HUT73739A - Integrative dna segment compraising gene encoding insecticidal protein - Google Patents

Description

EXTRACT

The present invention relates to insecticides of microbial origin.

More particularly, the present invention relates to DNA fragments encoding an insecticidal protein, and methods for producing hybrid microorganisms using the DNA fragments of the present invention having a greater insecticidal activity and a broader host range of insecticidal activity. The invention also encompasses vectors comprising the DNA fragments of the invention, host cells transformed with the vectors, and insecticidal compositions comprising the host cells of the invention.

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Our file number is 82517-2703 / PA

PUBLICATION LITERATURE

THE

Representative:

DANUBIA PATENT AND TRADEMARK OFFICE LTD

A DNA fragment encoding an insecticidal protein

The present invention relates to insecticides of microbial origin.

More particularly, the present invention relates to DNA fragments encoding an insecticidal protein, and methods of producing hybrid microorganisms using the DNA fragments of the present invention having a greater insecticidal activity and a broader host range of insecticidal activity.

The invention also encompasses vectors comprising the DNA fragments of the invention, host cells transformed with the vectors, and insecticidal compositions comprising the host cells of the invention.

The present invention is useful for protecting plants against insect infestations.

In recent years, researchers have shown considerable interest in the use of Bacillus thuringiensis (B.t. \ and B.t.) to prevent insect infestation of plants by biological methods. These bacteria produce a toxic crystal, called parasporal crystal, when sporulated by insects.

The B.t. strains are classified into different serotypes or subspecies based on their lagellar antigens. Most B.t. the tribe is active against certain members of the order of scaly cubs,

Our file number is 82517-2703 / PA

-2, for example, against the larvae of butterflies and butterflies, but some strains are also toxic to members of the orders of bivalves and aphids, such as mosquito larvae and beetle larvae. Some toxic crystal producing strains have so far shown no toxic activity.

The B.t. its crystalline inclusion dissolves in the larval midgut and releases one or more insecticidal crystalline proteins, also known as δ-endotoxins, which have a molecular weight of 27 to 140 kD. Most crystalline proteins are protoxins that are converted into a toxic toxic polypeptide in the middle of the insect. Generally, in the literature, crystal proteins are referred to as Cry and genes encoding crystal proteins are designated cry.

To date, more than 40 cry genes have been characterized. A grouping system for cry genes has been published by Hofte and Whitely, Microbiol. Rev., 53, 242 (1989)], in which a system divides genes into four classes and a few subclasses based on structural and host similarities between the encoded proteins. The four major classes include the scaly-specific (I), the scaly-bipedal (II), the scaly-specific (III), and the bivalve-specific (IV) genes.

The scaly-specific genes (cryl) encode proteins of the molecular weight 130-140 kD, which are B.t. accumulate in bipyramidal crystalline inclusions during its sporulation. The cryl genes can be distinguished from other cry genes by their homology (> 50% amino acid identity) in their sequence. Three of these genes, cryIA (a), cryLA (b) and crylA (c), have more than 80% amino acid identity and are therefore considered as a separate subgroup. The most recently discovered crylB, crylC and crylD genes differ from each other and separately

They also differ from the crylA genes. CrylA, CrylB and CrylC proteins have so far been detected in crystal preparations isolated from 29 strains belonging to serotype 11 using 35 monoclonal antibodies [Hofte et al., Microbiol. Rev., 53, 242 (1989)].

The class of genes specific for scaly and dicotyledon contains genes encoding proteins of 56 kD, which form cubic inclusions. The first cryll gene was B.t. subsp. kurstaki strain HD-263, and Bacillus megateriumbwo. Expression. CrylIA protein producing cells were toxic to the scaly species Heliothis virescens and Lymantria dispar as well as to the larvae of the bivalve Aedes aegypti.

The genes belonging to a specific class of codons encode gene products that are effective against the species belonging to the codend, and the coded proteins have a molecular weight of about 70 kD. At least three strains of B.t. tenebrionis, B.t. san diego and B.t. EG2158. These strains produce crystals of rhomboidal form, which contain a major protein.

The bipartite-specific class of cryIIV genes consists of a rather heterogeneous group of bipartite-specific crystal protein genes. The cytA gene and four other genes are B.t. 72 megadalton (mD) in israelensis. The ciylVA, crylVB, crylVC and crylVD genes, respectively, encode proteins with molecular weights of 135, 128, 78 and 72 kD. These proteins, together with the 26 kD product of the cytA gene, are organized into an egg crystalline complex. The B.t. crystalline complexes of the same or similar protein composition were also found in morrison's PG-14 strain. The B.t. israelensis or recombinant E. coli or recombinant bacillus

Preparations containing cryoV class crystal proteins from strain-4 strains have been found to be toxic to varying degrees against larvae of certain mites.

Since Hofte and Whitely created their original classification system, several new cry genes have been cloned and their nucleotide sequences have been determined. Yamamoto and Powell publish another classification system [Bacillus thuringiensis Crystal Proteins, 3-42. p. in Advanced Engineered Pesticides, ed. Leo Kim (1993).

Various commercially available B.t. preparations are commonly used to protect various cultivated plants, shade trees and ornamental plants against infection by various insect species and are applied to plants by means of chemical insecticides. Methods for applying spray liquors are well known to those skilled in the art.

Formulations containing hybrid bacteria may be applied in the form of sprays, powders or baits, alone or in combination with parasites, predators, or other protective methods, for example, chemical insecticides, radiation-induced sterilization, chemical sterilizers, pheromones, and the like. The use of stress-inducing agents and methods may enhance the pathogenicity of the hybrid bacteria of the present invention or activate their chronic infection.

The B.t. known methods for transformation include protoplast fusion, protoplast transfection, transduction, electroporation, and conjugation-like processes.

-5A B.t. electroporation is frequently used for its transformation due to the simplicity, speed and efficiency of the process. In 1989, a B.t. commuter vector (shuttle vector). The applicability of this commuter vector was first demonstrated by a B.t. crystalline toxin gene was transferred to a crystal minus (Cry) B.t. strain called cryB, and the resulting transformant expressed a 130 kD molecular weight crystal protein.

The native B.t. Recently, several cloning and expression vectors have been developed from plasmids, for example, shuttle vectors containing B.t. israelensis 3.65 Md plasmid and pBR322; B.t. plasmids derived from kurstaki strains HD263, HD73 and HD1; and B.t. israelensis and E. coli vectors. Such a commuting vector has been used to construct a B.t. israelensis strain and thus broaden its insect spectrum to be active against both diphthongs and diphthongs.

A major problem with this technology is the instability or incompatibility between native and exogenous plasmids. It is often the case that one or more native B./. plasmids are unable to co-survive with an exogenous plasmid (s) introduced into the bacterium, and therefore the native plasmid is rapidly lost by segregation. This problem is problematic when native plasmids contain one or more cry genes that are important for the preservation of the transgenic B.t. strain. This problem can be overcome by removing the portion of the plasmid which causes incompatibility between the recombinant (or exogenous) and native plasmids.

• ·

-6 Various studies have uncovered a number of methods that are capable of delivering exogenous DNA stably to bacterial cells. A B.t. native-compatible vector was used to construct a cryIIIA gene active against a cover number in a B.t. kurstaki strain, and the transgenic strain thus produced proved to be highly active against larvae and retained its activity against wild type scaly larvae. Bacteria may also be transformed with plasmids incapable of autonomous replication and carrying a selectable marker gene, provided that the plasmid used contains a DNA segment that is homologous to a portion of the host cell chromosome. For the above example, it has been shown that a plasmid is able to interact with a host cell by a single crossover mechanism involving the introduced plasmid and homologous host sequences. The result is a plasmid integrated into the chromosome flanked by direct repeats of the homologous DNA segment. Such insertions are mutagenic in nature if both ends of the duplicated segment are within the same transcription unit.

Delecluse et al. homologous recombination was used to inactivate the gene encoding the cytA insecticidal protein, B.t. israelensis [Delectuse et al., J. Bacteriol. 173, 3374 (1991)]. An integration vector containing a partial cytA sequence was generated and homologous recombination was induced in B.t. israelensis with the cytA gene on its 72 mD plasmid. Beyond what has been said, Calogero et al. a B. subtilis integration vector containing B.t. the complete crylA (c) coding region of strain HD73 of kurstaki [Calogero S. et al., Appl. Env. Microbiol.

55, 446 (1989)]. The plasmid vector has been shown to express the HD73-cryIA (c) gene. The patent specification (WO / 93/03619) attached to PCT / US91 / 05930 discloses a recombinant B.t. discloses a bacterium containing a commuting vector carrying an insecticidal gene and discloses a method for producing a recombinant bacterium.

However, neither the general methods described nor the publications cited contain any indication that the transformed B.t. whether the bacterial stability problem can be solved by chromosomal integration.

It has been an object of the present invention to provide for the stable survival and expression of crystal genes by integrating the genes into the chromosome of B.t. strains. The present invention also provides a method for generating strains of a broader host spectrum and / or novel specification B.t. More particularly, the present invention relates to a method for producing B.t. strains having potent anti-spodopteric activity while retaining their activity against scaly arthropods.

The present invention provides a DNA segment comprising a DNA sequence encoding one or more insecticidal proteins which are capable of replication and expression in B.t. strains, and a DNA sequence that promotes DNA segment B.t. integration into chromosomal DNA by homologous recombination.

The invention further provides a DNA segment comprising a DNA sequence encoding one or more insecticidal proteins which are capable of replication and expression in B.t.

-8 ··· · · · · · · · · · · · · · · · · · · · · · · · · · · · · into the genome.

The invention further provides a hybrid vector comprising a plasmid or a shuttle vector and a DNA segment of the invention operably linked to the plasmid or vector sequences.

The invention further relates to hybrid B.t. host cells into which a DNA sequence encoding at least one insecticidal protein of the DNA segment of the invention is integrated into a chromosome.

The present invention further provides a method for producing a DNA segment of the invention, comprising: producing a DNA sequence homologous to a chromosomal B.t. DNA sequence; and then operably linking to said sequence a DNA sequence encoding at least one insecticidal protein such that, if

B.t. cells are transformed with a DNA segment or a hybrid vector that contains a DNA segment functionally linked, the DNA sequence encoding the insecticidal protein is expressed.

The invention further relates to a process for transforming B.t. host cells comprising: i) generating a DNA sequence capable of randomly generating B.t. integrate into chromosomal DNA; ii) operably linking the resulting DNA sequence to a DNA sequence encoding one or more insecticidal proteins which are capable of replication and expression in B.t. cells; iii) isolating the DNA segment; iv) Bacillus thuringiensis host cells are transformed with a DNA segment capable of randomly expressing B.t. integrate into the chromosome of host cells; and

(v) isolating the host cells expressing the DNA sequence encoding the insecticidal protein as transformed host cells.

The invention also relates to a process for the use of a hybrid B.t. host cells expressing at least one exogenous insecticidal peptide according to the method of B.t. host cells are transformed with the DNA segment of the invention or the hybrid vector described above, allowed for homologous recombination, and the DNA sequence encoding the insecticidal protein stably incorporated into B.t. cell chromosome and isolating the transformed host cell.

The present invention provides a broad host spectrum insecticidal composition comprising a hybrid host cell of the invention, a carrier, and optionally other insecticidal active ingredients. In a preferred embodiment of the present invention, B.t. the insecticidal spectrum of cells can be broadened so that B.t. cells are transformed as described above with a DNA sequence encoding an insecticidal protein which remains expressible.

The present invention further provides a method of protecting plants against insect damage, comprising contacting the plant or soil in the vicinity of the plant with an effective amount of the insecticidal composition of the invention.

The invention will now be further illustrated by the following examples, drawings, and sequences.

Figure 1 is a diagram of plasmid pSB210 containing a gram-negative origin of replication.

Figure 2 shows the derivation of plasmid pSB147 as well as the family tree.

Figure 10 shows plasmid pSB210.1, which contains the pSB210 sequences.

Figure 4 shows plasmid pSB210-2, which contains the pSB210.1 sequences.

Figure 5 is a map of plasmid pSB210.3.

Figure 6 shows a map of plasmid pSB147.

Figure 7 is a schematic representation of the construction of plasmid pSB136.

The sequences relevant to the present invention are summarized in Tables 2 and 5 and in Examples 3, 5 and 11.

The present invention represents a step forward in the prior art B.t. broadening the spectrum of insecticidal activity. A hybrid B.t. strains containing a gene encoding more than one insecticidal protein, and thus having a broader host spectrum, may be produced by introducing a gene encoding a foreign insecticidal protein into the chromosome of known bacterial strains. As stated above, the present invention relates to a process of B.t. broadening the host spectrum of the strains by increasing the number of different insecticidal crystal proteins produced by the strain.

Recombinant B.t. plasmids introduced into strains can cause instability of their own plasmids, and it is also possible that the introduced plasmids themselves will be unstable. This process often results in the loss of expression of crystal protein genes in the cells. The present invention overcomes this problem by introducing the crystal protein gene into the host cell chromosome,

- 11 for expression without interfering with the expression of crystal protein genes on the host cell's own plasmids.

Accordingly, the present invention provides a DNA segment comprising a DNA sequence encoding one or more insecticidal proteins; and a DNA sequence homologous to a B.t. chromosomal DNA sequence, the DNA sequence encoding the insecticidal protein is incorporated into said B.t. cell chromosome, it is replicated and expressed with it.

The DNA sequence encoding the insecticidal protein may be any DNA sequence encoding an insecticidal protein that B.t. can be expressed in bacterial cells. Suitable insecticidal DNA sequences for use in the present invention include, but are not limited to, cryIA (a), cryLA (b), crylA (c), crylB, crylC, cryD, crylE, crylF, crylIA, crylIB, crylIIA, crylIIB, crylIIC, crylVA, crylVB, crylVC, crylVD and cytA genes, of any subspecies and / or Bt and any other insecticidal protein gene known to be expressed in Bacillus species or in other Gram-positive bacteria, as well as any insecticidal protein gene from B. larvae or B. papillae. Also suitable DNA sequences include those encoding insecticidal proteins such as the α-amylase inhibitor, the proteinase inhibitor, and any other bacterial toxin.

As a suitable homologous DNA sequence, the DNA segment of the invention may comprise any DNA sequence that is substantially homologous to any B.t. species or strain, such as those listed in paragraph 1 above.

«· • * · '· ·· * * ·· ·» »« ·· ·

- from any of the strains listed in Table ¹² - with any chromosomal DNA fragment. The homologous DNA sequence enables and directs the integration of the DNA segment of the invention into host cell DNA by homologous recombination. When the DNA segment of the invention is introduced into the host cell as a circular covalently closed DNA segment, homologous recombination may occur as a single cross-over event between the host cell DNA and the homologous DNA sequence. When the DNA segment of the invention is introduced as a linear DNA segment, homologous recombination can occur as a double cross-over event between the host cell DNA and the homologous DNA sequences flanking the DNA encoding the desired insecticidal protein. As stated above, homologous DNA sequences may be used in the form of one or two flanking DNA sequences. The DNA segment of the invention may also be prepared in double-stranded or single-stranded form. The single-stranded form may be prepared by denaturing the double-stranded form with heat or chemicals using methods known in the art. The double-stranded form can be prepared by digestion of the desired sequence with restriction enzymes followed by ligation using methods known in the art.

The above homologous DNA sequence is homologous to a fragment of the bacterial chromosome ranging in size from about 5 bases to about 20 kb. Preferably, the sequence is homologous to a fragment of about 500 bases to about 10 kb. Preferably, the homologous sequence may be homologous to B.t. with an approximately 2250 bp fragment encoding phospholipase C. Particularly preferred are the homologous DNA sequences which are B.t. insecticide on host cell chromosome ·············································································································································································

-13 homologous to sequences other than its endogenous genes. If the DNA segment contains a DNA sequence which is homologous to both the 5 'and 3' flanking sequences of the bacterial sequence encoding the insecticidal protein, as will be appreciated by one of ordinary skill in the art, at the end of the homologous region. Using this approach, adding a single new gene can broaden the host cell's insecticide spectrum.

The DNA segment of the invention may further comprise an origin of replication from a gram-negative bacterium. Examples of replication origins include any replication origin that is functional in one or more of the following gram-negative bacterial species or strains: Neisseria, Veillonella, Brucella, Pasteurella, Hemophilus, Bordetella, Escherichia, Erwinia, Shigella, , Serratia, Azotobacter, Rhizobuim, Nitrosomonas, Nitrobacter, Thiobacillus, Pseudomonas, Acetobacter, Photobacterium, Zymomonas, Aeromonas, Vibrio, Desulfovibrio and Spirillum. If the DNA segment of the invention is cloned in a gram-negative bacterium, such as E. coli, and then the Bacillus thuringiensis transformed with the cloned segment, only DNA sequences encoding the insecticidal protein that are integrated into the host cell chromosome will survive. Because the origin of gram-negative replication is not functional B.t. host cells, B.t. the host cell will not replicate the DNA segment of the invention introduced as above and will not encode the insecticidal protein coding sequence. '' '' '' '· · ·· ···· * w,

- 14 expressed only when the DNA segment is integrated into the host cell chromosome.

The DNA segment of the invention may further comprise a selectable marker gene which is expressed in unicellular organisms other than gram-positive bacteria, a selectable marker gene which is expressed in a gram-positive bacterium, and / or a selectable marker gene that is expressed in a gram-negative and gram-positive bacterium. too. A selectable marker gene expressed in a unicellular host other than a gram-positive bacterium is defined as any DNA sequence capable of generating any phenotype in a single-cell host other than a gram-positive host that is capable of expressing the particular DNA sequence. A selectable marker gene that can be expressed in a gram-positive bacterium is defined as any DNA sequence capable of generating in a gram-positive bacterial host a phenotype capable of detecting or selecting a gram-positive host carrying that DNA sequence. A selectable marker gene that can be expressed in both a gram-negative and a gram-positive bacterium is defined as any DNA sequence capable of generating a phenotype in a gram-positive and gram-negative bacterial host capable of detecting host cells carrying that DNA, or to select. Examples of suitable marker genes include marker genes whose expression induces drug resistance, chemical resistance, amino acid auxotrophy or prototrophy, or other phenotypic variation suitable for selection of a mutant or recombinant living being. The presence of a selectable marker gene facilitates cloning and / or maintenance of the DNA segment of the invention in Gram-negative bacteria and promotes recombinant B.t. carrying the DNA segment of the invention. bacterial selection and / or detection.

The present invention further provides a DNA segment comprising at least one DNA sequence encoding an insecticidal protein capable of carrying B.t. bacterial host cells and a DNA sequence capable of randomly replicating B.t. integrate into the host genomic DNA. Randomly integrating DNA sequences for use in the present invention include, but are not limited to, insertion sequences or transposon sequences, which are capable of inserting or copying themselves and functionally linked DNA sequences into B.t. random chromosomal or plasmid DNA positions of the host cell. Examples of such sequences include transposons, such as the Tn917 transposon, Tn15545 and Tn916 transposons, all of which are described in Camilli et al. Camilli et al., J. Bacterial 172. 3738 (1990), and other gram-positive transposons known in the art may also be mentioned. Although DNA sequences or units of DNA that are randomly integrated are known in the art, the present invention is the first to use such sequences to insert insecticidal genes into gram-positive bacteria.

Preferably, the DNA segment comprising the transposon sequence of the invention may be carried by a plasmid vector. Suitable plasmids for mice · «·«

for example, the plasmids pTV51Ts and pLTV1, which are described below with other plasmids. In a preferred embodiment of the invention, temperature-sensitive plasmids are used to select host cells containing transposons. Plasmids such as pTV51Ts and pLTV1 are unable to replicate above a certain temperature. As stated above, a DNA segment on a temperature-sensitive plasmid will only survive at a non-permissive temperature in a host cell if that DNA segment is integrated into the host cell genome. Transpositional efficiency can be enhanced by targeting a marker gene in the transposon, for example, a gene encoding drug resistance, amino acid xtrophy or prototrophy, or the like.

In a preferred embodiment of the invention, the DNA segment of the invention is used to generate multiple transposition events, a single B.t. host cell. When using temperature-sensitive plasmid vectors, it is achieved that the DNA segment is repeatedly inserted into the genomic DNA of the host cell by rapid increase in temperature. The use of transposon elements carrying marker genes encoding resistance to an active agent increases the likelihood of multiple transposition events by contacting the host cells with a greater amount of that active agent. When the host cells are contacted with mitomycin C, the transposition frequency is also increased. Alternatively, the gram-positive host cell is transformed with a plurality of different transposon elements, each of which contains a unique selection marker gene.

• * · * 4 * -., ··· ··.: .. ··} ·, ·

In one embodiment of the invention, the transposon element of the invention comprises any regulatory sequence required for expression of the inserted DNA sequence encoding the insecticidal protein in the host cell. In other embodiments of the invention, the transposon element may be engineered to generate an operon or gene fusion in which the insecticidal protein coding sequence is placed under the transcriptional and / or translational control of the host cell DNA. Operons and gene fusions can be generated using Youngman's TN917-mediated operon and gene fusion methods 79-103. p. (1973)] and other methods known in the art.

The transposon element of the present invention may be used in a transformed B.t. host cells expressing at least one exogenous insecticidal protein by operably attaching to the transposon element of the invention a DNA sequence encoding at least one insecticidal protein capable of replicating and expressing in Bt cells, thereby providing a DNA segment. and, if Bt cells are transformed with the resulting DNA segment, the DNA sequence encoding the insecticidal protein is integrated into the Bt cell chromosome, where it can be expressed, Bacillus thuringiensis host cells are transformed with the resulting DNA segment, and the DNA segment is deleted. Bt cell chromosome, and transformed host cells are isolated. The B.t. a transposon element or DNA sequence capable of being randomly integrated into the genome can be prepared using techniques known in the art, such as restriction enzyme digestion, ligation,

- 18 cloning and / or chemical synthesis. The gene or DNA sequence encoding the insecticidal protein may be produced using similar methods known in the art and functionally linked to a DNA sequence capable of being randomly integrated.

Transformation can be accomplished, for example, by transfection, electroporation, transduction or conjugation. Transformed host cells can be isolated by selecting for the selectable marker gene contained therein. In a preferred embodiment of the invention, the randomly integrated DNA sequence comprises the transposon tM917.

The insecticide spectrum of B.t. cells may be broadened by maintaining expression of the DNA sequences encoding the endogenous insecticide protein of the cells and operably linked to one or more exogenous insecticide coding genes capable of integrating into the transposon element of B.t.

The DNA segment of the invention may also be produced in the form of a hybrid plasmid. One of ordinary skill in the art will recognize that any plasmid sequence capable of carrying the DNA segment of the present invention may be used to construct the hybrid plasmid. Particularly suitable for use in the present invention are the plasmids pSB210.1, pSB210.2, pSB210.3, pSB136 and pSB147, which are detailed in Examples 1, 6 and 11 below, but other plasmids may also be used.

The DNA segment of the present invention may also be produced in the form of a hybrid hybrid shuttle vector operable in gram-positive bacteria. For this purpose • ·· ··· ·· " ··· · ·· · · • · ·· · · · · ·

- all vectors capable of replicating independently in Gram-negative bacteria, yeast or any other single-cell host are suitable, in addition to being capable of replication in Gram-positive bacteria.

Such commuting vectors are well known in the art. The suitability of commuter vectors was first demonstrated by a B.t. crystal toxin gene was transferred to a crystal minus - #. /, strain cryB. The resulting transformant cells expressed the 130 kD crystal protein. In addition to the above, Lereclus et al. another commuter vector was constructed which included B.t. pHT1030. plasmid pUC18, and a B.t. The cryIA (a) gene isolated from strain 407 was introduced into the cryB strain [Lereclus, D., et al., FEMS Microbiol. Became. 49 (1988)]. When the vector was introduced into the cry derivative of strain 407, the expression of the toxin gene was significantly increased compared to expression in the wild-type strain, possibly due to the increased copy number of the gene. Miteva et al. produced a commuter vector that included the 3.65 Mdalton B.t. israelensis, and pBR322 [Miteva, V.I. et al., Arch. Microbiol. 150, 496 (1988)]. Commuter vectors have also been described that, on the one hand, B.t. kurstaki-derived HD263, HD73, HD1 and B.t. israelensis B.t. plasmids and E. coli vectors on the other hand. Such a commuter vector has been used to construct a B.t. israelensis strain and thus the insecticide spectrum of the bacterium was widened and the active species were all bipedal. · ·

-20 and both, Crickmore, N. et al., Biochem. J. 270 (1990)].

The invention also relates to B.t. host cells comprising at least one DNA sequence encoding the insecticidal protein of the invention, stably incorporated into their chromosome. Suitable B.t. host cells, including B.t. subspecies and their various strains, and B.t. strains, like any other B.t. a subspecies or strain known in the art. As used herein, the term host cell refers to B.t. bacterial vegetative and spores. Stable incorporation of the DNA segment of the invention into the host cell chromosome is understood to persist for many generations in the host cell chromosome, B.t. also in the sporulation and germination phases of host cells.

In a preferred embodiment of the invention, the transformed B.t. host cells contain a DNA sequence encoding more than one expressable exogenous insecticide protein, stably integrated into their chromosome. The multiple occurring coding sequences may comprise any combination of the coding sequences for the insecticidal proteins described above and may include more than one copy of the same insecticidal protein coding sequence. In a particularly preferred embodiment of the invention, the host cell of the invention is capable of expressing two or more different insecticidal proteins.

One skilled in the art will recognize that the DNA segment of the invention can be used in any exogenous DNA sequence B.t. for stable incorporation into host chromosomes. As used herein, the term exogenous DNA refers to any DNA that is a B.t. farmer • · "

It changes the chromosomal DNA of 21 cells when integrated into the host cell chromosome. The desired DNA sequence can be introduced into B.t. cells as described for the DNA sequences encoding the insecticidal protein of the invention. As stated above, the present invention relates to a B.t. host cells having an exogenous DNA sequence that is replicable and expressed by the host cell in its chromosome.

The DNA segment of the invention may be prepared by generating a B.t. a DNA sequence homologous to a bacterial chromosomal DNA sequence and to which a DNA sequence encoding at least one insecticidal protein is operably linked, such that when a Bacillus thuringiensis bacterium is transferred with the DNA segment, it encodes the insecticidal DNA.

The homologous DNA sequence may be prepared by B.t. known bacterial genomic libraries are screened. If the desired B.t. A bacterial genomic library is not available and can be prepared using methods known in the art. Methods of screening for genomic libraries are also known in the art. Once the desired sequence has been identified, it can be cleaved from the genome by restriction enzyme digestion. If the DNA or peptide sequence is known, the desired DNA fragments can be synthesized using methods known in the art. Alternatively, when no known DNA or peptide sequence is available, restriction fragments of genomic origin are used to randomly clone homologous fragments.

• · • τ ··· · ♦ · * · • · · · · · · · · ·. · ··· * »· ⁴

The DNA sequence encoding the insecticidal protein may be functionally linked to a homologous DNA sequence such that, when the DNA encoding the insecticidal protein is integrated into the chromosome of the host cell by homologous recombination, the DNA encoding the insecticidal protein will be Bacilli. In one embodiment of the invention, the DNA segment comprises regulatory sequences capable of transcription and translation of DNA encoding insecticidal protein B.t. host cells. Such regulatory sequences may include, but are not limited to, promoter, operator, repressor, and / or enhancer sequences, transcription initiation and termination sites, ribosome binding sites, translation start and stop codons, and / or other regulatory sequences known in the art. For example, the DNA sequence encoding the insecticidal protein may be functionally linked to regulatory sequences that regulate expression of the DNA encoding the insecticidal protein in the bacterial host cell from which it is derived.

The invention also encompasses DNA segments designed to generate operons or gene fusions within the host cell DNA. In the case of operon fusion, the DNA segment may comprise regulatory elements that are capable of directing the translation of the mRNA encoding the insecticidal protein. The homologous DNA sequence may be designed to integrate the DNA sequence encoding the insecticidal protein within an host cell into an operon such that, upon integration into the host cell chromosome, the host cell regulatory sequences direct the insecticidal coding protein. Transcription and translation of a DNA sequence. Suitable for producing operon and gene illusions

A description of the -23mas procedures can be found, among others, in Sambrook et al. Sambrook et al., 1989, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, NY.

Once created, the DNA segment of the invention can be isolated using techniques known in the art, such as centrifugation or agarose gel electrophoresis.

The DNA segment of the present invention may further comprise a selectable marker gene, which may be a gene that is expressed in unicellular host cells other than gram-positive host cells, a gene that can be expressed in gram-positive host cells, or a gene capable of expressing expressed in both gram-positive and gram-negative host cells. A selectable marker gene which is operable in a single-celled organism is used to clone and purify the DNA segment in that organism.

The selectable marker genes of the present invention can be operably linked to a DNA segment by inserting the marker gene next to the DNA segment or inserting the gram-positive selectable marker gene into the DNA segment such that when the DNA segment is B.t. is integrated into the host cell chromosome, the selectable marker gene is not adversely affected by the DNA sequence encoding the insecticidal protein B.t. expression in a host cell. In addition to the foregoing, it is important that the linkage of the Gram-positive selection marker gene does not adversely affect the function of the marker gene for selection in the host cell used for cloning (when a non-Gram positive host cell is used) and B.t. it must be expressed in a host cell. In a preferred embodiment of the invention, the gram-

The -24 positive selection marker gene also contains all the regulatory elements necessary for its expression. The Gram-positive selectable marker gene may also be selected to function as described above in an operon in the host cell DNA or fused to a gene.

The method of the invention may further comprise the step of operably linking an origin of replication to the DNA segment described above that is functional in the unicellular organism used for cloning. This organism may be an insect cell, a CHO cell, a gram-negative bacterium, a yeast, and the like. The origin of replication described above can be functionally linked to the DNA segment of the invention by positioning the origin of replication within the DNA segment without interfering with the function of any other element of the DNA segment, e.g., DNA encoding the insecticidal protein. and DNA sequences homologous thereto.

A B.t. a host cell having a DNA segment encoding at least one insecticidal protein stably incorporated in its chromosome can be prepared as follows:

i) preparing a DNA segment of the invention;

ii) a B.t. transforming the host cell with the DNA segment;

iii) allowing homologous recombination to occur and the DNA sequence encoding the insecticidal protein to be stably incorporated into the chromosome of the host cell; and iv) isolating the transformed host cell.

B.t. host cells may be transformed with the DNA segment using techniques well known in the art, e.g.

-25 ··· · ············································································································· · After such steps by electroporation, transfection, transduction, and conjugation, or a combination of the methods listed.

The present invention also provides a broad spectrum insecticidal composition comprising an insecticidal effective amount of the hybrid host cells of the invention and a carrier.

The composition of the invention contains about 10 ⁶ to 10 ¹³ hybrid microorganisms per gram of carrier, and more preferably about 10 ¹⁰ to 10 ¹¹ microorganisms. However, other amounts of the microorganism may be used. In the composition of the invention, Bt host cells may be present in either vegetative or spore form. Suitable carriers are well known in the art and the skilled artisan will be able to select carriers suitable for use in the invention. Carriers are typically inorganic compounds or compositions that do not interact with either the host cells contained in the insecticidal composition or the plants to be treated. However, carriers which can be metabolized by plants or by soil-dwelling organisms and are therefore biodegradable may also be used. The efficacy and durability of the insecticidal composition can be enhanced by the addition of carriers such as dispersants, adhesives, wetting agents, and, for example, maize flour additives, Loco® (amine stearate) spray, 1956, L-100 and--35 polybutenes, corn oil, Triton Β-1946, Cellosize QP 4400, boric acid, surfactant oils, Pinolene®, and other adjuvants known in the art. The active compounds are mixed and formulated according to techniques known in the art, and the

The composition of the present invention is formulated as a powder, liquid or aerosol. Most preferably, the hybrid host cell is added at a low temperature and the cells are thawed just before use.

The insecticidal compositions of the present invention may further comprise other insecticidal compounds. It is known that B.t. containing compositions are compatible with many chemical insecticides, for example, a summary of such agents can be found in Herfs and Pflanzenkrankh (Herfs, W., and Pflanzenkrankh, Z .: Pflanzenschutz 72, 584 (1965)). As stated above, the insecticidal compositions of the present invention may contain one or more of the insecticidal active ingredients listed in Herfs, or may include B.t. host cells or hybrids thereof prepared in accordance with the present invention.

The present invention also provides a method of protecting plants from insect damage by applying an effective amount of the insecticidal composition of the invention to the plant or to the soil surrounding the plant.

Methods of application of commercially available microorganism-containing insecticidal compositions, which are well known in the art, are also suitable for use with the insecticidal compositions of the present invention. Typically, the compositions of the invention are applied by delivering from about 10 ^{8 to} about 10 ¹⁶ hybrid microorganisms per moon, and more preferably from about 10 ^{13 to} about 10 ¹⁴ hybrid microorganisms. The compositions of the present invention are most preferably applied by spraying the plants and repeated application can be employed.

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The following examples are intended to illustrate the present invention in more detail, but are not intended to limit the scope of the invention unless otherwise indicated.

Example 1

Preparation of plasmids

Competent E. coli DH5a (Gibco BRL) and GM2163 (New England Biolabs) cells were prepared according to Alexander's procedure.

D.C .: Meth, Enzvmol 154, 41-63. (1987)]. The plasmids were isolated from E. coli cells according to the procedure of Bimboim and Doly (Bimboim and Doly, Nucl. Acids. Gap. 7, 1513 (1979)].

Plasmid pSB210.2, which contains the cry1C gene, the ermC gene of B. substilis, which codes for erythromycin resistance, and the phospholitase C region, described by Lechner, which serves as an integration target [Lechner et al., Mol. . Microbiol. 3, 621 (1989)] was prepared using a three step process. First, plasmid pSB210 shown in Figure 1 was constructed from pSB140 of Figure 2, described in more detail in Example 2, by inserting a multiple cloning site (MCS) between the EcoRI and HindIII sites of the plasmid. MCS was prepared by assembling the oligonucleotides KK14 and KK14B, the sequence of which is shown in Table 2 below, which was purified by oligonucleotide purification chromatography from Applied Biosystems according to the manufacturer's instructions.

• · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·–– ·

Table 2

Sequences of oligonucleotides

Designation Sequence (5 '-> 3') Hybridizing gene Sequence ID no.

PhosLo GGAACGCTACATACTAGTGATAGAGTAG phospholipase C 1 Phos4 GCTTGTACACCGCAACTGTTTTCGCATG phospholipase C 2 KK14B AGCTTGCGGCCGCGTCXjACCCCGGGCCATGGGGGCCG (MOS) 3 KK14 AATTCGGGCCCCCATGGCCCGGGGTCGACGCGGCCGCA (MCS) 4 GalPl CCACAGTTACAGTCTGTAGCrCAATTACC crylC 5 GaIP2 CCGCTACTAATAGAACCTGCACCA crylC 6 NHS20 CAATACATTATCCATGGAAAATTCCTCCTTAAATATCATG cryHA 7 NHS39 GAGCAATGAAAGAGTTAGGGCCCTGTTTAAGGTGTCATG cryHA 8 NHS42 GAGTGAATTATGGGGG cryHA 9 NHS43 ATTTTGTATTAAACGG cryHB 10 cryHA 1 ACTATTTGTGATGCGTATAATGTA cryHA 11 cryHA2 AATTCCCCATTCATCTGC cryHA 12 PG2 GAAATCGGCTCAGGAAAAGG ennC 13 PG4 CCTTAAAACATGCAGGAATTGACG ermC 14 PG5 CTATTGGTTGGAATGGCGTG CRMC 15 TYIAA GAGCCAAGCAGCTGGAGGAGTTTACACC cryIA (a) 16 TYIAC TCACTTCCCATCGACATCTACC cryIA (c) 17 TYIUN12 ATCACTGAGTCGCTTCGCATGTTTGACTTTCTC crvI-Type5 18 TY6 GGTCGTGGCTATATCCTTCGTGTCACAGC crvI-typc 19 ΤΎ13 ACAGAAGAATTGCTTTCATAGGCTC crvI-type 20 TY14 GAATTGCTTTCATAGGCTCCGTC crvI-typc 21 Tet3 CAACAAACGGGCCATAAGCTTGTATAAG bet 22 Tct4 GCCGTCTGTAACGGTACCTAAGG bet 23 CPOl.Rcv CACCCAGTTTGTACTCGCAGG bet 24

In step 2, the fos-C gene was inserted into pSB210. The fos-C region was amplified by PCR from total HD73 DNA using the Phos1 and Phos4 primer oligonucleotides described in Table 2 above. The PCR product was cloned into the SmaI site of plasmid pUC18 to give plasmid pSB139. The fos-C target region was isolated from plasmid pSB139 as a 2.2 kb blunt-ended Kpn1 / BamH1 fragment, which was purified from gel and ligated into plasmid pSB210, which had been previously purified with Msei and BamHl according to the manufacturer's instructions. The resulting plasmid, the structure of which is shown in Figure 3, was named pSB210.1.

• ·· ·· ·· • · «· · · · · · · · * ·· ·· • • · · ·« * ·· · · · · «· '·· · ^Λ

In the final step of -29Α, a crystal gene was introduced into the plasmid. Plasmid pSB210.2 contains the cryl-C gene on a 4.2 kb Apal / Not1 fragment derived from pSB619, which is described in detail in Example 3 below. Plasmid pSB619 was digested with ApaI and NotI and the 4.2 kb Apal / NotI fragment was isolated. The 4.2 kb Apal / NotI fragment was ligated into the plasmid pSB210.1, previously cleaved with the Apai and NotI enzymes, to generate plasmid pSB210.2, the structure of which is illustrated in Figure 4. Plasmid pSB210.3 contains the cry1C gene isolated from plasmid pSB013 as a 6 kb fragment (described in more detail in Example 4), which was isolated after digestion with ApaI and NotI. The 6 kb Apal / NotI fragment was purified by electroelution and ligated into plasmid pSB210.1, previously cleaved with Apai and NotI, to produce plasmid pSB210.3, the structure of which is shown in Figure 5. . Plasmid pSB3210.3 differs from plasmid pSB3210.2 in that the cry1C gene is located behind the crylIA promoter, and in pSB3210.2 the cry1C gene is under the control of the native cry1C promoter. In both plasmids, the cry1C gene is followed by the cryLAC terminator. Plasmid pSB147 was constructed as described in Example 5 below. This plasmid contains the phospholipase C region as an integration target, and also contains the cryIIIA operon and the ermC gene, which codes for erythromycin resistance. For the electroporation experiments, plasmid DNA isolated from dam ', dcnT, E. coli strain GM2163 was used (Woodcock, D.M., Nucleic Acids Slit. 17, 3469 (1989)].

· «« * * • * * * * *

Example 2

Construction of plasmid pSB140

Plasmid pSB901 was constructed to isolate the ermC gene, which encodes erythromycin resistance. Plasmid pSB901 was constructed as described by Monod et al. Monod et al., J. Bacteriol. 167, 138 (1981)], from the plasmid pM13 Bacilus subtilis, the ermC gene was isolated as a HindIII / ClaI fragment. The HindIII / ClaI fragment containing the ermC gene was ligated into pUC18, which was digested with Hindin and Steel. To replace the tetr gene in plasmid pBR322 with the ermC gene from pSB901, plasmid pBR322 was digested with Aval and treated with the Klenow fragment of E. coli DNA polymerase I and the linearized vector. we did it. Following treatment with the Klenow fragment, plasmid pBR322 was digested with HindIII and the large fragment resulting from the digestion was separated from the fragment containing the tetr gene. Plasmid pSB901 was first digested with SmaI and HindIII and the SmaI / HindIII fragment containing the ermC gene was isolated. The fragment containing the ermC gene was then ligated with the larger HindIII fragment of pBR322 to generate plasmid pSB140. The construction of plasmid pSB140 is outlined in Figure 2.

Example 3

Construction of plasmid pSB619

The cry1C gene isolated as an 8 kb EcoRI DNA fragment was cloned into the EcoRI site of the Lambda ZAP-II vector, purchased from Stratagene. The said gene was isolated from the USDA institute so that it was isolated from the USDA institute. ··· * * ♦

A plasmid preparation of the obtained Bacillus thuringiensis strain HD229 from Bacillus thuringiensis strain was digested with EcoRI and the resulting phage fragments were separated by gel electrophoresis and the phage fragments of approximately 8 kb in length were isolated from the gel. The cry1C gene may be prepared by the method of Honee et al. Honee, G., Van Dér Salm, T., and Visser, B. Nucl. Acids. Gap. 16, 6240 (1988)]. The cry1C clone was digested in two separate steps, first with HindIII and KpnI and in step 2 with KpnI and EcoRI. The digestion resulted in a 2.6 kb HindIII / Kpn1 fragment containing the N-terminal portion of the crylC sequence together with the promoter and a 2.3 kb Kpn1 / EcoRI fragment containing the C-terminal fragment. sequence and terminator. The crylC gene was reconstructed so that the 2.6 kb HindIII / KpnI ffagmen

It was ligated to the 2.3 kb Kpn / EcoRI fragment in pTZ19R vector from Pharmacia. A unique NcoI cleavage site was introduced into the cry1C translation translation site by genetic manipulation, and an additional EcoRI site was introduced after the stop codon. These restriction sites allowed the entire coding region of the cry1C gene to be isolated on a single continuous fragment. The 3.8 kb HindIII / EcoRI fragment containing the cry1C promoter and the entire coding region was then cloned into the pBluescript vector together with the 350 bp cryIA (C) terminator produced by pCR.

The cryIA (C) terminator was prepared by PCR using two of the following primer oligonucleotides synthesized based on the published sequence of crylA (C):

f Λ

-32first primer: gtctcatgcaaactcagg, sequence number: 25, second primer: ctctggcgctccatctac, sequence number: 26.

In the PCR reaction, a B.t. a cryIA (C) gene cloned from a strain of kurstaki HD73 was used. The PCR terminator was cloned into the Xba I site of the pBluescript KS + (Stratagene) vector after blunt-ended with the Klenow fragment and cloned in the same orientation as the T3 promoter. The HindIII / EcoRI fragment containing the cryI (C) promoter and coding region was then cloned into the HindIII and EcoRI sites of the pBluescript KS + vector. Cloning and sequencing of the cryIA (C) gene was performed by Adang et al. [Adang, M.J., Staver,

M.J., Roucheleau, T.A., Leighton, J., Barker, R.F. and Thompson, D.V .: Gene 36, 289 (1985)]. The resulting plasmid construct was named pSB619.

Example 4

Construction of plasmid pSB013

The B.t. kurstaki strain HD-1 was obtained from USDA. Total DNA was isolated from the HD-1 strain using the ASAP reagent kit according to Boehringer Mannheim instructions. The B.t. The 1800 bp DNA fragment containing the complete crylIA operon was prepared by PCR using kurstaki HD-1 total DNA. is shown in Table. Vent polymerase was used in the PCR reaction according to the manufacturer's instructions (New England Biolaps, Beverly, MA). The products of the PCR reaction were isolated from gel and ligated into HindIII-digested pTZ19R vector. A ♦ · ιί «··· t

··

The product of the -33 ligation reaction was used to transform competent E. coli DH5a cells. Transformants were selected on LB plates containing 75 pg / ml ampicillin (amp) and 40 mM Xgal. The plasmids contained in the transformants were analyzed by ApaI and NcoI digestion, and the orientation of the 1800 bp PCR fragment at the multiple cloning site of pTZ19R plasmid was determined by digestion with AflII. A plasmid containing the 1800 bp fragment in the desired orientation was named pSBOO9.

pSB070 is a plasmid similar to pSB619 which contains the cryIIIC coding region instead of the cry1C coding region. The construction of plasmid pSB070 can be achieved by using B.t. tenebrionis or B.t. the cryIIIA gene from san diego is described in Hermstadt, C. et al. cloned as described in Hermstadt, C., Gilroy, T.E., Sobieski, D.A., Bennett, B.D. and Gaertner, F.H., Gene 57, 37 (1987)].

The 3 kb HindIII fragment containing the cryIIIA gene was cloned into pTZ18R plasmid (Pharmacia). A plasmid was isolated in which the C-terminal end of the coding region of the cryIIIA gene was ligated to the multiple cloning site containing the EcoRI site. To clone the cryIIIA gene into pSB070, a unique Nco site was created by genetic manipulation using the ATG codon at the translational start site. After construction of the NcoI site, the cryIIIC coding region was excised from pTZ18R by digestion with NcoI and EcoRI and then cloned into plasmid pSB619, previously removed from the cryIIC coding region.

Both plasmid pSB009, which contained the crylIA operon fragment cloned into plasmid pTZ19R, and plasmid pSB070, which contained the crylAA coding region with the crylC promoter and crylAC terminator, were digested with both. The 5667 bp fragment of plasmid pSB070 and the 1800 bp fragment of plasmid pSB009 containing the crylIA operon were isolated. These fragments were ligated. Ligate-competent E. coli DH5oc cells were transformed and clones were selected on LB plates containing 75 pg / ml ampicillin at 37 ° C overnight. A plasmid was isolated from 12 clones and digested with AflII and NotI to identify the isolate containing the desired operon unit. The appropriate plasmid was named pSBO 10.

The cry1C gene was cloned into the crylIA operon unit of plasmid pSBO10. Plasmid pSB619 was constructed as described in Example 3 above. The full-length cry1C coding region was constructed by digesting pSB619 with NcoI, EcoRI and BglII and isolating the 3900 bp NcoI / EcoRI fragment. The vector containing the operon unit, pSBO10, was digested with NcoI and EcoRI and isolated. The 3900 bp cry1C fragment was ligated with the corresponding Nco I / Eco RI fragment of pSBO10. DH5a cells were transformed with the ligate and clones were selected on LB plates containing 75 pg / ml ampicillin. Plasmid DNA from 12 clones was analyzed by restriction digests (AflII + EcoRI and AflII + BglII). The plasmid containing the full-length cry1C gene, 3 'to the crylIA operon, was designated pSBO13.

• · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · speaking · fre & parking ▼

Example 5

Construction of plasmid pSB304

Plasmid pSB304 was constructed so that B.t. The crylIA operon from strain galleriae HD232 was cloned. The B.t. strain galleriae HD232 was obtained from USDA. In the process of cloning the operon, B.t. DNA isolated from galleriae HD232 cells was digested with HindIII and the approximately 5 kb fragments were purified by gel electrophoresis. Gel-purified fragments were ligated into HindIII-digested plasmid pTZ18R (Pharmacia) and transferred to E. coli DH5oc cells. Clones containing the crylIA operon were identified by hybridization with a crylIA-specific oligonucleotide (CCCATGGATAATGTATTGAATAG TGGAAG, SEQ ID NO: 27). A plasmid was selected which contained the crylLA and lacZ genes in the same orientation. Plasmid DNA was isolated from the selected clone, and the BamHI fragment containing the unwanted 5'-end sequences of the crylIA operon was removed from the plasmid to generate plasmid pSB304. Further information on the sequence of the crylIA operon and its 5 'end region can be found in Widner et al. Widner, W.R. and Whiteley, H.R., J. Bacteriol. 171, 965 (1989)].

Example 6

Construction of plasmid pSB147

Plasmid pSB140 was constructed as described in Example 2. In the next step, the cryIIIA operon was incorporated into pSB140. The cryIIIA operon was isolated from pSB304 plasmid prepared as described in Example 5 above. Plasmid pSB304 is a B.t. galleriae strain HD232

Contains a crylIA operon from -36 cloned on a BamHI / HindIII fragment inserted into pTZ18R. The plasmids pSB30 and pSB140 were digested with EcoRI and HindIII. The large pSB140 fragment was isolated, and the EcoRI / HindIII fragment containing the crylIA operon was ligated with the large pSB140 fragment to give plasmid pSB141.

In the next step, an integration target sequence was introduced into the vector. The target sequence was a DNA fragment containing B.t. from the USDA Institute. phosphatidylinositol-specific phospholipase C gene (plc) from strain HD73 of kurstaki. This DNA fragment was prepared using the polymerase chain reaction using total DNA isolated from strain HD73 as a template. The B.t. Total DNA was isolated from kurstaki HD73 strain using the ASAP reagent kit according to Boehringer Mannheim.

ATCC 10792 Registered B.t. The DNA sequence of the strain plc region was retrieved from the GenBank database accession number XI4178, which was sequenced by Lechner et al. Lechner, M., et al., Mol. Microbiol. 3, 621 (1989)]. The one in question

The 2254 base pair sequence fragment contained, in addition to the pcl gene, a 454 base pair sequence fragment 5 'to the gene and an 810 base pair sequence fragment 3' to the gene. The two PCR primers, Phos1 and Phos4 primers described in Table 2, were designed to hybridize to the plasmid flanking regions. Using these primer oligonucleotides, a polymerase chain reaction was performed on the total HD73 template, thereby generating a 2.2 kb DNA fragment.

-37 containing the plc region of the HD73 strain. The resulting PCR product was treated with the Klenow fragment of E. coli DNA polymerase I and then cloned into a SmaI-cleaved pUC18 vector to produce plasmid pSB139.

Plasmid pSB139 was digested with KpnI and BamHI and the plc region was separated from the vector sequences. Plasmid pSB141 was also cleaved with KpnI and BamHI and the resulting fragments were ligated with the fragment containing the isolated plc region. The resulting construct, plasmid pSB141.5, contained the pBR322-derived portion of plasmid pSB141 as well as the plc region from plasmid pSB139.

In the next step, plasmids pSB141 and pSB141.5 were used to construct a plasmid containing the ermC gene, the plasmid and the cryIIIA operon. Plasmid pSB141.5 was digested with BamHI. Plasmid pSB141 was digested with BamHI and the ffagmen containing the crylIA operon and the ermC gene was isolated. The cryIIIA / ermC-BamHI fragment was ligated with the linearized plasmid pSB141.5, thereby generating plasmid pSB147. A map of plasmid pSB147 is shown in Figure 6 and a schematic diagram of the construction of plasmid pSB147 is shown in Figure 2.

Example 7

Introduction of hybrid plasmid into B./. cells by electroporation

The crystal genes were incorporated into B.t. cells by introducing the respective plasmids into the cells by electroporation (electrotransformation). The plasmids did not contain a gram-positive origin of replication that would have been necessary for their replication in cells.

-38 Instead, the plasmids contained a DNA region that functioned as target sequences during integration into the chromosome, the Phos-Region; they also contained a selectable marker gene that conferred cell erythromycin resistance. The plasmid, which was electroporated at high concentrations into cells, was forced into the chromosome by a single cross-over event, which resulted in a duplication of the target sequence.

Chromosomal integrants were prepared from strain HD73 using the procedure described above using plasmids pSB147 and pSB210.2. However, the other strains tested were resistant to electroporation and therefore could not be transformed with such high efficiency that chromosomal integration can be induced. Competent cells were prepared by inoculating 100 ml of 0.5 M sucrose cerebral heart infusion medium (BHIS, Difco) with disposable white pigs with the appropriate cells and inoculating fresh LB plates incubated overnight. Cells were cultured in 1L culture flasks containing shuttle at 37 ° C and shaken at 300 rpm, measured at 600 nm to an OD = 0.2, after which the cells were kept on ice. All wash solutions were used cooled. The cells were then transferred to 250 ml sterile flasks and pelleted for 7 minutes at 6000 rpm, and the cell pellet was then washed with 1 x 1 volume followed by 2 x 0.1 volume of 0.5 M sucrose. , 5 mM HEPES, pH 7. The pellet was then resuspended in a final volume of 10 ml of HEPES-sucrose solution. Freshly prepared competent cells were used to electroporate the integrating plasmids.

• ·

Plasmid DNA was mixed with -39200 μΐ competent cells. For HD73 cells, the following heart rate parameters were used: kV = 1.25, pF = 3, and Ω = οο. After pulse extraction, cells were transferred to 5 ml BHIS medium in a 125 ml flask and shaken at 250 rpm for 3 hours at 30 ° C for regeneration. For samples for which vector integration was desired, cells were pelleted by centrifugation at 7000 rpm for 5 minutes and plated on 10 µg / ml erythromycin LB medium.

The efficiency of the electroporation procedure was checked with plasmid pSB098 DNA as a control plasmid. PSB098 is a shuttle vector containing plasmid pTZ19R (Pharmacia) on the one hand and pBCl0.1 on the other. PBCló.l is a vector of B. cereus originated by Kreft, J. Kreft, Mol. Gene. Genet. 162, 59 (1978)]. Plasmids pTZ19R and pBC16.1 were digested with EcoRI. The linearized plasmids were ligated into a plasmid to produce plasmid pSB098, which has opposite polarity genes for ampicillin and tetracycline resistance.

Compensated cells from strain HD73 were electrotransformed using plasmid DNA isolated from dam strain dcmGM2163. The results of the transformation experiments are shown below

Table 3 summarizes. In addition to the data presented, strain HD1-51 was transformed with plasmid 210.1 (experimental data not shown).

· ····· ·················································•

Table 3

Transformation efficiency of B. thuringiensis kurstaki strain HD73

plasmid Quantity (Ug) Quantity Transfectants Efficiency (transformant / pgpSB098DNS) (Μΐ) Strain HD73 number pSB210.2 9 10 3 3.5x10 ⁵ pSB147 15 10 8 2x10 ⁶

The efficacy value in the last column of the table was determined in each experiment by electroporation of 0.51 pg pSB098 DNA and calculated the number of colony forming units per pg DNA. Based on the value obtained, it was possible to estimate how the cells reacted to the given electroporation conditions. No transformant was generated with plasmid pSB210.3.

The B.t. kurstaki HD7 3 strain was obtained from ÜSD A (cultures of bacillus thuringensis are available from the U.S. Department of Agriculture, USDA / ARS Agricultural Reviews and Manuals: ARM-S-30, October 1982).

The transformants produced, also referred to herein as recombinants or transfectants, were checked by PCR for the presence of the genes to be introduced. HD73 recombinants containing pSB210.2 sequences (HD73 :: pSB210.2) were screened for the presence of the cry1C gene using the galpl and galp2 primers, and for the presence of the ermC gene, the PG2 and PG4 primers were used. oligonucleotides applied- • 9 · · · · · · · · · · · · · · · · · · · · · · · · · ·

-41 lbs. Of the 8 HD73 :: pSB147 clones tested, two were detected using the cryIIA-I and cryIIIA-II primers to show the presence of the crylIA gene and the presence of the ermC gene using the PG2 and PG4 primers. The sequences of the primer oligonucleotides are given in Table 2 above. Regarding the plasmid profile, the recombinant HD73 :: pSB210.2 was identical to the parental HD73 strain.

Example 8

Preparation of phages for packaging a hybrid plasmid in a phage head

Phage CP51 was obtained on a filter paper disk impregnated with infected spores of B. cereus strain 569 according to Thome (1978) supra. The strain was resuscitated by inoculating 25 ml of NBY medium containing 0.4% glycerol (8 g of Difco Nutrient broth, 3 g of Difco yeast extract) (NBYG) with one of the disks and 37 ° C. C was cultured for 16 hours. The culture was harvested, the cell debris pelleted at 10,000 rpm, the phage lysate sterilized by passing through a 0.45 µm filter and stored at 16 ° C.

The titre of the lysate was determined by assay against the phage-free isolate of strain 569. The lysate was diluted 10 times and 100 times in 1% peptone. About 10 ⁶ cells from strain 569 were mixed with 100 μΐ fághigítással and was administered 2 ml of TBAB ( "Difco Tryptose blood agar Base") soft agar. This was applied to Phage Assay Plate (PA) plates (per liter: 8 g Difco Nutrient broth, 59 g NaCl, 0.2 g MgSO ₄ x 7H ₂ O, 0.05 g MnSO ₄ x H ₂ O, 0.15 g CaCl ₂ x2H ₂ O, pH 5.9-6.0) layered, which were dried overnight at room temperature. Plates were incubated overnight at 30 ° C and plaques were counted.

To test for susceptibility to phage infection, approximately 10 ⁶ cells from each strain tested (SA11, SA12, S287 and HD73) were inoculated * ♦ 9 ···

-42 layers for PA sheets. The surface of the agar was gently touched with a dots of phage strain culture and the plates were incubated at 30 ° C. on the following day, clarification of the cell layer was observed in all four strains.

Example 9

Propagation of phages

6 ml of LB medium was inoculated with fresh HD73 :: pSB210.2 cells from overnight culture plate in a 20 mm tube. The culture was grown at 37 ° C for 4-6 hours, the optical density was determined, and the cells were diluted with LB to a concentration of 3x10 ⁶ cells / ml. NBYG plates were overlaid with 4 ml lágyagarral NBY-equilibrated with 0.5 ml containing 4x10 ⁶ CP51fágtörzstenyészetet plaque forming units (PFU), and either 1 xlO ⁶ or 3x10 ⁶ cells were added. The plates were incubated overnight at 30 ° C and the phages were collected in 5 ml of PA medium. The upper agar layer was scraped into PA medium and placed in 18 mm plastic tubes. Cell debris was pelleted. The lysate, designated CP210.2, was sterilized by passing through a 0.45 μιη filter and stored at 15 ° C.

To determine the titer, 5x10 ⁶ CFU (colony forming units) from HD73 strain were mixed with 100 μΐ dilutions of 10 ′ ² and 10 ^-4 dilutions of CP210.2 lysate and poured onto PA plates as a top coat with 2 ml NBY. After overnight incubation at 30 ° C, 1,200 plaques were observed on the plate containing 10 ^-4 dilutions, resulting in a titre of 1.2 x 10 ⁸ PFU / ml.

4 · ν · ♦ · • 4 · * ····

Example 10

Definition of conditions used in transduction

The methods used to treat phages were based on the method described by Thome (1978), supra. For each B / strain, the number of colony forming units (CFU) per milliliter and the titer of phage stock cultures in milliliter plaque forming units were determined using serial dilutions.

The titer of the CP51 phage stock was determined as follows. A volume of 0.1 ml of phage diluted in 1% Peptone and approximately 2 x ^{1 1} spore of B. cereus strain 569 were added to 2 ml of PA soft agar and the inoculated soft agar was plated onto PA agar plates. The supernatant plates were incubated at 30 ° C for 16-20 hours. Plaques were counted and the titer of phage strains in PFU / ml was calculated based on the dilutions used. Cell concentrations of Bt cultures were determined by standard methods in the art. The results are summarized in Table 4 below.

Table 4

The number of colony forming units (CFU) of ff./.cells and

determination of phage stock titer (PFU) CFU CFU / ml B. cereus strain 569 5x10 ⁷ B. thurmgiensisAöxzs HD73 8.6x10 ⁸ SA11 and SA12 ⁸ 2,6xl0 S287 Ι, ΙχΙΟ ⁷ PFU PFU / ml CP51: 8x10 ⁶ CP210.2: l, 2xl0 ⁸

Example 11

Construction of plasmid pSB136

Plasmid pSB136 (see Figure 7) is an integration vector that facilitates insertion of the cry1C gene into the Bt chromosome. The pSB136 vector contains the cry1C gene, an integration target sequence, a tetracycline resistance gene, and part of the pBR322 vector. The Bt aizawai HD229 crylC gene and the pBC16-1 B. cereus tetracycline resistance gene (batch ¹ ) were cloned. The integration target sequence was a known DNA fragment from the Bt kurstaki HD1 cryB chromosome.

Plasmid pSB136 can be used to insert the cry1C gene into any chromosome B strain that is not tetracycline resistant. However terminal going to the integration, the recipient-strain must have sequences homologous to the integration target and the strain must be efficiently traszformálni (DNA used for transformation pg 1> = 10 ⁴ transformant must reach).

Competent E. coli - DH5α cells were prepared according to Alexander's method (Alexander, 1978, supra). Transformation was performed using techniques well known in the art using "Library Efficiency DH5a Competent Cells" cells (Gibco, BRL, Life Technologies, Inc., Gaithersburg, MD).

Transformants were selected on LB medium containing 75 pg / ml ampicillin. Restriction enzyme digestions, ligations, ethanol precipitation, phenol extractions, kinase reactions, and treatment of DNA with T4 DNA polymerase, bovine intestinal phosphatase, and the E. coli DNA polymerase I Klenow fragment are described in Maniatis et al. [Maniatis T. et al., Molecular

-45 Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982).

B.t., described by Stahly et al. kurstaki HD1-crylB is an HD1 strain from which the plasmid has been removed ("plasmid cured") [Stahly

D. P. et al., Biochem. Biophys. Gap. Comm. 84, 581 (1978)]. The B.t. Total DNA from kurstaki HD1-crylB strain was isolated according to the following procedure. 200 ml of 2xTY medium was inoculated with B.t. strain and incubated at 30 ° C overnight at 200 rpm with shaker mixing. 200 mL of 2xTY medium was inoculated with 2 mL of the above culture and incubated at 300 rpm with shaker at 30 ° C. When the optical density of the culture at 600 nm reached an OD = 0.8-1, the cells were harvested by centrifugation at 10,000 rpm for 5 min at 4 ° C. Cells were washed with TES buffer (TE + 100 mM NaCl) and resuspended in 18 ml buffer containing 25% sucrose, 25 mM Tris-HCl, pH 8 and 25 mM EDTA. . To the sucrose solution was added 2 mL of 10 mg / mL lysozyme and mixed gently. The mixture was incubated at 37 ° C for 30-60 minutes and assayed for the presence of protoplasts. To the mixture was added 2.2 mL of 20% SDS, mixed gently and incubated for 15 minutes at 50 ° C. To this was added 5.5 mL of 5 M NaCl, mixed gently and incubated for 5 minutes at 50 ° C and then overnight at 4 ° C. The mixture was centrifuged at 10,000 rpm for 10 minutes at 4 ° C and the supernatant was poured into 2 tubes. Cold EtOH (28 mL, 56 mL total) was added to the tubes, mixed gently, incubated overnight at -20 ° C and centrifuged at 13,000 rpm for 30 min. The precipitate was recovered and washed with 70% EtOH. The precipitate was then dissolved in 10 mL (20 mL total) of TE buffer containing 1 M NaCl and incubated overnight at 4 ° C. Subsequently, the contents of the two tubes were placed in one tube. To the tube was added 200 μΐ of RNase at 1 mg / ml and

1200 μΐ of 10 mg / ml proteinase K was added and the mixture was centrifuged. The aqueous layer was collected and washed twice more with 20 mL of phenol / chloroform. To the aqueous layer was added chloroform (20 mL) and centrifuged. The aqueous layer was collected and dialyzed overnight against 2000 mL of TE buffer.

Plasmid DNA used for cloning was isolated from E. coli cells by alkaline lysis [Bimbomim H.C. and Doly, J., "Rapid Alkaline Extraction Procedure for Screening Recombinant Plasmid DNA", Nucl. Acids Slit. 7, 1513-1523 (1979)] or using Quiagen columns purchased from Quiagen Inc.

Plasmid pSB136 was constructed in four steps (refer to Figure 7). In the first step, the tetracycline resistance gene (which is functional in E. coli) from the plasmid pBR322 was replaced by the Tac gene functional in Bacillus. Next, an integration target sequence was added to the plasmid, which was a DNA fragment with unknown function isolated from the HD1-cryB genome. In the third step, a NotI linker was added to the plasmid to facilitate cloning of the cry1C gene. In the final cloning step, the cry1C gene was added to the integration vector. Each of the above steps is described in detail below.

Plasmid pSB206 was constructed by constructing the tet ^r gene from pBC16plamid (Bemhard K. et al., J. Bacteriol. 133, 897 (1978)] was cloned into pUC18. Plasmid pBC 16-1 is described by Kreft et al. (Kreft J. et al., Mol. Gene. Genet. 162, 59 (1978)], was constructed from plasmid pBC16 by removing an EcoRI fragment. The tet ^r gene was isolated from plasmid pBC16-1 using the polymerase chain reaction procedure using the Tet3 and Tet4 primer oligonucleotides described in Table 4 above. The Tet3 primer inserted a HindIII site five downstream of the tet ^r gene, the Tet4 primer.

And -47 oligonucleotide inserted a KpnI site 3 'to the tef gene. The PCR product was inserted into plasmid pUC18 at the HindIII site at the multiple cloning site.

Plasmid pSB206 was digested with SmaI and HindIII to remove the tet ^r gene. To remove the tef gene from pBR322, this plasmid was digested with Aval, treated with the Klenow fragment of E. coli DNA polymerase I to make blunt ends, and then digested with HindIII. The desired fragments were purified and the pBR322 vector was ligated with the tef gene derived from plasmid pSB206 to give plasmid pSB131.

Subsequently, an integration target sequence was added to pSB131. The source of the integration target sequence is isolated from the HD1-cryB genome,

It was a 1.1 kb DNA fragment. This ffag fragment was cloned into pUC18 and the construct was designated pSB132. The 1.1 kb DNA fag fragment from the HD1-cryBgenome was isolated as follows. Total DNA from HD1-cryB strain was digested with HaelII or EcoRV, the two digested mixtures were mixed and subjected to electrophoresis on a 0.8% agarose gel. Three size fractions were excised from the gel: (1) 0.5-0.9 kb; (2) 0.9-1.8 kb; and (3) fractions corresponding to 1.8-2.7 kb.

DNA fractions (1) and (2) were purified. The DNA from fraction (2) was ligated into SmaI digested pUC18 and the resulting clones were characterized by Eco RI / Hind III digestion. Plasmid pSB132 is one

It contained a 1.1 kb insert.

The 1.1 kb cyrB fragment was then transferred from pSB132 to pSB131. Plasmid pSB132 was digested with EcoRI,

It was filled up by treatment with the Klenow fragment of E. coli DNA polymerase I and digested with HindIII. Plasmid pSB131 was Sspl- and • · · · · · · · · · · · · · · · ··· ·

Digested with -48HindIII and purified from plasmid pSB132,

It was ligated with a 1.1 kb fragment to construct pSB134.

A NotI linker was added to pSB134 to facilitate the insertion of the cry1C gene. The NotI linker had the sequence pAGCGGCCGCT (New England Biolabs # 1125, SEQ ID NO: 28). Plasmid pSB134 was digested with BamHI and blunt-ended by treatment with the Klenow fragment of E. coli DNA polymerase I. Next, at a 200: 1 molar ratio, the NotIII linkers were ligated into the linearized plasmid pSB143 and the resulting construct was designated pSB134.5.

In the final step, the cry1C gene was added to pSB134.5. The cry1C gene was derived from plasmid pSB619, described in Example 3 above. Plasmid pSB619 carries the B.t. a crylC gene derived from aizawai strain HD229, preceded by its native promoter and B.t. kurstaki HD73 cryIA (c) terminator sequence. The crylC gene, together with the promoter and terminator, was cloned as an Apal / NotI unit in Bluescript KS (+). To isolate the cry1C gene, plasmid pSB619 was digested with Apai, filled with T4 DNA polymerase and digested with NotI. Plasmid pSB134.5 was digested with EcoRI, digested with treatment with the Klenow fragment of E. coli DNA polymerase I, then digested with NotI, purified from the vector portion of pSB619 and ligated into pSB134.5. plasmid pSB136.

-49 ♦ ··· · ·· · «• · · ♦ ···» »·········» ♦

Example 12

Delivery of the crylC gene B.t. kurstaki HD1-cryB and HD73 strains

Plasmid pSB136 is all. Prepared in the same manner as described above in Example 3a. This plasmid contains the tef gene from plasmid pBCl6 and the DNA fragment of unknown function of the HD1-cryB chromosome as an integration target. These fragments were ligated into pBR322.

The competent B.t. kurstaki HD1-cryB and HD73 cells were prepared according to the BHIS procedure described in Example 6 above. Following the electrical pulse, cells were transferred to 5 ml of BHIS medium for 3 hours at 37 ° C for regeneration. For HD1-cryB cells, the following heart rate parameters were used: kV = 1.05, pF = 25, and Ω = οο. For HD73 cells, the following heart rate parameters were used: kV = 1.25, pF = 3, and Ω = οο. Following electroporation, the whole culture was pelleted, resuspended in low volume medium and plated on selective medium. Plasmid DNA pSB098 was used as a standard to determine the efficiency of the transformation, and the efficiency was expressed as the number of colony forming units (CFU) per plasmid pSB098 in each experiment.

For HD1-cryB cells, when the cells were electroporated with 7.5 pg of pSB136, a tetracycline-resistant colony was obtained. The overall transformation efficiency of the cells was 6x10 ⁵ CFU / pg (using plasmid pSB098). This new recombinant strain CryB :: pSB136 was detected by PCR analysis as described in Example 13 to include the tetracycline resistance gene and the cry1C gene.

The CryB :: pSB136 strain was grown in CYS medium until sporulation. This culture contained 4x10 ⁸ spores per milliliter, for comparison, in the same experiment, the HD1-CryB strain per milliliter

-505x10 contained ⁸ spores. The tetracycline resistance gene was found to be 98% stable during sporulation and conversion to the vegetative form.

For HD73 cells, when cells were electroporated with 15 µg of plasmid pSB136, two colonies were obtained in an experiment in which the cells had an overall transformation efficiency of 2x10 ⁶ CFU / µg DNA. Both colonies, designated HD73 :: pSB136, were positive for the crylC gene and the tetracycline resistance gene by the PCR assay described in Example 13 below.

Example 13

PCR assay for CryB :: pSB136 and HD73 :: pSB136 recombinant strains

The presence of the introduced cry1C gene and tetracycline resistance marker in the recombinant strains CryB :: pSB136 and HD73 :: pSB136 was confirmed by PCR. Cells from fresh overnight culture plate for 10 minutes in 8 µl of 1x Taq polymerase buffer with the required primer oligonucleotides (0.5 µl, 20 gm stock solution) and dNTP (1.6 µl, 1.25 in a stock solution containing 1 mM). The cell debris was pelleted and 0.05 units of Taq polymerase in 2 µl, 1x Taq polymerase buffer were added to the tube. Two primer combination oligonucleotides were used to detect the presence of the tetracycline resistance gene. The combination of Tet3 and Tet4 is an approx

A 1.4 kb fragment was obtained, and the combination of Tet3 and CPO1.Rev resulted in a fragment of about 0.35 kb. For the detection of the cry1C gene, the primers galP1 and galP2 were used to obtain a 0.8 kb fragment. The sequences of the primer oligonucleotides are shown in Table 2 above.

Example 14 • ·

-51 Delivery of the crylC and crylIA genes B.t. kurstaki strain HD73

Plasmid pSB304 was prepared as described in Example 5 above. The crylIA gene was isolated from plasmid pSB304 as a NotI-EcoRI fragment. Plasmid pSB134.5 was constructed as described in Example 10 above. The cryIIIA NotI-EcoRI fragment was ligated from pSB134.5.2 into NotI and EcoRI digested pSB134.5.

Competent HD73 cells were prepared according to the BHIS procedure described in Example 6 above. Following the electrical pulse, the cells were regenerated in 5 ml of BHIS medium for 3 hours at 37 ° C. For HD73 cells, the following heart rate parameters were used: kV = 1.25, pF = 3 and Ω = ° ο. Following electroporation, the whole culture was pelleted, resuspended in a small volume of medium and plated on a selective medium. To determine the transformation efficiency, as described in Example 12, plasmid pSB098 DNA was used as a standard and the transformation efficiency was expressed as 1 pg CFU per plasmid pSB098.

Electroporation of HD73 cells with 20 pg of pSB134.5.2 resulted in ten colonies. Transformation efficiency was 1x10 ⁶ CFU / pg DNA. Two of the transformants were positive for tetracycline resistance and cryIIIA genes by the PCR assay described in Example 15 below. The two transformants were designated HD73 :: pSB134.5.2.

Example 75

PCR analysis of the recombinant strain HD73 :: pSB134,5,2

The presence of the introduced crylIA gene and tetracycline resistance marker in the HD73 :: pBS134.5.2 recombinants was confirmed by PCR as described in Example 13. For testing on the crylIA gene, crylIAl and • ·

-52cryIIA2 primer oligonucleotides were used to obtain an euy 0.57 kb fragment. The sequences of the primer oligonucleotides are given in Table 4 above.

Example 16

B. t. -transductions a

In order to transfer the crystal gene integrated into the chromosome of a strain into generalized transductions of a strain which is not easily transformed by electroporation. In these cases, a strain containing an integrated crystal gene, HD73 :: pSB210.2, was used as a DNA donor for transduction of several B./. Strains including SA11, SA12 and S287.

The generalized transduction procedure is widely used for the exchange of genetic information, has been well characterized for Margolin E. coli and typhimurium 5, and according to Thome, occasionally Bacillus thuringiensisno [Margolin: "Escherichia coli and Salmonella typhimurium", Cell. and Mole. Biol. (1987); Thome, "Transduction in Bacillus thuringiensis", Applied and Enviromental Microbiology, 35, 1109 (1978)].

The phage CP51 used in the generalized transduction procedure, isolated from soil and used in experiments to determine the chromosomal map of Bacillus cereus, was obtained from C. B. Thome [Thome

C.B .: "Transducing Bacteriophage For Bacillus Cereus", J. of Virol., 2, 657 (1968); and "Transduction of Bacillus Cereus and Bacillus Anthracis", Bacteriological Reviews 32, 358 (1968)]. Thome et al. Also tested the phage against other Bac / 7 / w.s strains, including B. thuringiensis (see above, Thome (1978)).

Cells derived from SAH, SA12, S287 and HD73 strains a

Cultured according to the procedure described in Example 8 and diluted to about 10 ⁷ CFU / ml.

Isolate 2 of the recombinant strain HD73 :: pSB210.2 was used for propagation of CP51 phages. The titre of the resulting lysate, called CP210.2, was determined and measured in 1.2 x 10 ⁸ plaque forming units (PFU / ml) per milliliter. A sterile HA filter (Millipore) was placed on the surface of an LB plate, and 100 µΐ of each of the phage lysates and cells of CP210.2 was pipetted onto the filter, and the liquid was gently dispersed using a sterile wire yoke. The plates were incubated for 3 hours at 37 ° C. The filters were transferred to LB plates containing erythrochromycin (10 pg / ml), returned to 37 ° C and allowed to multiply for 36 hours. The results of plate transduction experiments are summarized in Table 5 below.

Table 5

Results of transduction experiments

Tribe Quenched CFU Infection multiplicity Ery ^r colonies Efficiency HD73 lx10 ⁶ 10 2 l, 6x10 ' ⁷ SAH 4x10 ⁶ 3 4 3x10 ' ⁷ SA12 4x10 ⁶ 3 2 l, 6x10 ' ⁷ S287 lx10 ⁶ 40 3 2xl0 ^'8

Efficacy was expressed as the number of erythromycin resistant colonies per plaque forming unit. The strains HD73, SA11 and SA12 were vaccinated with 1.2 x 10 ⁷ plaque forming units. For strain S287, 4x10 ⁷ PFU were inoculated.

Example 17

Investigation of recombinant strains by polymerase chain reaction

PCR of each recombinant strain was performed using whole cells as described in Example 13. Erythromycin-resistant colonies, designated by the abbreviation "CP," were analyzed for gene content by PCR and compared with the wild-type strain. The results are summarized in Table 6 below.

Table 6

Gene content of transduced strains by PCR analysis

Tribe IA (a) IA (b) IA (c) IF IC ermC SA11WT + + + + - - SA11CP1 + + + + + + SA11CP2 + + + + + + SA11CP3 + + + + + + SA11CP4 + + + + + + SA12WT + + + + - - SA12CP1 + + + + + + SA12CP2 + + + + + + S287WT + + + + - - S287CP1 + + + + + + S287CP2 + + + + + +

WT = wild-type

The recombinants retained the crystal gene pool in the parent strains. Only the recombinant strains were positive for entry.

-55 specific primers specific for the cry1C and ermC genes. In the cryIA (b) gene assay, the TY6 and TY14 probes were first used (see Table 2 above), but this pair gave some degree of cross-reaction with the control plasmid pSB210.2, which contains only the cry1C gene . In subsequent experiments, TY14 primer oligonucleotide was replaced with TY13 as described in Table 2 above.

Example 18

Wild-type and recombinant ff. /.- Comparison of plasmids from strains

The plasmids from the recombinant B./. Strains were isolated by the alkaline lysis procedure of Bimbomim and Doly (1979, supra), modified as follows. The strains were plated onto tetracycline containing LBlemezekre, and incubated overnight at 30 ° C, cultured, and then fresh SA plates [lxSpizizen salt, 1% casamino acids, 5% glucose, 0.0005 mmol / 1 MnSO ₄ H ₂ O) was inoculated and cultured for 4 to 4 hours at 37 ° C. For each strain, 2-3 spiked cells were suspended on ice in 100 μΐ TESL buffer (100 mM Tris pH 8, 10 mM EDTA, 20% sucrose, 2 mg / ml lysozyme) and for 15 minutes, Incubate at 37 ° C. To the mixture was added 200 μΐ lysis solution (0.2 N / 1 NaOH, 1% SDS), mixed by gently tilting the tubes and incubating for 5 minutes at room temperature. To the tubes was added 150 μΐ of ice-cold potassium acetate solution and the mixture was mixed by tilting the tubes. The solution was then centrifuged at 15,000 rpm for 20 minutes at 4 ° C. The supernatant was recovered using a wide-lumen disposable transfer pipette, then added to 1 ml of 100% ethanol and mixed by tilting the tube. The mixture was then centrifuged at 15,000 rpm for 20 minutes at 4 ° C. The supernatant was removed by aspiration and the pellet was suspended in 1 ml of 70% ethanol.

-56 · · · · · · · · · · · · · ································································································ed. , centrifuged at room temperature. The supernatant was removed by suction and the pellet was dried under vacuum in a Speedvac for 2 minutes. The dried pellet was resuspended in 20 μΐ TE buffer, incubated on ice for about 15 minutes, and mixed gently by tapping the side of the tube.

The DNA was electrophoresed in 1xTAE buffer, 0.8% agarose, U = 70V and A = 32 mAmp for 3 hours. The plasmid profile of the recombinants was found to be the same as that of the corresponding wild-type parental strains, thus confirming that the recombinants do not carry unwanted plasmids.

Example 19

Comparison of the chromosomal DNA of wild-type and hybrid A./.

Chromosomal DNA in the integrants generated in the HD73 strain, in the transduced strains generated in the strains SA11 and SA12, and in the HD7 3 strain were analyzed in DNA-DNA hybridization experiments. Three ffagenes used as plasmid hybridization probes were isolated from pSB139:

(a) a generic phospholipase C (fos-C) hybridization probe, which overlaps the known fos-C sequence from the BamHI site to the Clal site at 1800 base pairs;

(b) a left Eco (E _L ) hybridization probe covering an 850 base pair sequence from the BamHI site to the EcoRI site;

(c) a better Eco (E _R ) hybridization probe, which is a 1427 base pair Eco RI fragment.

It contains chromosomal DNA from the wild-type strain HD73. Example 1 was isolated from 100 ml samples of culture in 2xTY medium (5 g yeast extract, 5 g tryptone, 2.5 g NaCl).

Chromosomal DNA was isolated from wild-type strains SA11, SA12 and HD73 as well as the corresponding transduced strains using the ASAP reagent kit available from Boehringer Mannheim according to the manufacturer's instructions. The chromosomal DNA was completely digested with EcoRI or Apaienz, separated in EtEbr-containing TBE buffer, 0.8% agarose gel, purified, denatured, neutralized, and then as described by Sambrook et al., Molecular Cloning. : Laboratory Manual, "Cold Spring Harbor Laboratory Press" (1989)] was transferred to "HyBond" nylon membranes overnight by capillary blotting in 20xSSC buffer. The DNA was fixed to the membranes by treatment with 0.4 M NaOH for 20 minutes. Southem hybridization was performed using the Amersham ECL kit according to the manufacturer's instructions.

Eco RI-digested DNA hybridization analysis of HD73 and HD73 :: pSB210.2 strains revealed an expected 2.4 and 4.3 kb from the pSB210.2 integration vector. size internal fragments but did not clearly detect chromosomal regions on either side of the integration site or flanking regions. The large variation in the intensity of the inner vector strips and later stripes, which proved to be flanking regions, suggested that multiple integration events were likely to occur. Further analysis of the same filter by the E _L hybridization probe, not only on the expected internal Eco RI fragments, but also on the chromosomal DNA of integrants and wild-type samples.

It also showed the presence of a 1.8 kb band. These results show

-58 that they are located 5 'to the EcoRI site in the fos-C gene

At 1.8 kb, there is an EcoRI cleavage site. Hybridization with the ER probe detected the presence of internal fragments in the samples from the integrants and the presence of an approximately 9 kb band in both the integrant and wild-type DNA, thereby identifying the 3 'downstream site of the fos-C internal EcoRI site. found the following EcoRI site.

The presence of identical EcoRI bands 5 'and 3' downstream of the fos-C EcoRI cleavage site in both wild-type and integrant strains demonstrates that incorporation was as expected in the chromosomal region corresponding to the phospholipase C gene.

Southem blot analysis of similar Apa-digested DNA samples confirmed that multiple integration occurred. When analyzing chromosomal DNA from an isolate that generated only one integration event, we would see only two bands, both wild-type 5 'or 3' downstream of the Apai cleavage site in the integration vector pSB210.2, would be up to the sequence of the Apa cleavage site. Multiple integration events result in the same 5'and 3'bands, and an additional inner band corresponding to the DNA between the Apai cleavage sites delivered by two tandem integrated vectors.

South blot analysis showed the presence of a 10.4 kb fragment, which corresponds to the total length of the integrated plasmid, suggesting multiple integration events in the region. However, if more than two integration events occurred, the flanking Apai chromosomal fragments were not observed and

-59meghatározhatók. Hybridization with probes by Southern blot analysis from SA11CP1,-SA11 CP2, SA12CP1 and SA12CP2 transduced strains, digested with EcoRI DNA E _L and E _R also showed the presence of 2.4 and 4.3 kb internal EcoRI bands, confirming that the DNA carried by the transducing particles was derived from the desired integrated fos-C site. These inner bands were not visible in the bands containing the wild-type SA1 and SA12 DNAs. The probes also hybridized to flanking bands of the same size, namely 1.8 kb to about 9 kb, as in the wild-type strains HD73 and HD-51 and their respective integrants. The strains SA11, SA12, HD73 and HD-51 have similar fos-C regions. The exact number of "cross-ovcr" events cannot be determined with these tests.

Example 20

Stability and viability of hybrid /7/.- strains

The stability of the recombinant strains SA11CP1 and SA12CP2 was analyzed. The stability of the introduced genes was determined by culturing the strains without spermatozoa and conversion to the vegetative form without antibiotic selection.

Recombinant strains SA11CP1 and SA12CP2 as well as wild-type SA11 and SA12 were plated on LB plates and incubated overnight at 30 ° C. A single colony from each plate was used to inoculate 100 ml of CYS culture medium in a 500 ml culture flask containing a deflector. The cultures were cultured at 30 ° C and 300 rpm. When the cultures reached OD 50.0 at 600 nm, they were diluted 1:10. Growth of the cultures was monitored every half hour to record growth curves. After moving from the log phase to the stationary phase, you ···

Cultures were cultured for an additional 48 hours. A 1:10 dilution of each sporulated culture was made and the dilutions were heat treated at 65 ° C for 45 minutes.

The cultures were assumed to contain about 10 ⁹ spores per milliliter. Samples were diluted and inoculated onto LB plates. The number of spores converted to vegetative form in the recombinant strains was compared with the number obtained in the wild-type strain. Fifty hundred colonies were replicated on erythromycin-containing LB medium and antibiotic-free LB medium and incubated overnight at 30 ° C. The percentage of colonies with a selectable marker versus the number of viable colonies was determined.

The newly introduced genes were found to be 100% stable during sporulation and conversion to the vegetative form, as determined by the persistent resistance to erythromycin and the presence of cry1C and ermC genes detected by PCR. No spontaneous erythromycin-resistant colonies were found. During the first 8 hours, the rate of growth of these recombinants in CYS medium was essentially the same as that of the corresponding parental strain. In SA11 (wild-type) and the recombinant SA11CP1 Strain Number of spores per ml for 9,3xl0 ⁷ and l, 5xl0 ^8, the SA12- (wild-type) and the recombinant SA12CP2 Strain Number of spores per ml 3,5x10 ⁷ and 3.4x10 ⁷ respectively. The introduced genes did not adversely affect the viability of the recombinant strains.

Example 21

Expression of a gene product in hybrid B./. strains

The gene expressed, to test the recombinant and parental strains, 10 ml CYS medium (per liter 10 g casitone, 5g glucose, 2g yeast extract, 1 g of KH2PO4, 1 ml of 50 mmol / 1 _MgCl2, 1 ml of 50 mmol / 1 _MnCl2 , 1 ml of 50 mmol / 1 _ZnSO4, 1 ml of 50 mmol / 1 FECH, 1 ml of 200 mmol / 1 CaCl ₂₎ culture prepared at 36 h, 30 ° C humidified incubator. Samples of 50 μΐ volume were mixed with an equal volume of 2x sample loading buffer [0.125 mmol / L TrisHCl (pH 8), 4% SDS, 0.005% “Bromophenol Blue”, 20% (v / v) glycerol, 4% (v / v) b -mercaptoethanol] and boiled immediately for 5 minutes. Samples of 2.5, 5 and 10 µl were plated on 10% acrylamide gel (Novex) and electrophoresed for 1.5 hours at U = 125 volts.

Expression of the introduced crystal genes was clearly detectable by SDS-polyacrylamide gel electrophoresis for all recombinant strains. In addition to the 130 kDa band detected in the wild-type strain for HD73 :: pSB147, a new band of about 65 kDa was seen. 65 kDa corresponds to the expected size of the crylIA protein. For HD73 :: pSB210.2, SA11CP1, SA11CP2, SA11CP3, SA11CP4, SA12CP1, SA12CP2, S287CP1 and S287CP2, an additional band was observed at 135 kDa, which is expected from the CrylC protein. size, it was not seen in wild-type strains HD73, SA11, SA12 and S287. The recombinant strains continued to express other Cry-type proteins expressed by wild-type parental strains, which were detected as a running band corresponding to 130 kDaltons.

Example 22

Biological assay to test for lethality of hybrid B.t. strains

For most bioassays, the samples were placed in 500 mL culture flasks with a spoon, in 100 mL Fish meal media (5.5% Fish meal, 4% starch, 0.1% NH4Cl, 0.125% KH2PO4 , 0.05% MgSO ₄ , 0.001% FeSO ₄ , 0.001% MnCl ₂ ) were cultured at 30 ° C for 72 hours at 300 rpm. For Biological Assay # 1087, samples were also cultured in 500 mL culture flasks containing spinner, in 100 mL Fisch Meal medium at 30 ° C, but in this case inoculated with fresh sponge spore and 6 hours at 30 ° C. A 5% inoculum prepared from 100 ml of "Fish meal" starting culture was used. The SA11 samples (control) and the corresponding recombinants were seeded after 41 hours of culture, and the SA12 samples (control) and the corresponding recombinants were seeded after 47 hours of culture. Each sample was assayed for protein expression by diluting 1:10 with water and treated similarly to CYS cultures as described in Example 18 above. After harvesting, the cultures were stored at 4 ° C.

Recombinant strains SA11, SA12 and S287 cultured in "Fish meal" medium were tested against Trichopulsia ni and Spodoptera exigua larvae according to the following procedure. Samples from recombinant strains were mixed with artificial insect nutrition containing 132 g / l wheat germ, 28 g / l casein, 11 g / l vitamin mixture (Moorehead & Co. Van Nuys, CA).

It contained 8.8 g / l salt mixture (BioServ, Frenchtown, NJ), 2.3 g / l sorbic acid, 1.1 g / l methyl paraben, 13 g / l agar and 1.5 ml / l formalin. The mixture was fed with larvae in the late phase of the third stage of larvae incubated at 25 ° C. Mortality was recorded after 4 days, and the _LC50 eth (larvae • · · · · · · · · · · · • _«β ·· · · · · · · · causing« ·% of destruction concentration) of was determined by probit analysis known in the art.

All recombinant strains showed greater activity at 5 '. exigua as the wild-type strains from which they came. There was a 1.6-2.2-fold increase in activity against Spodoptera exigua larvae.

The cry1C gene was introduced into known locations on several chromosomes of various strains of Bacillus thuringiensis by electrotransformation and phage lysate transduction techniques. In the case of recombinant strains produced by transduction, the expression of the cry1C gene was detected by SDS-PAGE and the Cry1C protein contributed to the bioactivity against exuiga 5 larvae. Insertion of the crylC gene into the chromosome did not induce instability of the originally existing plasmids and maintained itself stable during sporulation and conversion to the vegetative form.

Example 23

B.t., to determine the temperature required to inhibit pLTV1 replication

B. subtitlis strain PY1177 (pLTV1) was obtained from Dr. Phil Youngman and was described by Camilli et al., J. Bacteriol. 172, 3738 (1990)]. PLTV1 DNA was isolated from strain PY1177 according to the method of Bimbóim and Doly (1979, supra). As described in Example 7 above, using the electroporation technique with pLTV1 DNA, B.t. kurstaki HD7 3 strain was transformed and the transformant was named HD73 + pLTV1.

The temperature at which the pLTVI plasmid B.t. kurstaki strain HD73, two one

This was determined by subsequent heat treatment at various temperatures, of which the first was performed in a liquid medium and the second in a solid medium according to Bohall's method (Bohall NA: J. Bacteriology 167: 716 (1986)). A 10 ml culture flask containing LB-teti ₀ medium was inoculated with a single colon of HD73 + pLTV1. This primary culture was grown at 30 ° C, mixed with a deflector, at 300 rpm and measured at 600 nm to an OD = 0.4. Cells were harvested by centrifugation, washed with LB (antibiotic-free) medium to remove tetracycline and resuspended in 10 ml of antibiotic-free LB. The resuspended cells were inoculated with 10 ml (1%) LB culture preheated to 30, 37, 40 and 42 ° C. After inoculation, the cultures were maintained at the appropriate temperature at 600 nm to an OD = 0.6-0.8. Then, the cultures were diluted 10 ⁴ -10 ⁷ factor, and 100 μΐ aliquots of LB ery _p, seeded plates O5 dilutions. Each plate was incubated overnight at the incubation temperature of the appropriate primary culture. After overnight incubation, LB-ery is _0; Colonies from the colonies on 05 plates were replicated on four different LB plates, in particular, antibiotic-free, LB-ery ₀ , LB _{cm- 2} , and LB _te thi plates. The plates were incubated overnight at 30 ° C and the next day classified for antibiotic sensitivity.

The results of the experiments in which the primary culture was grown in LB medium containing tetracycline were as follows. 20% of the colonies tested permissive for replication of pLTV1 at 30 ° C were sensitive to erythromycin, chloramphenicol and tetracycline, suggesting loss of plasmid due to inhibition of replication. When cultured at 37 ° C, 98% of the colonies were sensitive to all three antibiotics. At 40 ° C, 94% of the colonies were sensitive to all three antibiotics, and at 42 ° C, 100% of the colonies were sensitive to all three antibiotics. In experiments where the liquid primary cultures did not contain tetracycline, heat-induced plasmid loss occurred at the same temperatures. At tetracycline-free cultures at permissive temperature of 30 ° C, 38% plasmid loss was observed (significantly higher than the 20% plasmid incubated at 30 ° C in LB + cultures). -losing). Plasmid loss in tetracycline-free cultures incubated at 37 ° C, 40 ° C and 42 ° C was 94%, 90% and 90%, respectively. It was concluded that at 37 ° C and above, replication of plasmid pLTV1 was inhibited under the conditions used in the experiment. Because the plasmid may be lost even at 30 ° C permissive for replication, plasmid pLTV1 appears to be somewhat unstable.

Example 24

Determination of appropriate conditions for transposition of the Tn917 transposon carrying pLTV1 in BT

A three-step, three-day procedure was used to obtain transposable colonies. This method was first tested with strain HD73 + pLTVI. First, 10 mL, _R ery, cm ₅ - and tet5-containing liquid LB medium was inoculated with a single colony-HD73 containing pLTVl plasmid, and 30 ° C, mixed with terelőidommal at 300 rev / min, 600 nm en cultured to OD = 0.7. The primary culture was then centrifuged and the pelleted cells were washed in 10 mL of LB medium to remove the antibiotic. Another two culture flasks and 100 to 100 ml of LB ery _r t and cnió Art containing (but not containing tetracycline) 100 μΐ, washed primary culture did! inoculated. One culture flask at 30 ° C

-66 (at permissive temperature) while the other was grown at 37 (non-permissive temperature) at 300 rpm overnight.

After overnight culture, the cultures were diluted and inoculated into LB medium and light cm ₅ -LB medium. The plates were incubated overnight at the same temperature as the corresponding secondary culture flasks. Following this second overnight heat treatment, colonies isolated from erythema cm ₅ -LB plates grown at 37 ° C were replicated on LB plate medium, ery cm ₅ -LB medium, ery ₀ -LB. plate medium, cm ₇ -LB plate medium and thio-LB plate medium were used to determine the percentage of colonies resistant to erythromycin and chloramphenicol but sensitive to tetracycline, indicating a transposition event.

The ery ^r cm ^r tet ^s -telepeket designated HD73 :: pLTVl-strain.

As expected, almost 100% of the HD73 colonies carrying the pLTV1 plasmid from the above experiments have been transposed from the pLTV1 plasmid to the lacZ gene. The ery ^r cm ^r tet ^s colonies -HD73 PCRanalízise carried LACNHS1 and LACNHS2 primers showed that the lacZ gene from plasmid pLTVl was present in all cases. According to further PCR analysis using TY6 and TY7 primer oligonucleotides, 38 of the 40 transposable colonies retained the native cryIA (c) gene. The sequences of the primer oligonucleotides are given in Table 7 below.

-67Ί. spreadsheet

Qligonucleotide sequences are SEQ ID NO

LACNHS1 GGCTTTCGCTACCT 29 GGAGAGACGCGCC CGC LACNHS2 CCAGACCAACTGG 30 TAATGGTAGAGAC CGGC TY6 GGTCGTGGCTATAT 31 CATTCGTGTCACAG C TY7 CCACGCTATCCAC 32 GATGAATGTTCCTT C NHS21 GATATTTTAGCTCA 33 TGATCTTTTCCTCC TATTAAC NHS37 CATTACGCATTTGG 34 AATACC

Plasmid pSB050 was designed for the purpose of cryIIIAoperon in B.t. gallerie from strain HD232 to the genome HD73. The B.t. gallerie strain HD232 was obtained from USDA. The entire crylIA operon from strain HD232 was isolated as a 4 kb BamHI-HincII fragment from pSB304 described in Example 5. Plasmid pLTV1 was cleaved with the Sma I and Bam HI restriction enzymes to

-68 to obtain a 20.6 kb fragment, the fag fragments were ligated with each other to create plasmid pSB050. The ligation product was transfected with dam, dcmGM2163 E. coli. Individual colonies of GM2163 containing the desired construct were first selected on LBamp75 plates, and the colonies were inoculated with a replicate imprint technique onto a number of different agar plates containing either no antibiotic or amp ₇₅ , cm ₇ , eryio or thio-containing plates. they were. Of the fifty-five colonies examined, five were resistant to tetracycline, ampicillin and erythromycin, which meant that the corresponding fragment was ligated. All other colonies were sensitive to tetracycline, suggesting a lack of plasmid pLTVI. Resistant colonies were further analyzed by PCR amplification of the 1400 bp spacer between the LACNHS1 and LACNHS2 primers. The sequences of the above primer oligonucleotides are shown in Table 7 above and correspond to the sequences in the lacZ gene contained in plasmid pLTV1. PCR amplification using NHS37 and NHS21 primers having the sequences shown in Table 7 above resulted in a region from the erythromycin gene to the end of the first open reading frame of crylIAoperon. DNA from resistant colonies was analyzed by BglII restriction enzyme analysis, and colonies that gave the expected PCR results and 2 kb, 5.3 kb and 16 kb BglII fragments were considered to contain the pSB050 plasmid.

Transformation of HD73 strain with plasmid pSB050 was accomplished by electroporation of 5 µg of host cells with pSB050 DNA isolated from GM2163 strain using the following parameters: kV = 1.2, pF = 3 and Ω = οο. Cells were allowed to regenerate permissively at 30 ° C in BHIS medium with shaker mixing at 300 rpm for 3 hours. Cells were then concentrated with 10 µg / ml tetracycline containing 4 µg / ml of tetracycline.

Inoculated onto -69oz LB plates and incubated overnight at 30 ° C. In tetracycline-resistant HD73 colonies, the presence of plasmid pSB050 was detected by PCR analysis of the LACNHS1 and LACNHS2 primer oligonucleotides (yielding 1400 bp) and the NHS37 and NHS21 primer oligonucleotides (yielding 600 bp of Perkin). using recommended terms. These isolates were designated HD73 + pSB050.

Example 26

The introduction of CrylIA into B.t. 50 MDalton plasmids of strain HD73 by transpositions of pSB050-derived Tn917 transposon

Some HD73 + pSB050 isolates obtained as described in Example 25 were used for transposition of the crylIA operon from the pSB050 plasmid into the large resident plasmid of the HD73 strain. The HD73 + pSB050 isolates were incubated at non-permissive temperature and selected for chloramphenicol and erythromycin resistance as well as for tetracycline sensitivity. The ery cm ^r tet ^{r s} -telepeket designated HD73 :: 050's, suggesting that the transposition event occurred. Transposition was accomplished by the 1400 bp PCR amplification between the NHS37 and NHS21 primer oligonucleotides with the expected 600 bp fragment (the ergene to the crylIA gene) and the LACNHS1 and LACNHS2 primer oligonucleotides. The presence of an intact crylA (C) coding region was also confirmed by PCR amplification with the TY6 and TY7 primers, the sequences of which are shown in Table 7 above.

-70 • · · · · ··· ♦ · · · 4 · * · ··· «β 4 -

Example 27

Expression of CrvIA (c) and CrylIA Genes in Transposed HD73 :: 050 B.t.

Microscopic examination of the HD73 + pBS050 and HD73 :: 50 strains at 1000x magnification showed that both strains produced two types of crystals. In HD73 + pBS050 and HD73 :: 50 cells, both the bipyramidal CryIA (c) crystals characteristic of the HD73 strain and the cuboidal CryIILA crystals were observed. Such cuboidal crystals were not visible in wild-type HD73 cells.

Expression of CrylA (c) and CrylIA proteins in strain HD73 :: 050 was analyzed by SDS-PAGE. A 100 µl sample of a sporulated culture (40-50 hours culture) was pelleted, suspended in 10 mM EDTA, mixed with 2x SDS sample loading buffer on ice and boiled for three minutes. Samples were run on pre-cast 10% Novex gel and stained with “Coomassie blue”.

SDS-PAGE analysis revealed the presence of 135 kDa CrylA (c) and 65 kDa CrylIA proteins. This was also confirmed by Western blot analysis using specific antisera against two separate biotons, CrylA (c) or CrylIA.

Example 28

Spore numbers and. B.t.-Stability of HD73 :: 050 strain

For general characterization of relative "health status" of cultures, the number of spores in ml culture was compared in samples from the following Bt strains: wild-type strain HD73 and hybrid strains HD73 + pLTV1, HD73 + pSB050, and HD73 :: 050. samples. Sporulated cultures are treated for 45 minutes at 65 ° C.

-71 needles to kill the remaining vegetative cells. Samples from further dilutions were plated on LB agar plates and colonies were counted the following day. The stability of transposed DNA in strain HD73 :: 050 was determined by plating the above spore dilutions on LBagar plates containing 10 pg / ml tetracycline, 1 pg / ml erythromycin and 7 pg / ml chloramphenicol or on antibiotic-free LB agar plates. The number of colonies growing with selection was compared with the number of colonies without selection. The spore number for the HD73 :: 050 strain was slightly lower than for the wild-type HD73 strain. The number of spores per milliliter was between 1 and 2 x 10 ⁸ . Most HD73 :: 050 (i.e., 97-100%) retained adequate er / cfef antibiotic resistance, indicating that the transposed region did not become unstable and did not cleave from DNA after transposition.

Example 29

Plasmid profile of strain HD73 :: 050 B./

The plasmid profile of strain HD73 :: 050 was determined to determine whether the transposition event occurred on the chromosome or large plasmid of strain HD73. The plasmid preparation method used was a slightly modified alkaline lysis procedure for Bimboim and Doly. and Doly (1979), supra], described above in Example 18. Plasmid profile analysis of DNA preparations from HD73 :: 050 and wild type HD73 strains showed that the transposon was inserted into two 50 MDalton plasmids present in the original HD73 strain. An increase in the molecular weight of the plasmid bands was observed on the gels, which corresponded to the molecular weight of the transposed DNA. Some HD73 :: 050 isolates contain a high copy number of 50 MDalton carrying the cryIA (c) gene

Ί2 * · · ♦ ♦ ♦ ♦ ♦ · · · · ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** ** volt volt volt volt volt volt ** ** volt In other isolates, the lower copy number plasmid of 50 MDaltons was the target of transposition. No change in molecular weight was observed for any of the other plasmids originally present in the strain HD73.

Example 30

Southem blot analysis of the transposition of HD73 :: 050 and HD73 :: pLTV R1 strains

Total DNA from wild-type strains HD73, HD73 :: pLTV1 and HD73 :: 050 was prepared using the ASAP reagent kit according to the instructions of "Boehringer Mannheim". The DNA was digested with excess BamHI and EcoRI overnight, electrophoresed on a 20 cm 0.8% TBE agarose gel for 18 hours and then overnight in a 20xSSC buffer in Bioassay Bioassay. -Rad transferred to an accessible "Zeta-Probe" membrane. Membranes were treated and hybridized with the probe according to the instructions provided with the Amersham ECL kit. The PCR product of the 1400 bp LacZ gene described above in Example 25 was used as a probe to hybridize the membrane-transferred DNA. The bands corresponding to HD73 :: 050 DNA were compared to those corresponding to the wild-type HD73 strain.

Southem blot analysis showed that the transposon was inserted at different sites in the 50 MDalton plasmids. Preferred transposition site was not observed. The restriction map of pLTV1 showed that the HD73 :: pLTV1 strain contained 9 kb or larger of the probe hybridizing to the probe. Strips of this size correspond to a portion of plasmid pLTV1 transposed into a plasmid of 50 MDaltons. Similarly, the HD73 :: pLTV1 DNA contained probes hybridizing to the probe of 14 kb or larger, which hybridized to the probe. THE

-73 In the Southem analysis, hybridized bands of the expected size were observed in all cases. None of the hybridized bands were of the same size, confirming that transposition of pLTV1 occurred at random sites in the HD73 genome.

Southem blot analysis of HD73 :: 050 isolate showed, as expected, bands corresponding to 12 kb or larger fragments after BaniHI digestion, and bands corresponding to 17 kb or larger fragments after Eco RI digestion. The difference in size of the hybridized bands in the HD73 :: pLTV1 and HD73 :: 050 strains is due to the presence of the crylIA gene in the transposed region of the HD73 :: 050 strain. The above confirms that the crylIA gene was randomly inserted into the genome of HD73 strain.

Example 31

KB.t. Loss of strain HD73 :: 050

HD73 :: Fresh overnight colonies of HD73 and wild type HD73 strains were inoculated individually into 500 ml of CYS medium described by Yamamoto, collected by centrifugation, and agitated for 55 hours at 300 rpm with a shaker. Cultured at 30 ° C (Yamamoto T .: "ÁCS Symposium Series" 432, 46-60 (1990)). Cells were resuspended in 1/20 volume A buffer (5 mM Tris pH 8.0, 0.025% Triton) and the cell lysate was electrophoresed on 8% Novex SDS-PAGE gels. The concentration of CryIA (c) protein was determined by densitometric scanning. Nineteen, two-fold dilutions were made in buffer A and the amount of CryIA (c) protein in each dilution was determined. Known amounts of the proteins were mixed with insect feed and added to the late phase of the third larval stage.

-ΊΔTrichoplusia ni and Spodoptera exigua larvae, the larvae were incubated for 4 days at 25 ° C and mortality was recorded.

The results of the bioassay showed that the HD73 :: 050 hybrid strain had higher activity than the wild-type HD73 strain. The results obtained for the T. ni by, the wild-type HD73 strain LC ₅₀ (the concentration which caused half of the larval death)

It was 3.5 ppm, while in the case of HD73 :: 050 defined strain LC = ₅ p was 2 ppm. The results of S. exigua showed that the wild-type HD73 strain had an LC50M75 ppm, while the HD73 :: 050 strain had an LC50 of 22 ppm.

SUMMARY OF THE INVENTION The invention is summarized in the following points 1 and 2 and is defined in the appended claims.

The present invention relates to a DNA segment comprising:

(a) a DNA sequence encoding one or more insecticidal proteins capable of replication and expression in Bacillus thuringiensis cells, and a sequence homologous to a sequence present in Bacillus thuringiensis chromosomal DNA, thereby directing the homologous DNA sequence into the aforementioned DNA segment. and thereby inserting said DNA sequence encoding said insecticidal protein into the chromosomal DNA of Bacillus thuringiensis; obsession

b) a DNA sequence encoding one or more insecticidal proteins capable of replication and expression in Bacillus thuringiensis cells, and a DNA sequence capable of being randomly integrated into the chromosomal DNA of Bacillus thuringiensis, wherein said DNA segment is an insecticidal DNA sequence; sequence is stably integrated into chromosomal DNA.

· · ·

The present invention relates to a process for the production of host cells of Bacillus thuringiensis, comprising:

a) generating a DNA sequence homologous to Bacillus thuringiensis chromosomal DNA or DNA capable of being randomly integrated into Bacillus thuringiensis chromosomal DNA;

b) operably linking said DNA sequence to one or more DNA sequences encoding an insecticidal protein;

c) recovering the DNA segments;

d) transforming the Bacillus thuringiensis strain to integrate the DNA segment into the chromosomal DNA; and

e) isolating a transformed host cell in which the DNA sequence encoding the insecticidal protein is stably integrated into the chromosomal DNA of the host cell and is capable of replication and expression in the host cell.

SEQUENCE LIST

DETAILS OF SEQ ID NO: 1:

CHARACTERISTICS OF THE SEQUENCE:

LENGTH: 28 base pairs

TYPE: nucleic acid

HOW FIBER: One

TOPOLOGY: linear

MOLECULATING TYPE: genomic DNA

HYPOTHETICAL: No

ANTISSE: No

DESCRIPTION OF SEQ ID NO: 1:

GGAACGCTAC ATACTAGTGA TAGAGTAG 28

DETAILS OF SEQ ID NO: 2:

CHARACTERISTICS OF THE SEQUENCE:

LENGTH: 28 base pairs

TYPE: nucleic acid

HOW FIBER: One

TOPOLOGY: linear

MOLECULATING TYPE: genomic DNA

HYPOTHETICAL: No

ANTISSE: No

DESCRIPTION OF SEQ ID NO: 2:

GCTTGTACAC CGCAACTGTT TTCGCATG 28

-77AI 3. DETAILS OF SEQ ID NO:

CHARACTERISTICS OF THE SEQUENCE:

LENGTH: 37 base pairs

TYPE: nucleic acid

HOW FIBER: One

TOPOLOGY: linear

MOLECULATING TYPE: genomic DNA

HYPOTHETICAL: No

ANTISSE: No

DESCRIPTION OF SEQ ID NO: 3:

AGCTTGCGGC CGCGTCGACC CCGGGCCATG GGGGCCG

DETAILS OF SEQ ID NO: 4:

CHARACTERISTICS OF THE SEQUENCE:

LENGTH: 38 base pairs

TYPE: nucleic acid

HOW FIBER: One

TOPOLOGY: linear

MOLECULATING TYPE: genomic DNA

HYPOTHETICAL: No

ANTISSE: No

DESCRIPTION OF SEQ ID NO: 4:

AATTCGGGCC CCCATGGCCC GGGGTCGACG CGGCCGCA 38 • · · · · · · · · · · · · · · · · · · · · · · · · · · ···

-78 SEQ ID NO: 5:

CHARACTERISTICS OF THE SEQUENCE:

LENGTH: 29 base pairs

TYPE: nucleic acid

HOW FIBER: One

TOPOLOGY: linear

MOLECULATING TYPE: genomic DNA

HYPOTHETICAL: No

ANTISSE: No

DESCRIPTION OF SEQ ID NO: 5:

CCACAGTTAC AGTCTGTAGC TCAATTACC 29

DETAILS OF SEQ ID NO: 6:

CHARACTERISTICS OF THE SEQUENCE:

LENGTH: 24 base pairs

TYPE: nucleic acid

HOW FIBER: One

TOPOLOGY: linear

MOLECULATING TYPE: genomic DNA

HYPOTHETICAL: No

ANTISSE: No

DESCRIPTION OF SEQ ID NO: 6:

CCGCTACTAA TAGAACCTGC ACCA

-Ί9 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · English

DETAILS OF SEQ ID NO: 7:

CHARACTERISTICS OF THE SEQUENCE:

LENGTH: 40 base pairs

TYPE: nucleic acid

HOW FIBER: One

TOPOLOGY: linear

MOLECULATING TYPE: genomic DNA

HYPOTHETICAL: No

ANTISSE: No

DESCRIPTION OF SEQ ID NO: 7:

CAATACATTA TCCATGGAAA ATTCCTCCTT AAATATCATG 40

DETAILS OF SEQ ID NO: 8:

CHARACTERISTICS OF THE SEQUENCE:

LENGTH: 39 base pairs

TYPE: nucleic acid

HOW FIBER: One

TOPOLOGY: linear

MOLECULATING TYPE: genomic DNA

HYPOTHETICAL: No

ANTISSE: No

DESCRIPTION OF SEQ ID NO: 8:

GAGCAATGAA AGAGTTAGGG CCCTGTTTAA GGTGTCATG 39

-80Α 9. DETAILS OF SEQ ID NO:

CHARACTERISTICS OF THE SEQUENCE:

LENGTH: 16 base pairs

TYPE: nucleic acid

HOW FIBER: One

TOPOLOGY: linear

MOLECULATING TYPE: genomic DNA

HYPOTHETICAL: No

ANTISSE: No

DESCRIPTION OF SEQ ID NO: 9:

GAGTGAATTA TGGGGG 16

DETAILS OF SEQ ID NO: 10:

CHARACTERISTICS OF THE SEQUENCE:

LENGTH: 16 base pairs

TYPE: nucleic acid

HOW FIBER: One

TOPOLOGY: linear

MOLECULATING TYPE: genomic DNA

HYPOTHETICAL: No

ANTISSE: No

DESCRIPTION OF SEQ ID NO: 10:

ATTTTGTATT AAACGG • *

-81 All. SEQ ID NO:

CHARACTERISTICS OF THE SEQUENCE:

LENGTH: 24 base pairs

TYPE: nucleic acid

HOW FIBER: One

TOPOLOGY: linear

MOLECULATING TYPE: genomic DNA

HYPOTHETICAL: No

ANTISSE: No

All. DESCRIPTION OF SEQ ID NO:

ACTATTTGTG ATGCGTATAA TGTA 24

DETAILS OF SEQ ID NO: 12:

CHARACTERISTICS OF THE SEQUENCE:

LENGTH: 18 base pairs

TYPE: nucleic acid

HOW FIBER: One

TOPOLOGY: linear

MOLECULATING TYPE: genomic DNA

HYPOTHETICAL: No

ANTISSE: No

DESCRIPTION OF SEQ ID NO: 12:

AATTCCCCAT TCATCTGC 18

-82Α 13. DETAILS OF SEQ ID NO:

CHARACTERISTICS OF THE SEQUENCE:

LENGTH: 20 base pairs

TYPE: nucleic acid

HOW FIBER: One

TOPOLOGY: linear

MOLECULATING TYPE: genomic DNA

HYPOTHETICAL: No

ANTISSE: No

DESCRIPTION OF SEQ ID NO: 13:

GAAATCGGCT CAGGAAAAGG 20

DETAILS OF SEQ ID NO: 14:

CHARACTERISTICS OF THE SEQUENCE:

LENGTH: 24 base pairs

TYPE: nucleic acid

HOW FIBER: One

TOPOLOGY: linear

MOLECULATING TYPE: genomic DNA

HYPOTHETICAL: No

ANTISSE: No

DESCRIPTION OF SEQ ID NO: 14:

CCTTAAAACA TGCAGGAATT GACG 24

15. DETAILS OF SEQ ID NO: 15

CHARACTERISTICS OF THE SEQUENCE:

LENGTH: 20 base pairs

TYPE: nucleic acid

HOW FIBER: One

TOPOLOGY: linear

MOLECULATING TYPE: genomic DNA

HYPOTHETICAL: No

ANTISSE: No

DESCRIPTION OF SEQ ID NO: 15:

CTATTGGTTG GAATGGCGTG 20

DETAILS OF SEQ ID NO: 16:

CHARACTERISTICS OF THE SEQUENCE:

LENGTH: 28 base pairs

TYPE: nucleic acid

HOW FIBER: One

TOPOLOGY: linear

MOLECULATING TYPE: genomic DNA

HYPOTHETICAL: No

ANTISSE: No

DESCRIPTION OF SEQ ID NO: 16:

GAGCCAAGCA GCTGGAGGAG TTTACACC 28

-84 • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · <br> <br> <br> <br> <br> to to 37 to have all of you

DETAILS OF SEQ ID NO: 17:

CHARACTERISTICS OF THE SEQUENCE:

LENGTH: 22 base pairs

TYPE: nucleic acid

HOW FIBER: One

TOPOLOGY: linear

MOLECULATING TYPE: genomic DNA

HYPOTHETICAL: No

ANTISSE: No

DESCRIPTION OF SEQ ID NO: 17:

TCACTTCCCA TCGACATCTA CC 22

DETAILS OF SEQ ID NO: 18:

CHARACTERISTICS OF THE SEQUENCE:

LENGTH: 33 base pairs

TYPE: nucleic acid

HOW FIBER: One

TOPOLOGY: linear

MOLECULATING TYPE: genomic DNA

HYPOTHETICAL: No

ANTISSE: No

DESCRIPTION OF SEQ ID NO: 18:

ATCACTGAGT CGCTTCGCAT GTTTGACTTT CTC · «····

-85Α 19. DETAILS OF SEQ ID NO:

CHARACTERISTICS OF THE SEQUENCE:

LENGTH: 29 base pairs

TYPE: nucleic acid

HOW FIBER: One

TOPOLOGY: linear

MOLECULATING TYPE: genomic DNA

HYPOTHETICAL: No

ANTISSE: No

DESCRIPTION OF SEQ ID NO: 19:

GGTCGTGGCT ATATCCTTCG TGTCACAGC

DETAILS OF SEQ ID NO: 20:

CHARACTERISTICS OF THE SEQUENCE:

LENGTH: 25 base pairs

TYPE: nucleic acid

HOW FIBER: One

TOPOLOGY: linear

MOLECULATING TYPE: genomic DNA

HYPOTHETICAL: No

ANTISSE: No

DESCRIPTION OF SEQ ID NO: 20:

ACAGAAGAAT TGCTTTCATA GGCTC 25

-86Α 21. DETAILS OF SEQ ID NO:

CHARACTERISTICS OF THE SEQUENCE:

LENGTH: 23 base pairs

TYPE: nucleic acid

HOW FIBER: One

TOPOLOGY: linear

MOLECULATING TYPE: genomic DNA

HYPOTHETICAL: No

ANTISSE: No

DESCRIPTION OF SEQ ID NO: 21:

GAATTGCTTT CATAGGCTCC GTC 23

DETAILS OF SEQ ID NO: 22:

CHARACTERISTICS OF THE SEQUENCE:

LENGTH: 28 base pairs

TYPE: nucleic acid

HOW FIBER: One

TOPOLOGY: linear

MOLECULATING TYPE: genomic DNA

HYPOTHETICAL: No

ANTISSE: No

DESCRIPTION OF SEQ ID NO: 22:

CAACAAACGG GCCATAAGCT TGTATAAG

DETAILS OF SEQ ID NO: 23

CHARACTERISTICS OF THE SEQUENCE:

LENGTH: 23 base pairs

TYPE: nucleic acid

HOW FIBER: One

TOPOLOGY: linear

MOLECULATING TYPE: genomic DNA

HYPOTHETICAL: No

ANTISSE: No

DESCRIPTION OF SEQ ID NO: 23:

GCCGTCTGTA ACGGTACCTA AGG 23

DETAILS OF SEQ ID NO: 24:

CHARACTERISTICS OF THE SEQUENCE:

LENGTH: 21 base pairs

TYPE: nucleic acid

HOW FIBER: One

TOPOLOGY: linear

MOLECULATING TYPE: genomic DNA

HYPOTHETICAL: No

ANTISSE: No

DESCRIPTION OF SEQ ID NO: 24:

CACCCAGTTT GTACTCGCAG G 21 • * · «4 · · · • 44 ·· 4444 ·

-88Α 25. DETAILS OF SEQ ID NO:

CHARACTERISTICS OF THE SEQUENCE:

LENGTH: 18 base pairs

TYPE: nucleic acid

HOW FIBER: One

TOPOLOGY: linear

MOLECULATING TYPE: genomic DNA

HYPOTHETICAL: No

ANTISSE: No

DESCRIPTION OF SEQ ID NO: 25:

GTCTCATGCA AACTCAGG 18

DETAILS OF SEQ ID NO: 26:

CHARACTERISTICS OF THE SEQUENCE:

LENGTH: 18 base pairs

TYPE: nucleic acid

HOW FIBER: One

TOPOLOGY: linear

MOLECULATING TYPE: genomic DNA

HYPOTHETICAL: No

ANTISSE: No

DESCRIPTION OF SEQ ID NO: 26:

CTCTGGCGCT CCATCTAC

-89Α 27. DETAILS OF SEQ ID NO:

CHARACTERISTICS OF THE SEQUENCE:

LENGTH: 29 base pairs

TYPE: nucleic acid

HOW FIBER: One

TOPOLOGY: linear

MOLECULATING TYPE: genomic DNA

HYPOTHETICAL: No

ANTISSE: No

DESCRIPTION OF SEQ ID NO: 27:

CCCATGGATA ATGTATTGAA TAGTGGAAG 29

DETAILS OF SEQ ID NO: 28:

CHARACTERISTICS OF THE SEQUENCE:

LENGTH: 10 base pairs

TYPE: nucleic acid

HOW FIBER: One

TOPOLOGY: linear

MOLECULATING TYPE: genomic DNA

HYPOTHETICAL: No

ANTISSE: No

DESCRIPTION OF SEQ ID NO: 28:

AGCGGCCGCT

-90 • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · he · he · English Home |

DETAILS OF SEQ ID NO: 29:

CHARACTERISTICS OF THE SEQUENCE:

LENGTH: 30 base pairs

TYPE: nucleic acid

HOW FIBER: One

TOPOLOGY: linear

MOLECULATING TYPE: genomic DNA

HYPOTHETICAL: No

ANTISSE: No

DESCRIPTION OF SEQ ID NO: 29:

GGCTTTCGCT ACCTGGAGAG ACGCGCCCGC 30

DETAILS OF SEQ ID NO: 30:

CHARACTERISTICS OF THE SEQUENCE:

LENGTH: 30 base pairs

TYPE: nucleic acid

HOW FIBER: One

TOPOLOGY: linear

MOLECULATING TYPE: genomic DNA

HYPOTHETICAL: No

ANTISSE: No

DESCRIPTION OF SEQ ID NO: 30:

CCAGACCAAC TGGTAATGGT AGAGACCGGC

-91 DETAILS OF SEQ ID NO: 31:

CHARACTERISTICS OF THE SEQUENCE:

LENGTH: 29 base pairs

TYPE: nucleic acid

HOW FIBER: One

TOPOLOGY: linear

MOLECULATING TYPE: genomic DNA

HYPOTHETICAL: No

ANTISSE: No

DESCRIPTION OF SEQ ID NO: 31:

GGTCGTGGCT ATATCATTCG TGTCACAGC

DETAILS OF SEQ ID NO: 32:

CHARACTERISTICS OF THE SEQUENCE:

LENGTH: 28 base pairs

TYPE: nucleic acid

HOW FIBER: One

TOPOLOGY: linear

MOLECULATING TYPE: genomic DNA

HYPOTHETICAL: No

ANTISSE: No

DESCRIPTION OF SEQ ID NO: 32:

CCACGCTATC CACGATGAAT GTTCCTTC • ···························································································oved

-92Α 33. SEQ ID NO: 33

CHARACTERISTICS OF THE SEQUENCE:

LENGTH: 35 base pairs

TYPE: nucleic acid

HOW FIBER: One

TOPOLOGY: linear

MOLECULATING TYPE: genomic DNA

HYPOTHETICAL: No

ANTISSE: No

DESCRIPTION OF SEQ ID NO: 33:

GATATTTTAG CTCATGATCT TTTCCTCCTA TTAAC 35

DETAILS OF SEQ ID NO: 34:

CHARACTERISTICS OF THE SEQUENCE:

LENGTH: 20 base pairs

TYPE: nucleic acid

HOW FIBER: One

TOPOLOGY: linear

MOLECULATING TYPE: genomic DNA

HYPOTHETICAL: No

ANTISSE: No

DESCRIPTION OF SEQ ID NO: 34:

CATTACGCAT TTGGAATACC

Claims (9)

PATENT CLAIMS CLAIMS 1. A linear or circular DNA segment comprising at least one DNA sequence encoding an insecticidal protein which is capable of replication and expression in Bacillus thuringiensis cells, characterized in that in the linear DNA segment, all 5 'of the insecticidal protein coding sequence. all 3 'regions contain nucleotide sequences that are homologous to a sequence in a Bacillus thuringensis chromosomal DNA, thereby enabling the DNA sequence encoding the insecticidal protein to integrate into the bacterial chromosomal DNA or circular DNA. A region 5 'or 3' to the coding sequence for a protein having an action against a protein having an effect is a nucleotide sequence which is homologous to a sequence in the chromosomal DNA of Bacillus thuringiensis and thus encoding the protein having an insecticidal action. it can integrate into the bacterial chromosomal DNA. The DNA segment of claim 1, wherein it contains a replication origin operable in Gram-negative bacteria and a selectable marker gene. 3. A hybrid vector, comprising a DNA segment according to claim 1 or 2. A host cell of Bacillus thuringiensis, characterized in that it comprises a DNA segment according to any one of the preceding claims. '· · ·· « 5. An insecticidal composition comprising an effective amount of an insecticidal host cell and a carrier according to claim 4. 6. A method for producing a transformed Bacillus thuringiensis host cell comprising: i) introducing the vector of claim 3 or the DNA segment of claim 1 or 2 into a strain of Bacillus thuringiensis, and ii) isolating the resulting transformants in which the DNA sequence encoding the insecticidal protein is stably incorporated into the chromosomal DNA of the host cell. is integrated and is able to express and replicate there. 7. The method of claim 6, wherein the transformed host cells are transduced and produced in recipient Bacillus thuringiensis cells by: i) contacting the Bacillus thuringiensis host cells of claim 6 with a transducing phage; ii) allowing the phage to replicate in the host cell, thereby integrating the DNA sequence encoding one or more insecticidal proteins into the chromosomal DNA of the host cell; and iii) introducing the DNA sequence encoding the insecticidal protein into Bacillus thuringiensis recipient cells from the phage such that the DNA sequence encoding the insecticidal protein is stably incorporated into and expressed in the chromosomal DNA of the recipient cells, 8. The method of claim 6 or 7, wherein the host cell and the recipient cell are Bacillus thuringiensis kurstaki cells. »« ·· The method according to any one of claims 6, 7 or 8, characterized in that the DNA sequence encoding the insecticidal protein is the crylC sequence or a sequence homologous thereto. The agent shall: Ρ95 03017 PUBLICITY r r _ Figure 1 DISCLOSURE COPIES 2/7 Ρ95 03017 ER EcoRJ Kp> Kpn! Hd-Hindi BnwBamHI ^ Sm Sma Cleavage sites in parentheses were eliminated during cloning Figure 2 Ρ95 03017 PUBLICATION LITERATURE 3/7 Figure 3 DISCLOSURE COPIES 4/7 Ρ95 03017 Eco RI Paternal Hind