KR101892957B1 - Strain of bacillus thuringiensis forming spores of high environmental friendliness and low in vivo persistence - Google Patents

Abstract

The present invention relates to Bacillus thuringiensis from which specific genes are removed to improve environmental friendliness of spores and decrease in vivo persistence of spores. The present invention provides a strain of Bacillus thuringiensis from which an sspA gene having a base sequence represented by SEQ ID NO: 1 and an sspB gene having a base sequence represented by SEQ ID NO: 2 are removed. According to the present invention, sparged spores are quickly removed in an environment to allow development of biological pesticides or simulation agents with environmental friendliness. Also, if a person inhales spores, they are quickly removed from the lung. This invention allows the development of biological pesticides or mimetic agents that are safe for humans.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a strain of Bacillus thuringiensis, which grows in an environmentally friendly manner and has a reduced in vivo remnant.

The present invention relates to a bacterium having a bacterium , such as Bacillus subtilis, in which a specific gene has been removed to improve the environmental friendliness of spores and to reduce the survival of spores thuringiensis . Specifically, the present invention relates to a strain which has a high sensitivity to ultraviolet rays, sunlight and heat and forms spores having low in vivo persistence.

Microorganisms of the genus Bacillus and Clostridium form spores under certain conditions, which are the most difficult to destroy among all organisms and are known to survive under adverse conditions. Most of the common microorganisms are resistant to ultraviolet rays, heat of about 100 ° C and stress such as drying, so special treatment such as high-pressure steam sterilization is necessary to effectively kill these spores.

Bacillus thuringiensis ) is a germ-positive bacillus of the genus Bacillus that forms spores. It is harmless to human body and Cry protein produced by spore formation has an insecticidal effect and has been widely used worldwide as a bio pesticide against harmful insects. Is an anthrax bacterium that is harmless to the human body but is similar in size to anthrax and spores and has various properties as a simulant of anthrax bacteria and is being developed as a simulator of anthrax bacteria for use in the biodefense field in the United States and the United Kingdom .

Bacillus thuringiensis is usually sprayed outdoors in the form of spores or used in laboratories. As mentioned earlier, spores are the most difficult to destroy in the form of living organisms, In the case of an unintentional leak outdoors, it has a problem that it persists in the natural world for a very long time.

In these cases, the Bacillus tritencenthos Thuringiensis spores were also detected after 4 years (Non-Patent Document 0001) and were detected even after 7 years after being sprayed on the cabbage field (Non-Patent Document 0002). Anthrax bacteria, a pathologically very close pathogen to Bacillus thuringiensis, has also been detected 40 years after spore spraying (Non-patent document 3).

This environmentally resistant spore has the advantage of being able to store easily for a long period of time, while unintentionally leaking into the environment or causing genetically modified Bacillus tingensis When living modified organisms (LMOs), such as thuringiensis , are intentionally applied to the environment for their intended use, it is desirable to die quickly in nature in order to minimize the impact on the ecosystem. However, There is a risk of disturbance of ecosystem such as remaining in a very long-term environment.

In addition, spores of Bacillus thuringiensis , known to be harmless to humans as equivalent to Biosafety level 1, are injected into the lung intratracheally in a mouse experiment for 70 days It has been reported that spores may remain in the lungs and cause an inflammatory reaction (Non-Patent Document 0004), and Bacillus cereus group ( Bacillus cereus group) together with Bacillus thuringiensis cereus group), an anthrax bacterium, has been reported to remain in the lung for at least 8 weeks when the spore is administered nasally (Non-Patent Document 0005). Bacillus thuringiensis ( B. Thuringiensis spores are left in the lungs for a long time and cause a health problem to the human body. However, there is no effective way to solve this problem.

SASP (Small Acid-soluble Spore Protein) is a DNA-binding protein that is produced in large amounts in forespore in spore formation period and protects spore DNA. (Ultraviolet rays, heat, hydrogen peroxide, etc.), and Bacillus subtilis ) (Non-Patent Document 0006). This SASP protein is distinguished by α / β type and γ type. In α / β type SASP gene, several genes coding in one bacterium are found. Their amino acid sequences are almost similar to each other, impossible. For example, four α / β-type SASP genes have been identified in Bacillus subtilis (Non-Patent Document 0007) and seven in Bacillus megaterium (Non-Patent Document 0008) The sequence is very similar. Two major α / β-type SASP genes were named sspA and sspB in Bacillus subtilis in which the SASP gene was most frequently studied. When these two strains were removed, the environmental resistance (UV, heat, hydrogen peroxide, freeze drying, etc.) It is known to be significantly lowered. Thus, Bacillus Turing Gen System (Bacillus thuringiensis ), two major α / β-type SASP genes corresponding to sspA and sspB in Bacillus subtilis can be removed. Thus, it is possible to produce a strain in which the environmental resistance of spores is significantly reduced, and the microorganism The resistance to spores is expected to be significantly reduced even for reactive oxygen species such as hydrogen peroxide, which is a major countermeasure against bacteria. The strain that forms a safe spore in the human body by greatly reducing the survival in vivo Of the total population. However, until now, Bacillus thuringiensis) does not bar the SASP gene in strains not attempt to remove the unknown about how to achieve these goals need to remove any of the SASP gene is similar to animal testing studies using spore removal of the SASP gene is also nonexistent situation.

 Van Cuyk S, Deshpandee, Hollander, Duval N, Ticknor L, Layshock J, Gallegos-Graves L, Omberg KM. 2011. Persistence of Bacillus thuringiensis subsp. kurstaki in urban environments following spraying. Appl Environ Microbiol 77: 7954-7961.  Hendriksen NB, Hansen UN. 2002. Long-term survival and germination of Bacillus thuringiensis var. kurstaki in a field trial. Can J Microbiol 48: 256-261.  Manchee RJ, Broster MG, Melling J, Henstridge RM, Stagg AJ. 1981. Bacillus anthracis on Gruinard Island. Nature 294: 254-255.  Barfod KK, Poulsen SS, Hammer M, Larsen ST. 2010. Sub-chronic lung inflammation after airway exposures to Bacillus thuringiensis biopesticides in mice. BMC Microbiol 10: 233.  Jenkins SA, Xu Y. 2013. Characterization of Bacillus anthracis persistence in vivo. PLoS One 8: e66177  Setlow P. 2006. Spores of Bacillus subtilis: their resistance to and killing by radiation, heat and chemicals. Journal of Applied Microbiology 101: 514-525.  Connors, M. J., et al., (1986) Cloning and nucleotide sequence of genes for three small, aicd-soluble proteins of Bacillus subtilis spores. J. Bacteriol. 166: 417-25  Setlow, P. (1988) Small, acid-soluble spore proteins of Bacillus species: structure, synthesis, genetics, function and degradation. Ann. Rev. Microbiol. 42: 319-338  Despalinsa, Marsit S, Oberto J. 2011. Absynte: a web tool to analyze the evolution of orthologous archaeal and bacterial gene clusters. Bioinformatics 27: 2905-2906.  Peng D, Luo Y, Guo S, Zeng H, Ju S, Yu Z, Sun M. 2009. Elaboration of an electroporation protocol for large plasmids and wild-type strains of Bacillus thuringiensis. J Appl Microbiol 106: 1849-1858.  Yousten AA, Rogoff MH. 1969. Metabolism of Bacillus thuringiensis in relation to spore and crystal formation. J Bacteriol 100: 1229-1236.

In order to achieve the above object, the present invention is homologous to the gene for the major genes for Bacillus Turing Gen System (B. thuringiensis) Bacillus subtilis (Bacillus subtilis) a (Small Acid-soluble Protein Spore) SASP identified in (orthologs) To dramatically increase the susceptibility of the spores produced by the strain to ultraviolet rays, sunlight and heat, and to dramatically reduce the survival of the spores in vivo.

In order to achieve the above object, the present invention provides the gene (sspA) and gene (sspB) consisting of the nucleotide sequence of SEQ ID NO: 2 consisting of the nucleotide sequence of SEQ ID NO: 1 are both removed Bacillus Turing Gen System (B. thuringiensis) strains Lt; / RTI >

The present invention also provides a strain having the characteristics of the species deposited with KCTC Accession No. KCTC18560P.

The present invention also provides a biocidal pesticide containing the strain.

The present invention also provides a mimetic agent comprising said strain.

According to the present invention, the sprayed spores can be rapidly removed from the environment, and it becomes possible to develop a biocidal pesticide or a mimetic agent having environmental friendliness.

In addition, when humans inhale spores of Bacillus thuringiensis , which is sprayed in large quantities, inhaled in the lungs for a long time may cause health problems such as inflammation, according to the present invention, It is possible to develop a strain capable of forming a spore that is safe for the human body.

1 is Bacillus Turing Gen System (Bacillus thuringiensis ) BMB171 and the amino acid sequences of sspA and sspB proteins in B. subtilis .
Figure 2 is a Bacillus subtilis (B. subtilis), Bacillus Turing Gen system using the amino acid sequence of the gene of sspA (B. thuringiensis) is a result of the new analysis of the genome and the plasmid teoni of BMB171 strain.
FIG. 3 is a result of a synaptic analysis of the genome and plasmid of B. thuringiensis strain BMB171 using the amino acid sequence of the sspB gene of B. subtilis .
4A to 4E are conceptual diagrams showing a process of performing gene deletion using a gene deletion vector and an I-SceI expression vector according to an embodiment of the present invention.
Figure 5 is a map of pAD02 vector expressing the I-SceI enzyme used for gene removal in accordance with an embodiment of the present invention.
Figure 6 is a map of the pAD05 vector used to remove the sspA gene (BMB171_C4286) in an embodiment of the invention.
Figure 7 is a map of the pAD07 vector used to remove the sspB gene (BMB171_C0753) in an embodiment of the invention.
FIG. 8 is a graph showing the results of ultraviolet sensitivity test using spores of three strains prepared according to the embodiment of the present invention and spores of a wild type strain as a control group.
9 is a graph showing experimental results of artificial sun photoperiodicity test using spores of BT-005 strain prepared according to an embodiment of the present invention and spores of a wild type strain as a control group.
10 is a result of the cell count measurement of spores of BT-005 strain prepared according to the embodiment of the present invention and spores of a wild-type strain as a control at 4 ° C and 37 ° C for 10 weeks.
FIG. 11 is a graph showing the results obtained by injecting spores of BT-005 strain prepared according to the present invention and wild type strain spores into the lungs of rats, collecting lungs at 1 week, 2 weeks and 4 weeks, Which is a graph showing the results of measurement of the number.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, wherein like reference numerals are used to designate identical or similar elements, and redundant description thereof will be omitted. In the following description of the embodiments of the present invention, a detailed description of related arts will be omitted when it is determined that the gist of the embodiments disclosed herein may be obscured. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the invention as claimed. , ≪ / RTI > equivalents, and alternatives.

Terms including ordinals, such as first, second, etc., may be used to describe various elements, but the elements are not limited to these terms. The terms are used only for the purpose of distinguishing one component from another.

As used herein, the term " synteny " refers to a phenomenon in which the order of orthologs is preserved when chromosomes of two or more species are compared, and " synteny analysis " And comparing the chromosomes of two or more species using Tanney, to analyze which homologous gene corresponding to a specific gene is located on a chromosome of another species.

The term spore as used herein is synonymous with an endospore formed in a microorganism so as to have resistance in an extreme environment such as high temperature, low temperature, ultraviolet ray, drying and high pressure.

Hereinafter, the strain producing spores with high environmental friendliness and low in vivo survival according to the present invention will be described in detail.

Examples of the Bacillus sp. Strain used in Examples and Experimental Examples of the present invention include Bacillus thuringiensis strain, specifically Bacillus thuringiensis BMB171 ( Bacillus thuringiensis strain), which lacks a plasmid having a Cry protein gene thuringiensis BMB171) was purchased from Bacillus Genetic Stock Center (BGSC).

In addition, Bacillus Turing Gen System (Bacillus The genome information of the thuringiensis strain BMB171 was determined using the GenBank CP001903.1 sequence known from GenBank of the National Center for Biotechnology Information (NCBI), which is a known database, and the specific nucleotide sequence of a gene such as BMB171_C4286 and BMB171_C0753 And protein information are all based on information known to the NCBI.

The method for producing the strain according to the present invention uses a recombinant method based on I-SceI meganuclease. Hereinafter, a method for producing a strain according to the present invention will be described in detail.

4A to 4E are conceptual diagrams illustrating a process of removing a specific gene of a genome of a strain using a gene deletion vector and an I-SceI megaclasma expression vector.

Referring to FIG. 4A, when the GeneA on the chromosome of the strain is to be removed, approximately 1.5 kb of the region on either side of the gene is cloned into the left homology arm and the right homology arm, respectively. The gene deletion vector used in this process has a replication origin that works only in E. coli and has a spectinomycin resistance gene that works in both E. coli and Bacillus, And an 18-bp I-SceI recognition sequence (5-TAGGGATAACAGGGTAAT-3, SEQ ID NO: 3) recognized by the clathrate. Since the I-SceI recognition sequence is composed of 18 bp, unlike the general restriction enzyme recognition site composed of 6 bp, it is extremely unlikely to be present on the chromosome, and is not present on the chromosome of most known microorganisms. It is also absent from the chromosome of the B. thuringiensis strain BMB171 used in the example.

As shown in FIGS. 4A and 4B, when a gene deletion vector is introduced into a strain, the gene deletion vector includes a left homology arm and a right homology arm having the same base sequence on the chromosome, Homologous recombination between the left homology arm of the vector and the left homology arm of the chromosome or the right homology arm of the vector and the right homology arm of the chromosome homologous recombination, single cross-over, and the entire vector is integrated into the target locus. In FIG. 4A, it is assumed that homologous recombination occurs between left homology arms. In contrast, homologous recombination between right homology arms is also possible.

As the gene removed vector is described above as used herein, because only gajigi origin of replication (replication origin) that works in E. coli the Bacillus Turing Gen System (B. thuringiensis) strains that can not maintain the state with the plasmid vector and the chromosome by homologous recombination , Only the strain in which the vector is inserted into the chromosome can be grown when cultured in the spectinomycin-containing medium after the transformation.

Therefore, the strain is transformed into a strain by the method of non-patent document 0010 and the like using a gene elimination vector, and cultured overnight in a Luria-Bertani (hereinafter referred to as LB) solid medium containing spectinomycin (200 ug / ml). The next day, the number of colonies grown in the medium can be selected and the presence or absence of insertion of the vector into the corresponding gene locus can be confirmed through a polymerase chain reaction (hereinafter also referred to as PCR) using a vector-based primer, Which can eliminate false positive colonies.

4C, colonies identified as having the gene deletion vector inserted therein were selected and electroporation of the pAD02 vector as an I-SceI meganuclease expression vector was carried out by the method of Non-Patent Document 0010, ml LB solid medium containing tetracycline and incubated overnight at 30 ° C.

The whole base sequence of the pAD02 vector used as the I-SceI megaclasma expression vector in Fig. 4C is shown in SEQ ID NO: 4, and the map of the vector is shown in Fig.

The pAD02 vector consisting of the nucleotide sequence of SEQ ID NO: 4 contains the sequence of slpA promoter of SEQ ID NO: 5 from the 408th to 778th bases in the nucleotide sequence of SEQ ID NO: 4, the sequence of 806th to 1507th nucleotides Comprising a temperature sensitive replication origin sequence comprising the I-SceI meganuclease gene sequence of SEQ ID NO: 6 and a Bacillus sequence of SEQ ID NO: 7 from 2802 to 3154 sequence And a replication origin sequence of SEQ ID NO: 9, which contains the tetracycline resistance gene sequence of SEQ ID NO: 8, which is an antibiotic resistance gene, and which functions in E. coli, from the 5547th to 6135th bases, , And an ampicillin resistance gene sequence of SEQ ID NO: 10, which is an antibiotic resistance gene, at nucleotides 6306 to 7166 The lure is.

As described above, the pAD02 vector has a temperature-sensitive replication origin that operates in Bacillus strains and can be maintained in the B. thuringiensis strain BMB171 used in the embodiment of the present invention described below. And can be easily removed when cultured at high temperature (40 ° C) without antibiotics. In addition, when a strain is cultured in a tetracycline-containing medium having a tetracycline resistance gene (Tet ^r ), a strain having a pAD02 vector can be selectively grown. In addition, this vector is pAD02 Bacillus Turing Gen System (B. thuringiensis) using slpA promoter (slpA promoter) derived from YBT-020 strain continuously expressed (constitutively) the I-SceI Mega New Cloud kinase.

The I-SceI meganuclease expressed in this manner recognizes and cleaves the I-SceI recognition sequence region of the gene deletion vector inserted into the chromosome, which causes double strand breakage in the host chromosome DNA break, which is a serious DNA damage that can kill a host if it is not repaired.

Thus, DNA repair is performed to recover the DNA. In this process, the frequency of homologous recombination at the corresponding site is increased. As shown in FIG. 4d, the left homology arm of the host located on the chromosome and the gene Homologous recombination occurs between the left homology arm of the replacement vector or the right homology arm of the host and the right homology arm of the gene replacement vector.

At this time, depending on whether homologous recombination occurs in which gene arm, two results are obtained. As shown in FIG. 4E, the gene is restored to its original state or the target gene is removed as shown in FIG. 4F. In this process, the vector-derived sequence is removed. The strains that have undergone DNA repair can be easily distinguished because they lose their resistance to spectinomycin due to the elimination of the vector-derived sequence. Among them, the strains in which the target gene is deleted are easily discriminated by colony PCR can do.

In addition, since the pAD02 vector expresses the I-SceI meganuclease at a very low level, the frequency of DNA repair and homologous recombination is low, and the recombinant target strain Can be finally selected.

In the replica plating method, 10 colonies obtained by transforming with the pAD02 vector were selected and diluted in 1 ml LB liquid medium containing tetracycline. Then, 5 ul of the culture was added to 5 ml LB containing 20 ug / ml tetracycline Liquid medium and incubate at 37 ° C and 200 rpm for 6 hours. Thereafter, the culture medium is appropriately diluted, and then the resulting mixture is spread on 10 to 20 sheets of LB solid medium containing 20 ug / ml of tetracycline and cultured overnight at 30 캜. The next day, the colonies grown in this medium were transferred to replicate platings on 20 LB solid medium containing 200 ug / ml spectinomycin using a velvet cloth and cultured at 37 캜 to obtain LB containing spectinomycin Select colonies that do not grow on solid medium.

At this time, the selected colonies are colonies in which DNA repair occurs on the chromosome and the vector-derived sequence and the spectinomycin resistance gene (Spc ^r ) are removed. These selected colonies can be detected in two ways as described above (FIGS. 4E and 4F), so that it can be confirmed by colony PCR that the target gene has been removed as intended.

Once the pAD02 vector has been removed, the pAD02 vector should be removed. The pAD02 vector used has a temperature-sensitive replication origin and can be easily removed by incubation without antibiotics at 40 ° C. When the pAD02 vector is removed, the tetracycline resistance gene Can not be grown in the tetracycline-containing medium. Therefore, it is possible to confirm that the pAD02 vector has been removed by culturing in a tetracycline-containing medium.

Specifically, the selected colonies are inoculated into 5 ml of LB broth without antibiotics and cultured at 40 ° C and 200 rpm for 6 hours. The culture medium is then diluted appropriately and then plated on 5 ml LB solid medium without antibiotics and cultured overnight at 30 ° C. Finally, to determine if the pAD02 vector was removed, several of the grown colonies were picked and streaked onto the tetracycline-containing LB solid medium and cultured to see if the pAD02 vector was removed.

On the other hand, pAD02, pAD05 and pAD07 vectors used in the present invention are self-modified vectors based on pUC19 vector.

Hereinafter, the present invention will be described more specifically based on examples and experimental examples. However, these examples and experimental examples are merely examples of the present invention, and the scope of the present invention is not limited to these examples.

The Bacillus subtilis strain used in the practice of the present invention is Bacillus sp. thuringiensis strain B. thuringiensis strain BMB171 was purchased from Bacillus Genetic Stock Center (BGSC), which has one chromosome and one plasmid (pBMB171).

And Bacillus The genome information of the thuringiensis strain BMB171 was obtained from GenBank CP001903.1 sequence known from GenBank of National Center for Biotechnology Information (NCBI), which is a well-known database, and the genetic information such as gene name and gene insertion site Both the specific base sequence and protein information are in accordance with information known to the NCBI.

As a result of BLAST analysis using genomic sequence of B. thuringiensis BMB171 and base sequence of sspA gene or sspB gene of Bacillus subtilis, 7 homologous genes were found, and their amino acid sequence The results are shown in Fig. The seven homology genes are BMB171_C0753, BMB171_C1155, BMB171_C1771, BMB171_C2802, BMB171_C2818, BMB171_C4286 and BMB171_P0269, of which BMB171_P0269 is located on the plasmid pBMB171 and the remaining 6 genes are located on the chromosome. As shown in FIG. 1, the amino acid sequences of seven SASP proteins of Bacillus thuringiensis are very similar to each other, and there are many different parts from the amino acid sequence of Bacillus subtilis. Therefore, β type SASP, and no information from the literature was available since deletion of these genes was not performed in Bacillus thuringiensis .

In this study, we tried to find out which genes are homologous to two major α / β type SASPs of Bacillus subtilis by Sintonian analysis. In the Synthony analysis, Absynte (the Archaeal and Bacterial Synteny Explorer) 0009) (http://archaea.u-psud.fr/absynte) was used. As a result of analyzing the genome of B. thuringiensis BMB171 using the sspA amino acid sequence (NCBI reference sequence NP_390835.1) of 168 strains of Bacillus subtilis in the above Absynte web server as a query sequence, As a result of analyzing the genome of B. thuringiensis BMB171 using the sspB amino acid sequence (NCBI reference sequence NP_388856.1) of 168 strains of Bacillus subtilis as a query sequence, .

As shown in FIG. 2, several genes around the sspA gene of Bacillus subtilis are conserved only around the BMB171_C4286 gene, which is annotated as sspB in the NCBI database among seven SASP genes of Bacillus thuringiensis . The BMB171_C4286 gene is presumed to be an ortholog of sspA of Bacillus subtilis, and the annotation of sspB NCBI is considered to be an error not based on experimental evidence.

As shown in FIG. 3, several genes around the sspB gene of Bacillus subtilis are conserved only around the BMB171_C0753 gene, which is annotated with sasP1 in the NCBI database among the seven SASP genes of Bacillus turingensis ( B. thuringiensis ) . Therefore, the BMB171_C0753 gene is presumed to be an ortholog of sspB of Bacillus subtilis, and the annotation of NCBI called sasP1 is considered to be an error not based on experimental evidence.

Based on the above synaptic analysis, the orthologs corresponding to the two major α / β type SASPs of Bacillus subtilis were estimated as BMB171_C4286 ( sspA ) and BMB171_C0753 ( sspB ), and the gene deletion vector pAD05 ) And pAD07 (Fig. 7), respectively.

Specifically, the pAD05 vector consisting of the nucleotide sequence of SEQ ID NO: 11 includes a first left homology arm sequence of SEQ ID NO: 12 and a nucleotide sequence of 1921 Th to 3437th bases, the first right homology arm sequence of SEQ ID NO: 13, the 3888th to 4466th bases, the replication origin sequence that operates in E. coli of SEQ ID NO: 9, , The 4590th to 4607th bases, the I-SceI recognition sequence of SEQ ID NO: 3, the 4814th to 5596th bases, and the spectinomycin resistance gene sequence of SEQ ID NO: 14, which is an antibiotic resistance gene. The first left homology region and the first right homology region are about 1.5 kb on both sides of the target gene BMB171_C4286 as described in the gene removal process shown in FIG.

Specifically, the pAD07 vector consisting of the nucleotide sequence of SEQ ID NO: 15 includes a second left homology arm sequence of SEQ ID NO: 16 with bases 402 to 1907 in the nucleotide sequence of SEQ ID NO: 15, To the 3409th base, a second right homology arm sequence of SEQ ID NO: 17, a 3860th to 4438th base, a replication origin sequence operative in the E. coli of SEQ ID NO: 9, , The 4562nd to 4579th bases, the I-SceI recognition sequence of SEQ ID NO: 3, the 4786th to 5568th bases, and the spectinomycin resistance gene sequence of SEQ ID NO: 14, which is an antibiotic resistance gene. The first left homology region and the first right homology region are about 1.5 kb sites on both sides of the target gene BMB171_C0753 as described in the gene removal process shown in FIG.

The BMB171_C4286 gene and the BMB171_C0753 gene were sequentially removed from the wild-type B. thuringiensis BMB171 using the gene deletion vectors pAD05 and pAD07. First, the BMB171_C4286 gene was removed using the pAD05 vector. This strain was named BT-003, and the BMB171_C0753 gene was removed from the BT-003 strain using the pAD07 vector to produce a strain in which both genes were removed. BT-005. In order to use as a control for the UV sensitivity test, BMB171_C0753 gene was removed from the wild-type B. thuringiensis BMB171 using the pAD07 vector in order to prepare a strain from which only the BMB171_C0753 gene had been removed. 004. Thus, BT-003 strain is a sspA gene deletion strain, BT-004 strain is a sspB gene deletion strain, and BT-005 strain is a strain lacking both sspA and sspB genes.

In the following experimental example is to demonstrate the Bacillus Turing Gen System (B. thuringiensis) and the environmental friendliness of increasing in vivo reduction of the remaining property BT-005 strain spores prepared in this invention by experiments.

Specifically, in Experimental Example 1, the strains B. thuringiensis BT-003, BT-004, B. thuringiensis BT-004, Bacillus thuringiensis BT- BT-005 (B. thuringiensis BT- 005) and to control the hoekseon smear (streaking) a wild-type Bacillus Turing Gen cis BMB171 (B. thuringiensis BMB171) in LB solid medium were cultured overnight at 30 ℃, in 5 ml LB medium the next day One colony was inoculated and shake cultured at 30 DEG C and 200 rpm for 8 hours and then added to 250 ml of GYS medium (Non-Patent Document 0011), which was a 25 ml sporulation medium containing 125 ml flask, And cultured at 30 DEG C and 200 rpm for 48 hours to produce spores. The resulting spores were harvested by centrifugation at 5,400 g for 5 min at 4 ° C, suspended in 25 ml of phosphate buffered saline (PBS), centrifuged at 5,400 g for 5 min at 4 ° C, I abandoned it. This procedure was repeated three more times, washed 4 times with PBS, and finally suspended in 5 ml of PBS. The cells were then diluted appropriately in PBS and plated on TSA (Trypticase soy agar) solid medium to measure cell numbers.

The four spores thus prepared were each diluted so that 220 CFU was obtained from 200 CFU (Colony Forming Unit) per TSA solid plate. Immediately after the staining, it was placed on the clean bench bottom and irradiated with an Ultraviolet-C lamp for a given time. After the irradiation, the cells were wrapped in a silver foil and cultured overnight in a 30 ° C incubator. The number of colonies that grew the next day was counted. At this time, the ultraviolet-C intensity at the clean bench bottom was 78 uW / cm ² when measured with an ultraviolet-C measuring instrument, and the irradiation time was 0 second, 20 seconds, 40 seconds, 60 seconds, 80 seconds, , And all experiments were independently performed three times and averaged. In addition, the relative ratio of the number of colonies at each irradiation time was obtained as a percentage compared to the number of colonies at 0 second. The standard deviation of the triplet was also obtained.

As a result, as shown in FIG. 8, the sensitivity of the remaining three strains to the ultraviolet light was increased as compared with that of wild-type spores (BMB171). Especially, the spores of BT-005 strain in which both sspA and sspB genes were removed The synergy effect was observed, and it was confirmed that the sensitivity to ultraviolet light was dramatically increased.

When the two genes were removed, it was confirmed that the susceptibility of the spores to ultraviolet-C was dramatically increased as reported in Bacillus subtilis. However, since ultraviolet-C does not reach the surface due to the atmosphere in the natural world, It can not be said that it is simulated. In Experimental Example 2, the relative susceptibility of BT-005 spp. To which two genes were removed was measured for artifical sunlight compared to wild type spores. The spores were prepared by the method described in Experimental Example 1 using B. thuringiensis BT-005 and B. thuringiensis BMB171 as a control. (Suntest CPS plus solar simulator, manufacturer Atlas Material Testing Solutions) equipped with a cooling assembly. The test conditions of the artificial solar tester were set at 35 ° C for black standard temperature and 400w / m ² for irradiation level. The temperature in the chamber was between 20 ° C and 22 ° C and the relative humidity was 57 % To 60%. 1.2 ml of spore suspension at a concentration of 2.0 × 10 ⁸ CFU / ml was injected into a well of a 6-well plate, and then a quartz plate with a thickness of 2 mm was placed in the chamber and artificial sunlight was irradiated for a given time. After the irradiation, 1 ml of the suspension was taken out, appropriately diluted in PBS, plated on a TSA solid plate culture medium, and cultured overnight at 30 ° C in an incubator. Experiments were repeated three times independently, and the number and standard deviation of viable spores were determined for each irradiation time.

As a result, as shown in FIG. 9, the number of wild-type spores (BMB171) decreased from 2.0 × 10 ⁸ CFU / ml to 5.9 × 10 ⁵ CFU / ml after 50 minutes of solar irradiation, And decreased dramatically from 2.0 × 10 ⁸ CFU / ml to 2.0 × 10 ¹ CFU / ml or less. As a result, it was confirmed that the spore of the strain BT-005 significantly increased the susceptibility to artificial sunlight as compared with that of the wild-type spore (BMB171), which is consistent with the result of the ultraviolet-C susceptibility test.

Next, in order to confirm the stability with respect to temperature, B. thuringiensis BT-005 was used in Experimental Example 3 and B. thuringiensis BMB171 was used as a control. Spores were prepared by the method described in Example 1, and spores were diluted in PBS at a concentration of 1.0 × 10 ⁷ CFU / ml in a 25 ml Pyrex glass bottle, sealed with a film, and incubated in a 4 ° C. refrigerator and a 37 ° C. incubator 1 ml of the suspension was taken every week, appropriately diluted in PBS, plated on TSA solid plate culture medium, and cultured overnight at 30 ° C in an incubator to count the number of cells. The number of cells was measured from 0 to 10 weeks, and three samples were prepared for each spore and temperature, and triplicate experiments were carried out. As shown in FIG. 10, when stored at 4 ° C. for 10 weeks, the number of cells of wild-type spores (BMB171) did not decrease at all, whereas the number of cells of BT-005 strain began to decrease from 7 weeks and stored at 37 ° C. for 10 weeks The spore of strain BT-005 showed a dramatic decrease in cell number compared with wild-type spore (BMB171). Thus, it was confirmed that the spores of strain BT-005 were vulnerable to heat.

Next, experimental example 4, to compare the remaining of the animals in the living body Bacillus Turing Gen system BT-005 (B. thuringiensis BT- 005) and a control group, wild-type Bacillus Turing Gen cis BMB171 (B. thuringiensis BMB171) the experiment Each spore was prepared by the method described in Example 1, followed by anesthesia with a 6-week-old female Balb / c mouse. An incision of 1 cm to 2 cm was made in the neck region and the trachea was exposed to 2.0 × 10 ⁸ CFU / ml concentration (1.0 x 10 < ⁷ > CFU per individual) of the suspension of spores prepared as described above was intratracheal instillation using a flexible tube attached to a syringe. After injection, the skin was closed with 3-0 silk suture and disinfected with povidone iodide. Twenty four mice per spore of each strain were intraperitoneally intratracheal instillated and eight mice were used to measure the number of cells remaining in the lungs at 1, 2, and 4 weeks after injection . The mice were anesthetized, the thoracic cavity opened, the lungs were harvested, weighed, weighed, homogenized, serial diluted in PBS, and plated on TSA solid plate media. Thereafter, the cells were cultured overnight at 30 ° C in an incubator, and then the number of cells was measured. The results were expressed in terms of CFU per gram of lung weight, and the difference between the mean values of the other groups was analyzed. Statistical calculations were performed using GraphPad Prism (GraphPad software) and P values less than 0.05 were considered significant.

As shown in FIG. 11, the number of cells per tissue g was 1.2 × 10 ⁷ CFU, 9.8 × 10 ⁵ CFU, and 4.6 × 10 ⁴ CFU at 1 week, 2 weeks, and 4 weeks in wild type spores, The spore showed a dramatic reduction of 1.6 × 10 ⁴ CFU per gram of tissue per gram at week 1, a remarkable reduction of 2.9 × 10 ⁰ CFU (2.9 CFU) at 2 weeks and a 0 CFU at 4 weeks (BT-005, results of 2 and 4 weeks are too small to be shown on the graph, so the result of 1 week, 2 weeks, 4 weeks of BMB171 in A, and only results of 1 week of BMB171 and BT-005 in B Respectively. In comparison with the decrease in the number of cells obtained by storing spores of BT-005 at 37 ° C. for 4 weeks in the above Experimental Example 3 (FIG. 10), the decrease in cell number in vivo of BT-005 showed a dramatic difference, It was confirmed that the decrease in cell number was not caused by body temperature (37 ° C), and it was assumed that this decrease was due to the immune response of the living body.

The spores of Bacillus thuringiensis with the two genes sspA (BMB171_C4286 gene) and sspB (BMB171_C0753 gene) of the present invention removed from the above experiment showed remarkable sensitivity to ultraviolet rays, artificial sunlight and heat And it was confirmed that the survival in vivo was dramatically decreased. The strain produced in the present invention was a strain in which two genes described in SEQ ID NO: 1 and SEQ ID NO: 2 were deleted from B. thuringiensis BMB171, and was deposited at the Korea Institute of Bioscience and Biotechnology (KCTC) Bacillus Turing Gen cis to (Bacillus thuringiensis BT-005 (Accession No .: KCTC18560P).

The genes of SEQ ID NO: 1 and SEQ ID NO: 2 showed strains of the BMB171 strain used in the Examples among the strains of B. thuringiensis , but the genes of B. thuringiensis species the various strains belonging to the species are almost identical in structure of the genome, so that sspA of other strains of Bacillus thuringiensis predicted by applying the synaptic analysis described in the present invention sspB homology genes are also included within the scope of the present invention.

The term "BMB171_C4286", "BMB171_C0753", "BMB171_C1155", "BMB171_C1771", "BMB171_C2802", "BMB171_C2818", "BMB171_P0269" as the genes described in the present specification represents a locus tag indicating a specific gene, It is used in place of the gene symbol, and information on this is known from the database such as GenBank of the National Center for Biotechnology Information (NCBI) through the homepage (http://www.ncbi.nlm.nih.gov) Information and sequence. And such methods will be apparent to those skilled in the art.

It will be apparent to those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof.

In addition, the above detailed description should not be construed in all aspects as limiting and should be considered illustrative. The scope of the present invention should be determined by rational interpretation of the appended claims, and all changes within the scope of equivalents of the present invention are included in the scope of the present invention.

<110> ADD <120> DELETION OF TWO GENES OF BACILLUS THURINGIENSIS THAT CONFERS          ENVIRONMENTAL-FRIENDLINESS / REDUCTION OF IN VIVO PERSISTENCE ON          SPORES AND DOUBLE-KNOCKOUT STRAIN <130> PT20170070 <160> 17 <170> KoPatentin 3.0 <210> 1 <211> 198 <212> DNA <213> Artificial Sequence <220> <223> sspA (BMB171_C4286) ORF <400> 1 atgtcacgta gcacaaataa attagcggtt cctggtgctg aatcagcatt agaccaaatg 60 aaatacgaaa tcgctcaaga gtttggtgtt caacttggag ctgatgcaac agctcgcgct 120 aacggttctg ttggtggtga aatcactaaa cgtctagttt cactagctga gcaacaatta 180 ggcggttacc aaaaataa 198 <210> 2 <211> 204 <212> DNA <213> Artificial Sequence <220> <223> sspB (BMB171_C0753) ORF <400> 2 atggcaaacc aaaattcttc aaatcaatta gtagtaccag gtgcaacagc tgcaatcgat 60 caaatgaagt acgaaatcgc tcaagaattt ggtgtacaat taggagcaga ttctacggct 120 cgcgctaacg gttcagttgg tggagaaatc acaaaacgtc tagttgcaat ggctgagcaa 180 agccttggcg gattccacaa ataa 204 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> I-SceI recognition sequence <400> 3 tagggataac agggtaat 18 <210> 4 <211> 7366 <212> DNA <213> Artificial Sequence <220> PAD02 (I-SceI expression vector) <400> 4 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctctgt cgattaatgt 420 cgtaatatct ctatagttag atgttgtaat tggaaagctt tttctgttat agttgtaaat 480 ggttataaga agtttgttat aaatgtaatt ctgacgcgtt tttctattca tttaaaatat 540 tctgattttt agatctggat cctttaaaat gaaagtcgaa aaaacgatcg tttcgaattg 600 tgaaaattca cttttttgtc gaatttaagg ttttaaatta gatgaaaaga gtgtatgatt 660 ctttaatacg ggatattcta ttattctgta taaggaatac tttaaccaca tttatatcta 720 ctagtttcaa aaataaatag aatatctatt taaaacagta tggaactagt attacatttc 780 tagataaaag agatggaggt aacttttgca tcaaaaaaac caggtaatga acctgggtcc 840 gaactctaaa ctgctgaaag aatacaaatc ccagctgatc gaactgaaca tcgaacagtt 900 cgaagcaggt atcggtctga tcctgggtga tgcttacatc cgttctcgtg atgaaggtaa 960 aacctactgt atgcagttcg agtggaaaaa caaagcatac atggaccacg tatgtctgct 1020 gtacgatcag tgggtactgt ccccgccgca caaaaaagaa cgtgttaacc acctgggtaa 1080 cctggtaatc acctggggcg cccagacttt caaacaccaa gctttcaaca aactggctaa 1140 cctgttcatc gttaacaaca aaaaaaccat cccgaacaac ctggttgaaa actacctgac 1200 cccgatgtct ctggcatact ggttcatgga tgatggtggt aaatgggatt acaacaaaaa 1260 ctctaccaac aaatcgatcg tactgaacac ccagtctttc actttcgaag aagtagaata 1320 cctggttaag ggtctgcgta acaaattcca actgaactgt tacgtaaaaa tcaacaaaaa 1380 caaaccgatc atctacatcg attctatgtc ttacctgatc ttctacaacc tgatcaaacc 1440 gtacctgatc ccgcagatga tgtacaaact gccgaacact atctcctccg aaactttcct 1500 gaaataactg caggcatgca agcttcagaa cggattgttg atgattacga aaatattaag 1560 agcacagact attacacaga aaatcaagaa ttaaaaaaac gtagagagag tttgaaagaa 1620 gtagtgaata catggaaaga ggggtatcac gaaaaaagta aagaggttaa taaattaaag 1680 cgagagaatg atagtttgaa tgagcagttg aatgtatcag agaaatttca agatagtaca 1740 gtgactttat atcgtgctgc gagggcgaat ttccctgggt ttgagaaagg gtttaatagg 1800 cttaaagaga aattctttaa tgattccaaa ttcgagcgtg tgggacagtt tatggatgtt 1860 gtacaggata atgtccagaa ggtcgataga aagcgtgaga aacagcgtac agacgattta 1920 gagatgtaga ggtactttta tgccgagaaa actttttgcg tgtgacagtc cttaaaatat 1980 acttagagcg taagcgaaag tagtagcgac agctattaac tttcggttgc aaagctctag 2040 gatttttaat ggacgcagcg catcacacgc aaaaaggaaa ttggaataaa tgcgaaattt 2100 gagatgttaa ttaaagacct ttttgaggtc tttttttctt agatttttgg ggttatttag 2160 gggagaaaac ataggggggt actacgacct cccccctagg tgtccattgt ccattgtcca 2220 aacaaataaa taaatattgg gtttttaatg ttaaaaggtt gttttttatg ttaaagtgaa 2280 aaaaacagat gttgggaggt acagtgatgg ttgtagatag aaaagaagag aaaaaagttg 2340 ctgttacttt aagacttaca acagaagaaa atgagatatt aaatagaatc aaagaaaaat 2400 ataatattag caaatcagat gcaaccggta ttctaataaa aaaatatgca aaggaggaat 2460 acggtgcatt ttaaacaaaa aaagatagac agcactggca tgctgcctat ctatgactaa 2520 attttgttaa gtgtattagc accgttatta tatcatgagc gaaaatgtaa taaaagaaac 2580 tgaaaacaag aaaaattcaa gaggacgtaa ttggacattt gttttatatc cagaatcagc 2640 aaaagccgag tggttagagt atttaaaaga gttacacatt caatttgtag tgtctccatt 2700 acatgatagg gatactgata cagaaggtag gatgaaaaaa gagcattatc atattctagt 2760 gatgtatgag ggtaataaat cttatgaaca gataaaaata attacagaag aattgaatgc 2820 gactattccg cagattgcag gaagtgtgaa aggtcttgtg agatatatgc ttcacatgga 2880 cgatcctaat aaatttaaat atcaaaaaga agatatgata gtttatggcg gtgtagatgt 2940 tgatgaatta ttaaagaaaa caacaacaga tagatataaa ttaattaaag aaatgattga 3000 gtttattgat gaacaaggaa tcgtagaatt taagagttta atggattatg caatgaagtt 3060 taaatttgat gattggttcc cgcttttatg tgataactcg gcgtatgtta ttcaagaata 3120 tataaaatca aatcggtata aatctgaccg atagattttg aatttaagag tgtcacaaga 3180 cactcttttt tcgcaccagc gaaaactggt ttaagccgac tgcgcaaaag acataatcga 3240 ttcacaaaaa ataggcacac gaaaaacaag ttaagggatg cagtttatgc atcccttaac 3300 ttacttatta aataatttat agctattgaa aagagataag aattgttcaa agctaatatt 3360 gtttaaatcg tcaattcctg catgttttaa ggaattgtta aattgatttt ttgtaaatat 3420 tttcttgtat tctttgttgg ggatccacgc gtcttaaggc ggccgcggta ccgggcccgt 3480 cccgctcgag ccggccatat tgttgtataa gtgatgaaat actgaattta aaacttagtt 3540 tatatgtggt aaaatgtttt aatcaagttt aggaggaatt aattatgaag tgtaatgaat 3600 gtaacagggt tcaattaaaa gagggaagcg tatcattaac cctataaact acgtctgccc 3660 tcattattgg agggtgaaat gtgaatacat cctattcaca atcgaattta cgacacaacc 3720 aaattttaat ttggctttgc attttatctt tttttagcgt attaaatgaa atggttttga 3780 acgtctcatt acctgatatt gcaaatgatt ttaataaacc acctgcgagt acaaactggg 3840 tgaacacagc ctttatgtta accttttcca ttggaacagc tgtatatgga aagctatctg 3900 atcaattagg catcaaaagg ttactcctat ttggaattat aataaattgt ttcgggtcgg 3960 taattgggtt tgttggccat tctttctttt ccttacttat tatggctcgt tttattcaag 4020 gggctggtgc agctgcattt ccagcactcg taatggttgt agttgcgcgc tatattccaa 4080 aggaaaatag gggtaaagca tttggtctta ttggatcgat agtagccatg ggagaaggag 4140 tcggtccagc gattggtgga atgatagccc attatattca ttggtcctat cttctactca 4200 ttcctatgat aacaattatc actgttccgt ttcttatgaa attattaaag aaagaagtaa 4260 ggataaaagg tcattttgat atcaaaggaa ttatactaat gtctgtaggc attgtatttt 4320 ttatgttgtt tacaacatca tatagcattt cttttcttat cgttagcgtg ctgtcattcc 4380 tgatatttgt aaaacatatc aggaaagtaa cagatccttt tgttgatccc ggattaggga 4440 aaaatatacc ttttatgatt ggagttcttt gtgggggaat tatatttgga acagtagcag 4500 ggtttgtctc tatggttcct tatatgatga aagatgttca ccagctaagt actgccgaaa 4560 tcggaagtgt aattattttc cctggaacaa tgagtgtcat tattttcggc tacattggtg 4620 ggatacttgt tgatagaaga ggtcctttat acgtgttaaa catcggagtt acatttcttt 4680 ctgttagctt tttaactgct tcctttcttt tagaaacaac atcatggttc atgacaatta 4740 taatcgtatt tgttttaggt gggctttcgt tcaccaaaac agttatatca acaattgttt 4800 caagtagctt gaaacagcag gaagctggtg ctggaatgag tttgcttaac tttaccagct 4860 ttttatcaga gggaacaggt attgcaattg taggtggttt attatccata cccttacttg 4920 atcaaaggtt gttacctatg gaagttgatc agtcaactta tctgtatagt aatttgttat 4980 tacttttttc aggaatcatt gtcattagtt ggctggttac cttgaatgta tataaacatt 5040 ctcaaaggga tttctaaatc gttaagggat caactttggg agagagttca aaattgatcc 5100 tttttttata acaggcctcc gctcgaaagc ttggcgtaat catggtcata gctgtttcct 5160 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt 5220 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc 5280 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg 5340 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 5400 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 5460 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 5520 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 5580 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 5640 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 5700 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat 5760 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 5820 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 5880 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 5940 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt 6000 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 6060 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 6120 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 6180 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc 6240 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 6300 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca 6360 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct 6420 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca 6480 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 6540 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg 6600 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct 6660 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa 6720 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta 6780 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc 6840 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg 6900 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa 6960 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg 7020 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc 7080 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg 7140 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat 7200 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata 7260 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc 7320 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtc 7366 <210> 5 <211> 371 <212> DNA <213> Artificial Sequence <220> <223> The slpA promoter <400> 5 tgtcgattaa tgtcgtaata tctctatagt tagatgttgt aattggaaag ctttttctgt 60 tatagttgta aatggttata agaagtttgt tataaatgta attctgacgc gtttttctat 120 tcatttaaaa tattctgatt tttagatctg gatcctttaa aatgaaagtc gaaaaaacga 180 tcgtttcgaa ttgtgaaaat tcactttttt gtcgaattta aggttttaaa ttagatgaaa 240 agagtgtatg attctttaat acgggatatt ctattattct gtataaggaa tactttaacc 300 acatttatat ctactagttt caaaaataaa tagaatatct atttaaaaca gtatggaact 360 agtattacat t 371 <210> 6 <211> 702 <212> DNA <213> Artificial Sequence <220> <223> I-SceI meganuclease <400> 6 ttgcatcaaa aaaaccaggt aatgaacctg ggtccgaact ctaaactgct gaaagaatac 60 aaatcccagc tgatcgaact gaacatcgaa cagttcgaag caggtatcgg tctgatcctg 120 ggtgatgctt acatccgttc tcgtgatgaa ggtaaaacct actgtatgca gttcgagtgg 180 aaaaacaaag catacatgga ccacgtatgt ctgctgtacg atcagtgggt actgtccccg 240 ccgcacaaaa aagaacgtgt taaccacctg ggtaacctgg taatcacctg gggcgcccag 300 actttcaaac accaagcttt caacaaactg gctaacctgt tcatcgttaa caacaaaaaa 360 accatcccga acaacctggt tgaaaactac ctgaccccga tgtctctggc atactggttc 420 atggatgatg gtggtaaatg ggattacaac aaaaactcta ccaacaaatc gatcgtactg 480 aacacccagt ctttcacttt cgaagaagta gaatacctgg ttaagggtct gcgtaacaaa 540 ttccaactga actgttacgt aaaaatcaac aaaaacaaac cgatcatcta catcgattct 600 atgtcttacc tgatcttcta caacctgatc aaaccgtacc tgatcccgca gatgatgtac 660 aaactgccga acactatctc ctccgaaact ttcctgaaat aa 702 <210> 7 <211> 353 <212> DNA <213> Artificial Sequence <220> <223> Temperature sensitive replication origin <400> 7 ttacagaaga attataatgcg actattccgc agattgcagg aagtgtgaaa ggtcttgtga 60 gatatatgct tcacatggac gatcctaata aatttaaata tcaaaaagaa gatatgatag 120 tttatggcgg tgtagatgtt gatgaattat taaagaaaac aacaacagat agatataaat 180 taattaaaga aatgattgag tttattgatg aacaaggaat cgtagaattt aagagtttaa 240 tggattatgc aatgaagttt aaatttgatg attggttccc gcttttatgt gataactcgg 300 cgtatgttat tcaagaatat ataaaatcaa atcggtataa atctgaccga tag 353 <210> 8 <211> 1377 <212> DNA <213> Artificial Sequence <220> <223> tetracycline resistance gene <400> 8 gtgaatacat cctattcaca atcgaattta cgacacaacc aaattttaat ttggctttgc 60 attttatctt tttttagcgt attaaatgaa atggttttga acgtctcatt acctgatatt 120 gcaaatgatt ttaataaacc acctgcgagt acaaactggg tgaacacagc ctttatgtta 180 accttttcca ttggaacagc tgtatatgga aagctatctg atcaattagg catcaaaagg 240 ttactcctat ttggaattat aataaattgt ttcgggtcgg taattgggtt tgttggccat 300 tctttctttt ccttacttat tatggctcgt tttattcaag gggctggtgc agctgcattt 360 ccagcactcg taatggttgt agttgcgcgc tatattccaa aggaaaatag gggtaaagca 420 tttggtctta ttggatcgat agtagccatg ggagaaggag tcggtccagc gattggtgga 480 atgatagccc attatattca ttggtcctat cttctactca ttcctatgat aacaattatc 540 actgttccgt ttcttatgaa attattaaag aaagaagtaa ggataaaagg tcattttgat 600 atcaaaggaa ttatactaat gtctgtaggc attgtatttt ttatgttgtt tacaacatca 660 tatagcattt cttttcttat cgttagcgtg ctgtcattcc tgatatttgt aaaacatatc 720 aggaaagtaa cagatccttt tgttgatccc ggattaggga aaaatatacc ttttatgatt 780 ggagttcttt gtgggggaat tatatttgga acagtagcag ggtttgtctc tatggttcct 840 tatatgatga aagatgttca ccagctaagt actgccgaaa tcggaagtgt aattattttc 900 cctggaacaa tgagtgtcat tattttcggc tacattggtg ggatacttgt tgatagaaga 960 ggtcctttat acgtgttaaa catcggagtt acatttcttt ctgttagctt tttaactgct 1020 tcctttcttt tagaaacaac atcatggttc atgacaatta taatcgtatt tgttttaggt 1080 gggctttcgt tcaccaaaac agttatatca acaattgttt caagtagctt gaaacagcag 1140 gaagctggtg ctggaatgag tttgcttaac tttaccagct ttttatcaga gggaacaggt 1200 attgcaattg taggtggttt attatccata cccttacttg atcaaaggtt gttacctatg 1260 gaagttgatc agtcaactta tctgtatagt aatttgttat tacttttttc aggaatcatt 1320 gtcattagtt ggctggttac cttgaatgta tataaacatt ctcaaaggga tttctaa 1377 <210> 9 <211> 589 <212> DNA <213> Artificial Sequence <220> <223> Replication origin (pUC19) <400> 9 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 60 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 120 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 180 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 240 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 300 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 360 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 420 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac 480 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 540 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaa 589 <210> 10 <211> 861 <212> DNA <213> Artificial Sequence <220> <223> ampicillin resistance gene <400> 10 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 60 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca 120 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc 180 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc 240 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg 300 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta 360 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc 420 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt 480 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg 540 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct 600 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc 660 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct 720 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac 780 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc 840 tcactgatta agcattggta a 861 <210> 11 <211> 5853 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > pAD05 (sspA deletion) <400> 11 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cacacggtgt atatgtgtca 420 acgaaatctg cttgttcttc aaaagcaaat gaagtgagca gagtgttagt gtcaatggga 480 gtaccgcatg cagcagctgc atgcgctatt cgtattagtt tggcaccaga aaacacaatg 540 gaagaagtaa aacaatttga aggtattgta aaagagacga tgccaaaatt atatgaagtg 600 atgaggtaaa gaaattatgt tgaaatatga atatatttta gtgcgttacg gagaaatgac 660 gacaaaaggt aagaaccgtt ctaagtttgt aagcacatta aaagataacg tgaagttcaa 720 actgaaaaaa ttcccaaata ttaaaatcga tgcaacacat gatcgtatgt acatccagtt 780 aaatggtgaa gatcatgaag cagtctctga aagattgaag gatgtattcg gtattcataa 840 atttaactta gcgatgaaag taccatcaga attagaagac attaaagaag gtgcattagc 900 agctttctta caagtaaaag gtgatgtgaa aacatttaaa attactgtac accgttctta 960 taagcatttc ccaatgagaa cgatggaatt attacctgag attggcggac acattctaga 1020 aaatacagaa gatattacag tagatgttca taatccagat gtaaatgtac gtgtagagat 1080 ccgcagtggc tatagctata tcatgtgcga tgagcgtatg ggagctggcg gtttaccagt 1140 tggcgttggc ggaaaagtaa tggtacttct ttctggtggt attgatagcc cagtagcagc 1200 ttacttaacg atgaaacgcg gcgtatctgt ggaagcagtt cacttccata gtccaccttt 1260 cacaagtgag cgtgcaaaac aaaaagtaat cgatttagcg caagagttaa cgaagtattg 1320 taaacgagta acgcttcacc ttgttccgtt tacagaagtg caaaaaacga ttaataaaga 1380 aatcccatct agctattcaa tgacggttat gcgccgtatg atgatgcgta ttacagaacg 1440 tattgctgag gagcgtaacg cacttgcaat tacgactggt gaaagtcttg gacaagtagc 1500 aagccaaacg ttagatagta tgcatacgat taacgaagta acaaactacc cagttattcg 1560 tccgcttatt acgatggata aattagagat tattaaaatc gctgaagaaa tcggcacgta 1620 tgatatttca attcgtccat atgaagattg ctgtactgta ttcacaccag caagcccagc 1680 gacgaagccg aaacgtgaaa aagcaaatcg ttttgaagcg aaatacgatt tcacaccatt 1740 gatcgatgaa gctgtagcga acaaagaaac aatggtatta caaacggtag aagtagtggc 1800 ggaagaagaa aaatttgaag aacttttcta aaaccataaa ttaccatatc taaaacgtgt 1860 atgaagtcat gttatttatc acactctata ctcacaagga ggtgatataa catgggtacc 1920 taatcccata tgatggctta gaaagagcgg gttctcccgc tctttctttt tttgtgttgt 1980 gtcgctattt gtcggtaagt cgatgtaatt tcgtttacta attcgacaga actgaaaaat 2040 ttgtcaaaag aagagggttc aaaattaact gaaaattctt tattatataa aggaggcata 2100 aagaaaaggg aggatatttt atgaagcgag aagaattgtt ggcgccaccg tcttataatt 2160 tagtttctga aatagagaaa tatacaggtg ataaagggaa gttagcttta atttggcaag 2220 atgataaagg gaatcgaagg gaagttacat acgctgaatt aatgcaaggt gcgaataaaa 2280 ttggaaacgc ttttataaaa agtggtttgc aaaaagggga caagcttctc attatgatgc 2340 cgcgtttaat tgaagcgtac atgacgtata ttgctgctat taaagcagga tttgtcgtaa 2400 ttccaagctc ggaaatgtta cgtaaaaaag atatagaata tcgaattgga catggagaag 2460 taaaagcgat agtaagctat gagccgtata ttaggcagtt tgatgatata gaagcgatgg 2520 aatctcttcg aaaatttgtc ctgagcgagc agtcagtaga tggatgggtt aatttaaaaa 2580 cagcgttaga aacagagagt gacatgttag aaatggcgaa gacagataaa gaagatatgg 2640 tctttttatc ttacacgtca ggaacgacag gaaatccaaa aggtgtcgtt catacacatg 2700 cgtgggcata cgctcattta cgtacgagcg ctccaaattg gctcggaatt gaagagaatg 2760 atattgtatg ggcaacagct agtccaggct ggcaaaagtg gatttggagc ccgtttttag 2820 caactttagg atcaggggca acaggttttg tatatcacgg taagtttgaa ccgaaaacgt 2880 acctaaactt attagacgat aataaggtga atgtactttg ttgtacaccg actgagtata 2940 gattaatggc aaaggtagaa gatttaagcc agtacaattt agaggcatta catagcgctg 3000 tatcggcagg tgagccgtta aatagagaag tcattgaaac gttcaaaaag cactttcata 3060 ttacagtgag agacggctat gggcaaacgg aaaatacatt acttgttggt gtaatgaaag 3120 ggatggatat tagaccagga tcaatgggga aaccaacgcc aggtaaccac gtcgatatcg 3180 taaatgagga aggtactcca gttaaagtag gcgaagttgg tgatattgcg gttcacattg 3240 aaacgccggc cctctttaaa caatattaca aagacgatga gcgtacagcg atgcaatttc 3300 gcggtgatta ttatattaca ggtgataaag cgaagaaaga tgaagacggc tacttctggt 3360 ttgaagggcg cggcgatgat atcattatta gctctggtta tacaatcggg ccgtttgaag 3420 tagaggatgc acttgtagga tcctctagag tcgacctgca ggcatgcaag cttggcgtaa 3480 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata 3540 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta 3600 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa 3660 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 3720 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 3780 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 3840 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 3900 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 3960 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 4020 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 4080 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 4140 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 4200 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 4260 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 4320 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 4380 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 4440 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 4500 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 4560 aaaaggatct tcacctagat ccttttgggt agggataaca gggtaatcat gcaaatgtca 4620 ctaatattaa taaactatcg aaggaacaat ttctttctat tttcaatagt tacaaattgt 4680 ttcactaaat taaagtaata aagcgttctc taatttcaca agaggacgct ttattcttcc 4740 caaaaattgt tcaatattta tcaataaatc agtagtttta aaagtaagca cctgttattg 4800 caataaaatt agcctaattg agagaagttt ctatagaatt tttcatatac ttaacgagtg 4860 ctttcacctt tgaatatagt ccttcccact tatcatcaca ctctccccga tagccttttc 4920 tagctatatc cagtaaagtt acatgctctt taggtaaaag aggtatagcc cattctgcag 4980 cgacatcttt cgaggtaatt tcaccagtag tcactgtttg ccacattcga gctagggtta 5040 aaattacatt acgctcatca ccttttatcc cctcaattag ttctggcaaa gaatccttaa 5100 ttgctcttcg aatatctgtc aaaggtacgg agacaagtat acttgaagaa tcaggaccaa 5160 atagagaaat actattcttt cttgcttgtg ctaaaacaat agccaaatca ggatcatagc 5220 ttggttcctg aatttgtcca ttctcaaatt cacccctgag ccactcaccg tatataaatt 5280 ctctttttgg aggatattgc caagggacaa cttcactcct atttataacc gtaacttcaa 5340 gtggtctaac agaatccgta tttccaatct ttcctgatat agtcattagt ctttctgtta 5400 gtttttttcg agttaattga ggtaaactat gattcacgac gactagaaca tctacatcgc 5460 tgttaatgcg taaaccacca tttactgctg aaccaaatag atatactcca actattgaac 5520 ttccaaataa atcttttacg atttttaatg tttgaatcgc ttgatttggt atttttccgt 5580 taatcaaatt gctcatgatt tcacctcgtt gattatgttc atataaagtt tatattgata 5640 ctcaatttac ttaccctaga ttggacatat acttaaatta ctgttcaata aagctgaccg 5700 ttagcgttta agtacatcct ttcacaattt gtctacagat taataattat tctttattat 5760 acagatcgat cctctagcgc gccgacgtct aagaaaccat tattatcatg acattaacct 5820 ataaaaatag gcgtatcacg aggccctttc gtc 5853 <210> 12 <211> 1513 <212> DNA <213> Artificial Sequence <220> <223> Left homology arm 1 <400> 12 acacggtgta tatgtgtcaa cgaaatctgc ttgttcttca aaagcaaatg aagtgagcag 60 agtgttagtg tcaatgggag taccgcatgc agcagctgca tgcgctattc gtattagttt 120 ggcaccagaa aacacaatgg aagaagtaaa acaatttgaa ggtattgtaa aagagacgat 180 gccaaaatta tatgaagtga tgaggtaaag aaattatgtt gaaatatgaa tatattttag 240 tgcgttacgg agaaatgacg acaaaaggta agaaccgttc taagtttgta agcacattaa 300 aagataacgt gaagttcaaa ctgaaaaaat tcccaaatat taaaatcgat gcaacacatg 360 atcgtatgta catccagtta aatggtgaag atcatgaagc agtctctgaa agattgaagg 420 atgtattcgg tattcataaa tttaacttag cgatgaaagt accatcagaa ttagaagaca 480 ttaaagaagg tgcattagca gctttcttac aagtaaaagg tgatgtgaaa acatttaaaa 540 ttactgtaca ccgttcttat aagcatttcc caatgagaac gatggaatta ttacctgaga 600 ttggcggaca cattctagaa aatacagaag atattacagt agatgttcat aatccagatg 660 taaatgtacg tgtagagatc cgcagtggct atagctatat catgtgcgat gagcgtatgg 720 ggctggcgg tttaccagtt ggcgttggcg gaaaagtaat ggtacttctt tctggtggta 780 ttgatagccc agtagcagct tacttaacga tgaaacgcgg cgtatctgtg gaagcagttc 840 acttccatag tccacctttc acaagtgagc gtgcaaaaca aaaagtaatc gatttagcgc 900 aagagttaac gaagtattgt aaacgagtaa cgcttcacct tgttccgttt acagaagtgc 960 aaaaaacgat taataaagaa atcccatcta gctattcaat gacggttatg cgccgtatga 1020 tgatgcgtat tacagaacgt attgctgagg agcgtaacgc acttgcaatt acgactggtg 1080 aaagtcttgg acaagtagca agccaaacgt tagatagtat gcatacgatt aacgaagtaa 1140 caaactaccc agttattcgt ccgcttatta cgatggataa attagagatt attaaaatcg 1200 ctgaagaaat cggcacgtat gatatttcaa ttcgtccata tgaagattgc tgtactgtat 1260 tcacaccagc aagcccagcg acgaagccga aacgtgaaaa agcaaatcgt tttgaagcga 1320 aatacgattt cacaccattg atcgatgaag ctgtagcgaa caaagaaaca atggtattac 1380 aaacggtaga agtagtggcg gaagaagaaa aatttgaaga acttttctaa aaccataaat 1440 taccatatct aaaacgtgta tgaagtcatg ttatttatca cactctatac tcacaaggag 1500 gtgatataac atg 1513 <210> 13 <211> 1517 <212> DNA <213> Artificial Sequence <220> <223> Right homology arm 1 <400> 13 taatcccata tgatggctta gaaagagcgg gttctcccgc tctttctttt tttgtgttgt 60 gtcgctattt gtcggtaagt cgatgtaatt tcgtttacta attcgacaga actgaaaaat 120 ttgtcaaaag aagagggttc aaaattaact gaaaattctt tattatataa aggaggcata 180 aagaaaaggg aggatatttt atgaagcgag aagaattgtt ggcgccaccg tcttataatt 240 tagtttctga aatagagaaa tatacaggtg ataaagggaa gttagcttta atttggcaag 300 atgataaagg gaatcgaagg gaagttacat acgctgaatt aatgcaaggt gcgaataaaa 360 ttggaaacgc ttttataaaa agtggtttgc aaaaagggga caagcttctc attatgatgc 420 cgcgtttaat tgaagcgtac atgacgtata ttgctgctat taaagcagga tttgtcgtaa 480 ttccaagctc ggaaatgtta cgtaaaaaag atatagaata tcgaattgga catggagaag 540 taaaagcgat agtaagctat gagccgtata ttaggcagtt tgatgatata gaagcgatgg 600 aatctcttcg aaaatttgtc ctgagcgagc agtcagtaga tggatgggtt aatttaaaaa 660 cagcgttaga aacagagagt gacatgttag aaatggcgaa gacagataaa gaagatatgg 720 tctttttatc ttacacgtca ggaacgacag gaaatccaaa aggtgtcgtt catacacatg 780 cgtgggcata cgctcattta cgtacgagcg ctccaaattg gctcggaatt gaagagaatg 840 atattgtatg ggcaacagct agtccaggct ggcaaaagtg gatttggagc ccgtttttag 900 caactttagg atcaggggca acaggttttg tatatcacgg taagtttgaa ccgaaaacgt 960 acctaaactt attagacgat aataaggtga atgtactttg ttgtacaccg actgagtata 1020 gattaatggc aaaggtagaa gatttaagcc agtacaattt agaggcatta catagcgctg 1080 tatcggcagg tgagccgtta aatagagaag tcattgaaac gttcaaaaag cactttcata 1140 ttacagtgag agacggctat gggcaaacgg aaaatacatt acttgttggt gtaatgaaag 1200 ggatggatat tagaccagga tcaatgggga aaccaacgcc aggtaaccac gtcgatatcg 1260 taaatgagga aggtactcca gttaaagtag gcgaagttgg tgatattgcg gttcacattg 1320 aaacgccggc cctctttaaa caatattaca aagacgatga gcgtacagcg atgcaatttc 1380 gcggtgatta ttatattaca ggtgataaag cgaagaaaga tgaagacggc tacttctggt 1440 ttgaagggcg cggcgatgat atcattatta gctctggtta tacaatcggg ccgtttgaag 1500 tagaggatgc acttgta 1517 <210> 14 <211> 783 <212> DNA <213> Artificial Sequence <220> <223> Spectinomycin resistance gene <400> 14 atgagcaatt tgattaacgg aaaaatacca aatcaagcga ttcaaacatt aaaaatcgta 60 aaagatttat ttggaagttc aatagttgga gtatatctat ttggttcagc agtaaatggt 120 ggtttacgca ttaacagcga tgtagatgtt ctagtcgtcg tgaatcatag tttacctcaa 180 ttaactcgaa aaaaactaac agaaagacta atgactatat caggaaagat tggaaatacg 240 gattctgtta gaccacttga agttacggtt ataaatagga gtgaagttgt cccttggcaa 300 tatcctccaa aaagagaatt tatatacggt gagtggctca ggggtgaatt tgagaatgga 360 caaattcagg aaccaagcta tgatcctgat ttggctattg ttttagcaca agcaagaaag 420 aatagtattt ctctatttgg tcctgattct tcaagtatac ttgtctccgt acctttgaca 480 gatattcgaa gagcaattaa ggattctttg ccagaactaa ttgaggggat aaaaggtgat 540 gagcgtaatg taattttaac cctagctcga atgtggcaaa cagtgactac tggtgaaatt 600 acctcgaaag atgtcgctgc agaatgggct atacctcttt tacctaaaga gcatgtaact 660 ttactggata tagctagaaa aggctatcgg ggagagtgtg atgataagtg ggaaggacta 720 tattcaaagg tgaaagcact cgttaagtat atgaaaaatt ctatagaaac ttctctcaat 780 tag 783 <210> 15 <211> 5825 <212> DNA <213> Artificial Sequence <220> <223> pAD07 (sspB deletion) <400> 15 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgtagaacgg tttcttggca 420 cgtataaatc agtgtacatg aaaccaatac acgggagctt tggtagaaat attcatcaaa 480 tattttattc tcaaacagag aattgctact actgtcgtta tcgcgaaaat gaagaaaata 540 aactgagaaa gtatcaatca ttagaaacgc ttctaaacca cgtattaaaa ggacatgatt 600 taaaaaagtt tatcgtacag caaggtattt cgttacttcg cttcgatggg caacctgttg 660 atttccgcat tcatacaaat aaaaaccatt tcggtaattg gatagttagt gcaatcgtcg 720 ccaaaatcgc cggcaaaggc agtttaacaa cgcacgtaaa tagtggcggt gatacaaaat 780 tattacaaga gctttttcca gattcaacga aacaagttca aattgaaaat aaattaaaac 840 atactgcctt acaaataagt tacgcgctcg atgaacaagt aactggaaat attggagaaa 900 tcggttttga tatcggttta gacacaaagg aaaatccgtg gctctttgaa gccaactcta 960 aacctgggcg aactgtattc caagataaaa aattaaaaga gcaaagtgaa ctgacgcgcc 1020 agttatttta cgaatatgcc gtctacttaa ctgaacattc tttacgtgat acaaaagaaa 1080 aaatgtctca aataaaatca ggggcttcat caaacaccga acatatccct tctcctatga 1140 ttcactcaca aatacaaaaa caaaaacttc cacctagata ataaggtgga agttttctat 1200 ttacacaagt tttactacaa cctcagtttc cacgttacat actctgcaat aaaaaatatg 1260 attgcaatat tcatttgaat tattatgcgt taaaccatct actgcactta ataattcttg 1320 atccatatag gcactataat cgtctatgta atctacagac ttcccataat cttggagtat 1380 actttggcaa ttttgacaag aatatgtttg tgattctaat gcgttacaaa gcgggcaaat 1440 ccccatcatg attcctccat acgttatcgt ttataagcct ctctttttta gagagaagag 1500 agcaacctat ccagacatag gagcgcccac tccatcccta cttaaaaata gaactatgaa 1560 gttgctaccg atttcatttt tatttcaata aatcataaat aagaagctaa taattagcct 1620 acaagtagca ggctacaatg aaaaaattat atttaatatc ccccttagtt tggcctgttt 1680 tcttaaaaat acaacgatta ctcttatttc ataatttacg aaatcaatac gtttttttga 1740 caaaatttac aaattattat tttgtatatc tttctaggta acatccatac tatgagtaca 1800 aacttaatta ccgatggatt agcgccaacc ggggcgataa tctggttcaa ctccagctag 1860 tccaaccaaa aaaactttta taactctaag gaggatatat tcctatgggt acctaaatat 1920 atatggcaaa aaggtctcaa ggcatatgcc ttgagacctt ccttaattta ttgcaacatt 1980 tgtaaaaact tcttcgtgcg ttcttctttt ggatttgtaa atacttcttc tggtgtacct 2040 tgttcgacaa cgacaccacc atccatgaaa ataacacggt tcgcaatttg atgtgcaaaa 2100 cgcatttcgt gcgttacaat aaccatcgtc atgccctctt tagcaagttc tttcattact 2160 ttcagaactt cttgaacgag ttcaggatca agcgctgacg tcggctcatc aaatagtaac 2220 acttccggct ccatcgcaag tgcgcgcgca attccaacgc gctgttgttg tccgcctgat 2280 aattggaacg gatataaatc cactttatct gctaaaccaa ctttttcaag gaaatagttt 2340 gcttttttct tcgcttcttc tttccccatc tttttcactg taacgagccc ttccattaca 2400 ttttgaagtg ctgttaaatg cgggaataaa ttatggtgct ggaataccat acctgtttgc 2460 gtacgaagat taacaatatc tttcttcgtt actttttgag agaagttaag ctctttatta 2520 ccgatacgaa tattaccagc gtttggcgtt tctaatacat tgagacaacg taagaatgtc 2580 gttttaccag atccagacgg cccaataata acaacaacct cgcccttctc aactgttaaa 2640 tctatatgct ttagtacggt attatctccg aaactttttt gtaagtgctg aattgaaatc 2700 ataaaaacaa aaactccttt tgtaaaagga ttattttaat gtatagcgtt ctgaacgctt 2760 ttctaacatc tgttgtacga ttgataataa gaaacaaata acccagtaaa taagacctgc 2820 ttcaaaataa acaattaaaa actcgtagtt catcgccgca atttcctgtg cttttcggaa 2880 catttctgtt actaaaatta acgatgctaa tgaagtatcc ttcactaagc taataaatgt 2940 atttgaaagc ggcgggattg atacgcgcgt tgcttgtggt aaaataacac gttttaatgc 3000 ttgtggatat gtcatcccaa ttgtataagc tgcttcccac tgccctttcg gaatggaaag 3060 gatagaagca cgaataattt cagatgcata tgcaccgaca tttaatgaaa atccaacaac 3120 tgctgctgta tatggctcaa cttcaatatt aagagttgga agaccataga aaataataa 3180 taactgtaca agaagtggtg ttccgcgaat gatagataca tagatacgag caatccattg 3240 taaaatacga ctacctgaaa tacgtgcgag cgctgttaac gtcgcaagta taagaccgat 3300 aataaatgta ataagcgtta atggaattgt tgtaaaaaca gcttccttca gcataggcat 3360 gaaggaagtc tgcataatat ctatccaagt agacaatcga tctgaaactg gatcctctag 3420 agtcgacctg caggcatgca agcttggcgt aatcatggtc atagctgttt cctgtgtgaa 3480 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct 3540 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc 3600 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg 3660 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc 3720 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag 3780 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa 3840 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc 3900 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc 3960 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg 4020 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt 4080 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc 4140 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc 4200 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag 4260 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg 4320 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa 4380 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag 4440 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact 4500 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttgg 4560 gtagggataa cagggtaatc atgcaaatgt cactaatatt aataaactat cgaaggaaca 4620 atttctttct attttcaata gttacaaatt gtttcactaa attaaagtaa taaagcgttc 4680 tctaatttca caagaggacg ctttattctt cccaaaaatt gttcaatatt tatcaataaa 4740 tcagtagttt taaaagtaag cacctgttat tgcaataaaa ttagcctaat tgagagaagt 4800 ttctatagaa tttttcatat acttaacgag tgctttcacc tttgaatata gtccttccca 4860 cttatcatca cactctcccc gatagccttt tctagctata tccagtaaag ttacatgctc 4920 tttaggtaaa agaggtatag cccattctgc agcgacatct ttcgaggtaa tttcaccagt 4980 agtcactgtt tgccacattc gagctagggt taaaattaca ttacgctcat caccttttat 5040 cccctcaatt agttctggca aagaatcctt aattgctctt cgaatatctg tcaaaggtac 5100 ggagacaagt atacttgaag aatcaggacc aaatagagaa atactattct ttcttgcttg 5160 tgctaaaaca atagccaaat caggatcata gcttggttcc tgaatttgtc cattctcaaa 5220 ttcacccctg agccactcac cgtatataaa ttctcttttt ggaggatatt gccaagggac 5280 aacttcactc ctatttataa ccgtaacttc aagtggtcta acagaatccg tatttccaat 5340 ctttcctgat atagtcatta gtctttctgt tagttttttt cgagttaatt gaggtaaact 5400 atgattcacg acgactagaa catctacatc gctgttaatg cgtaaaccac catttactgc 5460 tgaaccaaat agatatactc caactattga acttccaaat aaatctttta cgatttttaa 5520 tgtttgaatc gcttgatttg gtatttttcc gttaatcaaa ttgctcatga tttcacctcg 5580 ttgattatgt tcatataaag tttatattga tactcaattt acttacccta gattggacat 5640 atacttaaat tactgttcaa taaagctgac cgttagcgtt taagtacatc ctttcacaat 5700 ttgtctacag attaataatt attctttatt atacagatcg atcctctagc gcgccgacgt 5760 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt 5820 tcgtc 5825 <210> 16 <211> 1506 <212> DNA <213> Artificial Sequence <220> <223> Left homology arm 2 <400> 16 gtagaacggt ttcttggcac gtataaatca gtgtacatga aaccaataca cgggagcttt 60 ggtagaaata ttcatcaaat attttattct caaacagaga attgctacta ctgtcgttat 120 cgcgaaaatg aagaaaataa actgagaaag tatcaatcat tagaaacgct tctaaaccac 180 gtattaaaag gacatgattt aaaaaagttt atcgtacagc aaggtatttc gttacttcgc 240 ttcgatgggc aacctgttga tttccgcatt catacaaata aaaaccattt cggtaattgg 300 atagttagtg caatcgtcgc caaaatcgcc ggcaaaggca gtttaacaac gcacgtaaat 360 agtggcggtg atacaaaatt attacaagag ctttttccag attcaacgaa acaagttcaa 420 attgaaaata aattaaaaca tactgcctta caaataagtt acgcgctcga tgaacaagta 480 actggaaata ttggagaaat cggttttgat atcggtttag acacaaagga aaatccgtgg 540 ctctttgaag ccaactctaa acctgggcga actgtattcc aagataaaaa attaaaagag 600 caaagtgaac tgacgcgcca gttattttac gaatatgccg tctacttaac tgaacattct 660 ttacgtgata caaaagaaaa aatgtctcaa ataaaatcag gggcttcatc aaacaccgaa 720 catatccctt ctcctatgat tcactcacaa atacaaaaac aaaaacttcc acctagataa 780 taaggtggaa gttttctatt tacacaagtt ttactacaac ctcagtttcc acgttacata 840 ctctgcaata aaaaatatga ttgcaatatt catttgaatt attatgcgtt aaaccatcta 900 ctgcacttaa taattcttga tccatatagg cactataatc gtctatgtaa tctacagact 960 tcccataatc ttggagtata ctttggcaat tttgacaaga atatgtttgt gattctaatg 1020 cgttacaaag cgggcaaatc cccatcatga ttcctccata cgttatcgtt tataagcctc 1080 tcttttttag agagaagaga gcaacctatc cagacatagg agcgcccact ccatccctac 1140 ttaaaaatag aactatgaag ttgctaccga tttcattttt atttcaataa atcataaata 1200 agaagctaat aattagccta caagtagcag gctacaatga aaaaattata tttaatatcc 1260 cccttagttt ggcctgtttt cttaaaaata caacgattac tcttatttca taatttacga 1320 aatcaatacg tttttttgac aaaatttaca aattattatt ttgtatatct ttctaggtaa 1380 catccatact atgagtacaa acttaattac cgatggatta gcgccaaccg gggcgataat 1440 ctggttcaac tccagctagt ccaaccaaaa aaacttttat aactctaagg aggatatatt 1500 cctatg 1506 <210> 17 <211> 1496 <212> DNA <213> Artificial Sequence <220> <223> Right homology arm 2 <400> 17 taaatatata tggcaaaaag gtctcaaggc atatgccttg agaccttcct taatttattg 60 caacatttgt aaaaacttct tcgtgcgttc ttcttttgga tttgtaaata cttcttctgg 120 tgtaccttgt tcgacaacga caccaccatc catgaaaata acacggttcg caatttgatg 180 tgcaaaacgc atttcgtgcg ttacaataac catcgtcatg ccctctttag caagttcttt 240 cattactttc agaacttctt gaacgagttc aggatcaagc gctgacgtcg gctcatcaaa 300 tagtaacact tccggctcca tcgcaagtgc gcgcgcaatt ccaacgcgct gttgttgtcc 360 gcctgataat tggaacggat ataaatccac tttatctgct aaaccaactt tttcaaggaa 420 atagtttgct tttttcttcg cttcttcttt ccccatcttt ttcactgtaa cgagcccttc 480 cattacattt tgaagtgctg ttaaatgcgg gaataaatta tggtgctgga ataccatacc 540 tgtttgcgta cgaagattaa caatatcttt cttcgttact ttttgagaga agttaagctc 600 tttattaccg atacgaatat taccagcgtt tggcgtttct aatacattga gacaacgtaa 660 gaatgtcgtt ttaccagatc cagacggccc aataataaca acaacctcgc ccttctcaac 720 tgttaaatct atatgcttta gtacggtatt atctccgaaa cttttttgta agtgctgaat 780 tgaaatcata aaaacaaaaa ctccttttgt aaaaggatta ttttaatgta tagcgttctg 840 aacgcttttc taacatctgt tgtacgattg ataataagaa acaaataacc cagtaaataa 900 gacctgcttc aaaataaaca attaaaaact cgtagttcat cgccgcaatt tcctgtgctt 960 ttcggaacat ttctgttact aaaattaacg atgctaatga agtatccttc actaagctaa 1020 taaatgtatt tgaaagcggc gggattgata cgcgcgttgc ttgtggtaaa ataacacgtt 1080 ttaatgcttg tggatatgtc atcccaattg tataagctgc ttcccactgc cctttcggaa 1140 tggaaaggat agaagcacga ataatttcag atgcatatgc accgacattt aatgaaaatc 1200 caacaactgc tgctgtatat ggctcaactt caatattaag agttggaaga ccatagaaaa 1260 taataaataa ctgtacaaga agtggtgttc cgcgaatgat agatacatag atacgagcaa 1320 tccattgtaa aatacgacta cctgaaatac gtgcgagcgc tgttaacgtc gcaagtataa 1380 gaccgataat aaatgtaata agcgttaatg gaattgttgt aaaaacagct tccttcagca 1440 taggcatgaa ggaagtctgc ataatatcta tccaagtaga caatcgatct gaaact 1496

Claims (4)

B. thuringiensis strain for pesticide use, wherein the sspA gene consisting of the nucleotide sequence of SEQ ID NO: 1 and the sspB gene consisting of the nucleotide sequence of SEQ ID NO: 2 are all removed and the spore viability is reduced.
The sspA gene consisting of the nucleotide sequence of SEQ ID NO: 1 and the sspB gene consisting of the nucleotide sequence of SEQ ID NO: 2 were all removed to reduce the in vivo persistence of the spores. B. thuringiensis deposited with the deposit number KCTC18560P ) Strain.
B. thuringiensis strain for a mimetic agent in which the sspA gene consisting of the nucleotide sequence of SEQ ID NO: 1 and the sspB gene consisting of the nucleotide sequence of SEQ ID NO: 2 are all removed to reduce the in vivo persistence of spores.
The sspA gene consisting of the nucleotide sequence of SEQ ID NO: 1 and the sspB gene consisting of the nucleotide sequence of SEQ ID NO: 2 were all removed to reduce the in vivo persistence of the spores. B. thuringiensis for the mimetic agent deposited with the deposit number KCTC18560P ) Strain.

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