said inducing complex.

5. A method according to claim 3 or 4 wherein the polynucleotide encoding the said response protein comprises the sequence depicted in SEQ ID No. 5.

. A method according to claim 5 wherein the polynucleotide encoding the said response protein comprises the complement of one which binds to SEQ ID No. 5 at a temperature of between 60┬░C and 65┬░C in 0.3 strength citrate buffered saline containing 0.1% SDS followed by rinsing at the same temperature with

0.3 strength citrate buffered saline containing 0.1%o SDS wherein the said polynucleotide still encodes a protein which is capable of forming an inducing complex with the said chemical inducer.

7. A method according to any one of claims 2 to 6 wherein the polynucleotide encoding the said inducible promoter contains the region depicted as SEQ ID No. 21.

8. A method according to any one of claims 1 to 7 wherein the said eukaryotic cell is a plant cell.

9. A method according to claim 8 wherein the plant cell is from a plant selected from the group consisting of: melons, mangoes, soybean, cotton, tobacco, sugarbeet, oilseed rape, canola, flax, sunflower, potato, tomato, alfalfa, lettuce, maize, wheat, sorghum, rye, bananas, barley, oat, turf grass, forage grass, sugar cane, pea, field bean, rice, pine, poplar, apple, peaches, grape, strawberries, carrot, lettuce, cabbage, onion, citrus or nut plants.

10. A method according to any one of claims 1 to 9 wherein the said chemical inducer is N-(3-oxo)hexanoyl-L-homoserine lactone or a functional equivalent thereof.

11. A method according to any one of claims 8 to 10 wherein the said chemical inducer is applied to the plant cell as part of an agriculturally acceptable formulation.

12. A method according to any one of claims 2 to 1 1 wherein the polynucleotide encoding the said response protein is bounded by a promoter and a terminator sequence.

13. A method according to claim 12 wherein the promoter is inducible.

14. A method according to claim 13 wherein the promoter is selected from the group consisting of: Ale A/ R switch system, the GST switch system and the ecdysone switch.

15. A method according to claim 12 wherein the promoter is constitutive or developmentally regulated.

16. A method according to claim 15 wherein the promoter is selected from the group consisting of: Cauliflower mosaic virus 35S/19S, maize Ubiquitin and

Arabidopsis Ubiquitin 3.

17. A method according to any one of claims 1 to 16 wherein the said target gene encodes ╬▓-glucuronidase; Bacillus thuringenesis toxin; barnase or barstar.

18. A method of providing plants containing an inducible target gene comprising: (a) inserting into a plant cell which cell provides for production of a response protein a polynucleotide defining an inducible promoter sequence operably liked to said target gene; and (b) regenerating morphologically normal fertile plants thereof; and

(c) applying to the population of regenerants a chemical inducer or a functional variant thereof capable of binding to said response protein, whereby said chemical inducer binds to said response protein to form an inducing complex which binds to and induces said inducible promoter thereby initiating transcription of said target gene; and

(d) selecting those plants which are expressing the said target gene.

19. A method according to claim 18 wherein the said inducible promoter sequence comprises the nucleotide sequence depicted as SEQ ID No. 2 or a functional variant thereof and the response protein comprises the amino acid sequence depicted as SEQ ID No 16 or a functional variant thereof and the said chemical inducer is N-(3-oxo)hexanoyl-L-homoserine lactone or a functional equivalent thereof.

20. A method according to claim 18 wherein the said inducible promoter sequence contains the nucleotide sequence depicted as SEQ ID No. 10 or a functional variant thereof and the response protein comprises the amino acid sequence depicted as SEQ ID No 17 or a functional variant thereof and the said chemical inducer is N-(3-oxo)dodecanoyl-L-homoserine lactone or a functional equivalent thereof.

21. A method according to claim 18 wherein the said inducible promoter sequence contains the nucleotide sequence depicted as SEQ ID No. 12 or a functional variant thereof and the response protein comprises the amino acid sequence depicted as SEQ ID No 18 or a functional variant thereof and the said chemical inducer is N-(3-oxo)octanoyl-L-homoserine lactone or a functional equivalent thereof.

22. Plants produced according to a method of any one of claims 18 to 21.

23. Plants according to claim 22 which plants are selected from the group consisting of: melons, mangoes, soybean, cotton, tobacco, sugarbeet, oilseed rape, canola, flax, sunflower, potato, tomato, alfalfa, lettuce, maize, wheat, sorghum, rye, bananas, barley, oat, turf grass, forage grass, sugar cane, pea, field bean, rice, pine, poplar, apple, peaches, grape, strawberries, carrot, lettuce, cabbage, onion, citrus or nut plants.

24. A DNA construct which comprises a first polynucleotide region which comprises an inducible promoter sequence operably liked to and capable when induced of initiating transcription of a target gene and a second polynucleotide region which provides for the production of a response protein wherein upon contact of the said response protein with a chemical inducer the said response protein binds the chemical inducer to produce an inducer complex which binds the said inducible promoter sequence thereby initiating transcription of the said target gene.

25. A DNA construct according to claim 24 wherein the said first polynucleotide comprises the nucleotide sequence depicted as SEQ ID No. 2 or a functional variant thereof and the second polynucleotide region comprises a polynucleotide which encodes the amino acid sequence depicted as SEQ ID No. 16 or a functional variant thereof.

26. A DNA construct according to claim 24 wherein the said first polynucleotide comprises the nucleotide sequence depicted as SEQ ID No. 10 or a functional variant thereof and the second polynucleotide region comprises a polynucleotide which encodes the amino acid sequence depicted as SEQ ID No. 17 or a functional variant thereof.

27. A DNA construct according to claim 24 wherein the said first polynucleotide comprises the nucleotide sequence depicted as SEQ ID No. 12 or a functional variant thereof and the second polynucleotide region comprises a polynucleotide which encodes the amino acid sequence depicted as SEQ ID No. 18 or a functional variant thereof.

28. A DNA construct according to any one of claims 24 to 27 wherein the said second polynucleotide region comprises a promoter operably linked to the polynucleotide encoding the said response protein.

29. A DNA construct according to claim 28 wherein the said promoter is inducible.

30. A DNA construct according to claim 28 wherein the said promoter is constitutive or developmentally controlled.

31. Use of a DNA construct according to any one of claims 24 to 30 in the production of a plant containing a target gene of which the expression may be controlled by the application of a chemical inducer to the said plant.

32. A method of screening compounds in a bioassay comprising applying an amount of a chemical to a plant which plant contains a polynucleotide comprising an inducible promoter operably linked to a region encoding a reporter protein and which plant is also capable of producing a response protein and testing the said plant for the production of the said reporter protein.

33. A method of selectively controlling pests in a field which field comprises crop plants and pests wherein the plants are those obtained according to claim 18 and the said target gene encodes a target protein which is capable of controlling the said pests said method comprising applying to the plants an amount of a chemical inducer which is sufficient to bind to the said response protein to produce the said inducing complex which is capable of initiating transcription of the target gene which provides for the production of the target protein in an amount which is sufficient to control the said pests.

34. A method of providing a plant which contains a target gene which is inducibly controlled comprising: (a) inserting into a first plant cell a polynucleotide comprising a first inducible promoter operably linked to a target gene and regenerating a first morphologically normal fertile plant therefrom; (b) inserting into a second plant cell a polynucleotide comprising a promoter operably linked to a region encoding a response protein which is capable of binding to a chemical inducer to produce an inducing complex which is then capable of binding the said inducible promoter to allow for the initiation of transcription of the said target gene and regenerating a second morphologically normal fertile plant therefrom;

(c) cross pollinating said first plant with the said second plant or said second plant with said first plant and harvesting the seed therefrom; and

(d) growing said seed and applying to the resultant plants an amount of said chemical inducer which provides an inducing complex capable of binding the said inducible promoter to allow for the initiation of transcription of the said target gene.