KR100470907B1 - Transposon for bidirectional intramolecular genome deletions, construction of novel microorganism and identification of nonessential genes using the same - Google Patents

Abstract

The present invention relates to the removal of any site of a chromosome using an intramolecular translocation and a selection marker of a transposon, and more particularly, to the translocation enzyme recognition site (OE) of the transposon. And inserting the transposon and the selection markers into the microorganism by inserting the transposon and the transposase expression vector into the microorganism to remove any part of the microbial chromosome in both directions, thereby selecting genes unnecessary for growth under a given growth condition. At the same time, it relates to a new method for preparing new microbial mutants with some genes removed.

Description

TRANSPOZON used for intramolecular bidirectional chromosome removal, microbial mutant strain production and growth non-essential gene selection method using the same

The present invention relates to the removal of any site of a chromosome by using an intramolecular translocation and a selection marker of a transposon, and more particularly, the translocation enzyme recognition site (OE) of a transposon ) And a transposon containing a selection marker and the transposon and a transposase expression vector in both directions to remove any portion of the microbial chromosome, thereby selecting a gene that is unnecessary for growth under a given growth condition, A new method of making new microbial mutants removed.

In general, techniques for studying the function of a gene by inserting the transposon into the chromosome and losing the gene function at the position where the transposon is inserted are already known. However, no method of arbitrarily removing the inserted periphery using the inserted transposon has been used. In addition, techniques for removing peripheral DNA fragments using transposons on a vector have been known, but technology for inserting non-essential genes by inserting them into a chromosome of an organism to select non-essential growth genes and preparing microbial mutant strains from which chromosomes have been removed Nothing has been reported yet.

The present invention improves the conventional method of removing specific regions of a chromosome through several trials and errors in preparing a microorganism mutant strain from which specific regions of a chromosome have been removed due to the genetic function of the microorganisms which have not yet been fully revealed. It is an object of the present invention to provide a method for easily selecting and removing genes unnecessary for survival in a chromosome and a transposon used therein.

The present invention relates to the removal of any site of the chromosome using the intramolecular translocation of the transposon and the selection marker, and more particularly, the transposon comprising the translocation enzyme recognition site and the selection marker of the transposon and the transposon and translocation enzyme expression vector. The present invention relates to a novel method for producing a new microbial mutant strain in which a part of a gene is removed while selecting genes unnecessary for growth under given growth conditions by bidirectionally removing an arbitrary portion of a microbial chromosome.

The present invention provides a method of preparing a transposon comprising a transposon recognition site and a selection marker of a transposon and any chromosomal variant wherein the chromosomal portion is removed, comprising the following steps:

(1) preparing a transposon comprising a transposon translocation enzyme recognition site and a selection marker,

(2) inserting the transposon at any position of the microbial chromosome and confirming the inserted position,

(3) inserting a translocation enzyme expression vector into the chromosome to remove left and right chromosomal portions of the inserted transposon,

(4) identifying the removed chromosomal portion and selecting growth essential genes.

In the transposon according to the present invention and step (1), the transposon comprises a translocation enzyme recognition site of Tnγδ and a translocation enzyme recognition site of Tn5 as the transposon translocation enzyme recognition site, and Bacillus subtilis as the selection marker. Negative selection markers such as the sacB gene and the tetracycline repressor gene (tetR) of Tn10 and positive selection markers such as the chloramphenicol resistance gene (Cm ^R ). In this case, the Tn5 translocation enzyme recognition site is for insertion into the chromosome of the transposon, the Tnγδ translocation enzyme recognition site is for intramolecular translocation, and the selection marker is for selecting the mutant strain into which the transposon is inserted.

In one embodiment of the present invention, the transposon according to the present invention is a translocation enzyme recognition site of Tn5 consisting of 19 base pairs at both ends, tetR and sacB genes used as negative selection markers, and Cm ^R which is commonly used as positive selection markers. It includes a gene, and the translocation enzyme recognition site of Tnγδ consisting of 39 base pairs at both ends of the Cm ^R gene. More specifically, the Tn5 translocation enzyme recognition site (OE) having the sequence of SEQ ID NO: 2, the tetR gene having the sequence of SEQ ID NO: 4, the Tnγδ translocation enzyme recognition site having the sequence of SEQ ID NO: 3, and the sequence of SEQ ID NO: 5 5 '→ 3' direction of the Cm ^R gene, Tnγδ translocation enzyme recognition site which is reverse complementary to sequence of SEQ ID NO: 3, sacB gene having sequence of SEQ ID NO: 6, and Tn5 translocation enzyme recognition site which is reverse complementary to sequence of SEQ ID NO: 2 In order to prepare a transposon including one by one, it was named TnRIBD. At this time, the parts excluding the Tn5 translocation enzyme recognition site, the Tnγδ translocation enzyme recognition site, and the selection marker site, which are essential components of the transposon according to the present invention, may have various sequences and lengths depending on the type of the vector used in the preparation of the transposon. Can be.

According to one embodiment of the invention, the transposon TnRIBD can be obtained by treating restriction enzyme XhoI from a pRIBD vector (see Figure 2).

As described above, the method for preparing TnRIBD from a pRIBD vector may include the following steps:

(i) pDELTA2 (Wang, G. et al 1993. pDUAL:. a transposon-based cosmid cloning vector for generating nested deletions and DNA sequencing templates in vivo Proc Natl Acad Sci US A. 90:. 7874-8) sacB vector OE of Tnγδ, tetR in Tn10, Cm in pKClox (Yoon, YG et al. 1998. Cre / loxP-mediated excision and amplification of large segments of the Escherichia coli genome.Genet Anal. 14: 89-95) vector Amplifying the ^R gene by performing PCR,

(ii) inserting OEs of sacB, tetR, Cm ^R , and Tnγδ into a linear pMOD vector using a ligase to prepare a pRIBD vector, and

(iii) cutting the pRIBD vector with restriction enzyme XhoI to prepare linear TnRIBD.

The base sequence of the transposon TnRIBD prepared in this manner is as follows, which is shown in SEQ ID NO: 1.

TnRIBD Sequence

1 CTCGAG CTGT CTCTTATACA CATCT CAACC CTGAAGCTAT CTTCCGAAGC AATAAATTCA

∥ Tn5 OE → ∥

61 CGTAATAACG TTGGCAAGAC TGGCATGATA AGGCCAATCC CCATGGCATC GAGTAACGTA

121 ATTACCAATG CGATCTTTGT CGAACTATTC ATTTCACTTT TCTCTATCAC TGATAGGGAG

181 TGGTAAAATA ACTCTATCAA TGATAGAGTG TCAACAAAAA TTAGGAATTA ATG ATGTCTA

                  ∥ ← tetR

241 GATTAGATAA AAGTAAAGTG ATTAACAGCG CATTAGAGCT GCTTAATGAG GTCGGAATCG

301 AAGGTTTAAC AACCCGTAAA CTCGCCCAGA AGCTAGGTGT AGAGCAGCCT ACATTGTATT

361 GGCATGTAAA AAATAAGCGG GCTTTGCTCG ACGCCTTAGC CATTGAGATG TTAGATAGGC

421 ACCATACTCA CTTTTGCCCT TTAGAAGGGG AAAGCTGGCA AGATTTTTTA CGTAATAACG

481 CTAAAAGTTT TAGATGTGCT TTACTAAGTC ATCGCGATGG AGCAAAAGTA CATTTAGGTA

541 CACGGCCTAC AGAAAAACAG TATGAAACTC TCGAAAATCA ATTAGCCTTT TTATGCCAAC

601 AAGGTTTTTC ACTAGAGAAT GCATTATATG CACTCAGCGC TGTGGGGCAT TTTACTTTAG

661 GTTGCGTATT GGAAGATCAA GAGCATCAAG TCGCTAAAGA AGAAAGGGAA ACACCTACTA

721 CTGATAGTAT GCCGCCATTA TTACGACAAG CTATCGAATT ATTTGATCAC CAAGGTGCAG

781 AGCCAGCCTT CTTATTCGGC CTTGAATTGA TCATATGCGG ATTAGAAAAA CAACTTAAAT

841 GTGAAAGTGG GTCTTAA AAG CAGCATAACC TTTTTCCGTG ATGGTAACTT CACGGTAACC

   tetR →

901 AAGATGTCGA GTTAACCACC CATCGATGAT AAGCTGTCAA ACATGAGAAT TCGGTGAATC

961 CCATAAATTC CCCGGATC GG GGTTTGAGGG CCAATGGAAC GAAAACGTAC GTTAAGG ATC

∥ ← Tnγδ OE → ∥

1021 TCTATAGTGT CACCTAAATC GGACGCGCGC TGGTGGTACC TCCTTAGTTC CTATTCCGAA

1081 GTTCCTATTC TCTAGAAAGT ATAGGAACTT CGGCGCGCCT ACCTGTGACG GAAGATCACT

1141 TCGCAGAATA AATAAATCCT GGTGTCCCTG TTGATACCGG GAAGCCCTGG GCCAACTTTT

1201 GGCGAAAATG AGACGTTGAT CGGCACGTAA GAGGTTCCAA CTTTCACCAT AATGAAATAA

1261 GATCACTACC GGGCGTATTT TTTGAGTTGT CGAGATTTTC AGGAGCTAAG GAAGCTAAA A

∥ ←

1321 TGGAGAAAAA AATCACTGGA TATACCACCG TTGATATATC CCAATGGCAT CGTAAAGAAC

                            ← CmR →

1381 ATTTTGAGGC ATTTCAGTCA GTTGCTCAAT GTACCTATAA CCAGACCGTT CAGCTGGATA

1441 TTACGGCCTT TTTAAAGACC GTAAAGAAAA ATAAGCACAA GTTTTATCCG GCCTTTATTC

1501 ACATTCTTGC CCGCCTGATG AATGCTCATC CGGAATTACG TATGGCAATG AAAGACGGTG

1561 AGCTGGTGAT ATGGGATAGT GTTCACCCTT GTTACACCGT TTTCCATGAG CAAACTGAAA

1621 CGTTTTCATC GCTCTGGAGT GAATACCACG ACGATTTCCG GCAGTTTCTA CACATATATT

1681 CGCAAGATGT GGCGTGTTAC GGTGAAAACC TGGCCTATTT CCCTAAAGGG TTTATTGAGA

1741 ATATGTTTTT CGTCTCAGCC AATCCCTGGG TGAGTTTCAC CAGTTTTGAT TTAAACGTGG

1801 CCAATATGGA CAACTTCTTC GCCCCCGTTT TCACCATGGG CAAATATTAT ACGCAAGGCG

1861 ACAAGGTGCT GATGCCGCTG GCGATTCAGG TTCATCATGC CGTTTGTGAT GGCTTCCATG

1921 TCGGCAGATG CTTAATGAAT ACAACAGTAC TGCGATGAGT GGCAGGGCGG GGCGTAA GGC

CmR → ∥

1981 GCGCCATTTA AATGAAGTTC CTATTCCGAA GTTCCTATTC TCTAGAAAGT ATAGGAACTT

2041 CGAAGCAGCT CCAGCCTACA GATCTGGCCG CTAATACGAC TCACTATAGG GAACTGAC CC

 ∥ ←

2101 TTAACGTACG TTTTCGTTCC ATTGGCCCTC AAACCCC AAT TCGTCAGACT TACGGTTAAG

           Tnγδ OE → ∥

2161 CAGTCTGAAT GAATTCGAGC TCGCCGGGGA TCCTTTTTAA CCCATCACAT ATACCTGCCG

2221 TTCACTATTA TTTAGTGAAA TGAGATATTA TGATATTTTC TGAATTGTGA TTAAAAAGGC

2281 AACTTTATGC CCATGCAACA GAAACTATAA AAAATACAGA GAATGAAAAG AAACAGATAG

2341 ATTTTTTAGT TCTTTAGGCC CGTAGTCTGC AAATCCTTTT ATGATTTTCT ATCAAACAAA

2401 AGAGGAAAAT AGACCAGTTG CAATCCAAAC GAGAGTCTAA TAGAATGAGG TCGAAAAGTA

2461 AATCGCGCGG GTTTGTTACT GATAAAGCAG GCAAGACCTA AAATGTGTAA AGGGCAAAGT

2521 GTATACTTTG GCGTCACCCC TTACATATTT TAGGTCTTTT TTTATTGTGC GTAACTAACT

2581 TGCCATCTTC AAACAGGAGG GCTGGAAGAA GCAGACCGCT AACACAGTAC ATAAAAAAGG

2641 AGAC ATGAAC GATGAACATC AAAAAGTTTG CAAAACAAGC AACAGTATTA ACCTTTACTA

∥ ← sacB

2701 CCGCACTGCT GGCAGGAGGC GCAACTCAAG CGTTTGCGAA AGAAACGAAC CAAAAGCCAT

2761 ATAAGGAAAC ATACGGCATT TCCCATATTA CACGCCATGA TATGCTGCAA ATCCCTGAAC

2821 AGCAAAAAAA TGAAAAATAT CAAGTTCCTG AATTTGATTC GTCCACAATT AAAAATATCT

2881 CTTCTGCAAA AGGCCTGGAC GTTTGGGACA GCTGGCCATT ACAAAACGCT GACGGCACTG

2941 TCGCAAACTA TCACGGCTAC CACATCGTCT TTGCATTAGC CGGAGATCCT AAAAATGCGG

3001 ATGACACATC GATTTACATG TTCTATCAAA AAGTCGGCGA AACTTCTATT GACAGCTGGA

3061 AAAACGCTGG CCGCGTCTTT AAAGACAGCG ACAAATTCGA TGCAAATGAT TCTATCCTAA

3121 AAGACCAAAC ACAAGAATGG TCAGGTTCAG CCACATTTAC ATCTGACGGA AAAATCCGTT

3181 TATTCTACAC TGATTTCTCC GGTAAACATT ACGGCAAACA AACACTGACA ACTGCACAAG

3241 TTAACGTATC AGCATCAGAC AGCTCTTTGA ACATCAACGG TGTAGAGGAT TATAAATCAA

3301 TCTTTGACGG TGACGGAAAA ACGTATCAAA ATGTACAGCA GTTCATCGAT GAAGGCAACT

3361 ACAGCTCAGG CGACAACCAT ACGCTGAGAG ATCCTCACTA CGTAGAAGAT AAAGGCCACA

3421 AATACTTAGT ATTTGAAGCA AACACTGGAA CTGAAGATGG CTACCAAGGC GAAGAATCTT

3481 TATTTAACAA AGCATACTAT GGCAAAAGCA CATCATTCTT CCGTCAAGAA AGTCAAAAAC

3541 TTCTGCAAAG CGATAAAAAA CGCACGGCTG AGTTAGCAAA CGGCGCTCTC GGTATGATTG

3601 AGCTAAACGA TGATTACACA CTGAAAAAAG TGATGAAACC GCTGATTGCA TCTAACACAG

3661 TAACAGATGA AATTGAACGC GCGAACGTCT TTAAAATGAA CGGCAAATGG TATCTGTTCA

3721 CTGACTCCCG CGGATCAAAA ATGACGATTG ACGGCATTAC GTCTAACGAT ATTTACATGC

3781 TTGGTTATGT TTCTAATTCT TTAACTGGCC CATACAAGCC GCTGAACAAA ACTGGCCTTG

3841 TGTTAAAAAT GGATCTTGAT CCTAACGATG TAACCTTTAC TTACTCACAC TTCGCTGTAC

3901 CTCAAGCGAA AGGAAACAAT GTCGTGATTA CAAGCTATAT GACAAACAGA GGATTCTACG

3961 CAGACAAACA ATCAACGTTT GCGCCGAGCT TCCTGCTGAA CATCAAAGGC AAGAAAACAT

4021 CTGTTGTCAA AGACAGCATC CTTGAACAAG GACAATTAAC AGTTAACAAA TAA AAACGCA

sacB → ∥

4081 AAAGAAAATG CCGATATCCT ATTGGCATTT TCTTTTATTT CTTATCAACA TAAAGGTGAA

4141 TCCCATAAAT TCCCCGGATC CTCTAGAGTC GATGATGGTT G AGATGTGTA TAAGAGACAG

∥ Tn5 OE → ∥

4201 CTCGAG

As described above, in the transposon according to the present invention, the nucleotide sequence and length of the remaining portions except for the translocation enzyme recognition sites of Tn5 and Tnγδ, Cm ^R , tetR, and sacB gene regions are determined according to the type of vector used in the preparation of the transposon. If the nucleotide sequence includes all of the translocation enzyme recognition sites of Tn5 and Tnγδ, the selection marker (Cm ^R , tetR, sacB ) and the nucleotide sequence is preserved, the remaining sites partially delete one or more bases, Insertion or substitution does not affect the function of the transposon. In addition, the translocation enzyme recognition site of Tnγδ is located between the Tn5 translocation enzyme recognition sites at both ends of the transposon TnRIBD, and each of the negative selection markers (tetR and sacB ) is located on the outside and has a positive selection marker (Cm ^R ) therein. You just have to.

In step (2), transposomes are formed by adding a translocation enzyme of Tn5 to the transposon TnRIBD, and the transposomes are transferred to a microbial strain using a conventional electrotroporation method. After inserting the transposon at any position of the microbial strain chromosome, the microorganism mutant strain into which the transposon is inserted is selected, and then the transposon insertion position within the selected mutant strain is confirmed. At this time, since the arbitrary insertion function of the translocation enzyme of Tn5 is activated by magnesium ions, the formation of the transposome is performed under the condition that there is no magnesium ion, and the arbitrary insertion of the transposon in the microorganism strain is performed under the condition where the magnesium ion is present. To perform. In addition, since the transposon has a chloramphenicol resistance gene, it is possible to select a strain into which the transpozone is inserted in a medium containing chloramphenicol. The position of the inserted transposon can be confirmed by direct genome sequencing.

In the step (3), by introducing a translocation enzyme expression vector into the mutant strain, Tnγδ translocation enzyme is continuously expressed in the microbial mutant strain in which the transposon is inserted into the chromosome region to be removed. Tnγδ translocation enzyme recognizes the translocation enzyme recognition site of Tnγδ to generate intramolecular potential in the left and right direction to remove the chromosome portion around the transposon (see FIG. 1).

In one embodiment of the invention, the translocation enzyme expression vector is a pXRD4043 cleaved with a restriction enzyme NaeI / Cla I to prepare a gene fragment of the cut end (blunt end) comprising the translocation enzyme-related gene tnpA (SEQ ID NO: 7) Then, it can be obtained by inserting a linear vector pEL3 having a cleavage end obtained by cleavage with the restriction enzyme BamHI using ligase. The translocation enzyme expression vector prepared by the above method was named pELTP, and this is shown in FIG. 3 and the sequence thereof is shown in SEQ ID NO: 8.

In step (4), as can be seen in Figures 4a and 5a, the chromosome size removed by PCR using a primer located within the transposon and a primer located a few kb to several tens of kb away from the transposon By measuring the approximate degree of chromosome removal can be estimated. At this time, in one embodiment of the present invention, the primers located inside the transposon and the primers located away from the transposon, 3C (SEQ ID NO: 12) and p15 (SEQ ID NO: 13) or 5C (SEQ ID NO: 14) and m10 Direct chromosomal sequence analysis using No. 15) shows the exact size of the deletion.

The gene sacB of Bacillus subtilis expresses levansucrase, which breaks down sucrose into glucose and fructose and synthesizes fructose into the polymer levan. However, because the levan is toxic to microorganisms, microorganisms with sacB gene do not survive in a medium containing sucrose. Therefore, when the translocation enzyme recognizes the recognition site and causes intramolecular translocation, the sacB gene is removed, and the strain from which the sacB gene is removed can live in a medium containing sucrose. At this time, not only sacB gene is removed, but other genes around it are also removed. Therefore, if there is no gene necessary for survival at the site of removal, even if the surrounding genes are removed, that is, the strain in which the part of the chromosome is removed will be able to live in the medium. This allows the genes in the removed part to be identified as not essential for survival.

Km ^R (ganamycin resistance gene) under the control of the tetracycline promoter of the λ attachment site of MG1655 λatt :: P _tet- Km ^R by a tetracycline repressor expressed in tetR within the transposon; kanamycin resistant gene expression is inhibited. Therefore, only strains from which the tetR gene has been removed through intramolecular translocation can live in a medium containing kanamycin. In this case, the chromosomal portion outside the tetR gene is also removed at the same time as the tetR gene. This can be used to obtain a microbial mutant strain from which a portion of the chromosome has been removed. In addition, genes present in the removed chromosome can be selected as non-essential genes under given growth conditions.

In the present invention, E. coli was used as the target microorganism, and was prepared based on the transposon Tn5 and Tnγδ. It has been reported that Tn5 can be inserted at any position of the microbial chromosome (Berg, DD and MM Howe. 1989. Mobile DNA. American Society for Microbiology, Washington, DC), and Tnγδ is reported to be capable of intramolecular translocation. (Wang, G. et al . 1993. pDUAL: A tranoposon-based cosmid cloning vector for generating nested deletions and DNA sequencing templates in vivo . Pro. Natl. Acad. Sci. USA 90, 7874-7878). It is also reported that Escherichia coli carrying the sacB gene of Bacillus subtilis cannot live in a medium containing 5% sucrose (Link, AJ et al . 1997. Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli : Application to open reading frame characterization.J Bacteriol. 179, 6228-37), the expression of tetracycline inhibitors did not express the kanamycin resistance gene under the control of the tetracycline promoter, and it has been reported that E. coli could not be killed in kanamycin media. (Koob, MD et al . 1994. Minimizing the genome of Escherichia coli . Motivation and strategy. Ann NY Acad Sci. 30, 1-3).

Therefore, the present invention inserts a translocation enzyme recognition site of Tn5 and Tnγδ into a transposon, inserts it at an arbitrary position in the microbial chromosome, removes the left and right chromosomal portions of the transposon inserted through the Tnγδ translocation enzyme, and removes the chromosome negatively. By using the markers sacB and tetR / P _tet -Km ^R , the microbial mutant strains were prepared by removing the chromosomal parts on the left and right sides of the transposon, and also the development of a technique for detecting a growth unnecessary gene.

Hereinafter, the present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples.

Example

Example 1

Preparation of transposon TnRIBD capable of insertion at any position of E. coli chromosome

1 shows chromosomal insertion of a linear transposon TnRIBD, which has OEs of sacB , Cm ^R , tetR, and Tnγδ and has a translocation enzyme recognition site (Tn5 OE) of Tn5 consisting of 19 base pairs at both ends thereof.

As can be seen in Figure 2, the transposon TnRIBD can be obtained by treating the restriction enzyme XhoI in the pRIBD vector.

The pRIBD vector was prepared by the following method.

First, the tetA and tetR genes amplified by Tn10 (Professor Michael Koob, University of Minnesota, USA) by PCR were cleaved using restriction enzyme NotI / ClaI (New England Biolabs Inc., MA, USA) and then restriction enzyme NotI Riga in a vector pKClox (Yoon, YG, et al., 1998, Cre / loxP-mediated excision and amplification of the Escherichia coli genome, Genetic Analysis 14, 89-95) containing linear γ-ori cleaved with / ClaI A new vector was created by insertion using an enzyme (New England Biolabs Inc.) and named pKCtet. The sacB gene amplified by pDELTA2 (Gibco BRL products, MD, USA) by PCR was digested with restriction enzyme BamHI (New England Biolabs Inc.), and then ligase was added to the linear vector pKCtet digested with restriction enzyme BamHI. After inserting to create a new vector, we named it pGtesa. In addition, a portion containing OE of Tnγδ amplified in pDELTA2 by PCR was cleaved using restriction enzyme EcoRI (New England Biolabs Inc.), and then ligase was used in the linear vector pGtesa cut with restriction enzyme EcoRI. Insert it into a new vector and name it pGtesa3. Then, the Cm ^R gene amplified in pKClox by PCR was cleaved using the restriction enzyme KpnI / BglII (New England Biolabs Inc.), and then ligase was used in the linear vector pGtesa3 cleaved with the restriction enzyme KpnI / BglII. And inserted into a new vector, named pGCtesa3. Vector pMOD (Epicentre technologies, WI, USA) was digested with restriction enzyme EcoRI / BamHI (New England Biolabs Inc.) and then made into a blunt end using T4 DNA polymerase (New England Biolabs Inc.). After that, it was self-ligation using ligase and named it pMb2. Tn5 translocation enzyme recognition site of pMb2 using a 5 ′ primer (SEQ ID NO: 9) having a 5′-GAATTCTCGAGCTGTCTCTTATACACATCTC-3 ′ sequence and a 3 ′ primer having a 5′-ACATGTCTCGAGCTGTCTCTTATACACATCTC-3 ′ sequence (SEQ ID NO: 10) After amplifying the DNA portion containing the sequence by PCR, it was inserted into a linear vector pUC19 (New England Biolabs Inc.) cut using the restriction enzyme EcoRI / AflIII (New England Biolabs Inc.) using ligase. A vector was created and named pMb3. The vector pGCtesa3 was linearized with the restriction enzyme HindIII (New England Biolabs Inc.), and then inserted into the linear vector pMb3 digested with the restriction enzyme HindIII using ligase to make a new vector, which was named pRIBDt. . After cleaving the vector pRIBDt with restriction enzyme EcoRV / NotI (New England Biolabs Inc.), DNA fragments containing OEs of tetR, Cm ^R , sacB , Tnγδ and Tn5 were digested with T4 DNA polymerase. ), Followed by self ligation using ligase, which was named pRIBD (see FIG. 2).

The vector pRIBD synthesized as described above was treated with restriction enzyme XhoI (New England Biolabs Inc.), extracted from an agarose gel, and used as a transposon TnRIBD.

Example 2

Insert the transposon TnRIBD at any position on the E. coli chromosome and confirm the insertion position

500ng of transposon TnRIBD and 10units of Tn5 translocation enzyme (1unit / μl, purchased from Epicentre technologies) were reacted with secondary distilled water to make a total of 20μl, followed by reaction at 37 ° C. for 1 hour to form a transposome. At this time, in order to suppress the arbitrary insertion function of the translocation enzyme, the reaction proceeded in the absence of divalent magnesium ions. Subsequently, 1 μl of the transposome was removed by conventional electroporation method (Bio-Rad, Bacterial electro-transformation and pulse controller instruction manual, Cat. No 165-2098) under reaction conditions in which magnesium ions exist. E. coli MG1655 λ _att :: P _tet -Km ^R strain (Koob, MD et al. 1994. Minimizing the genome of Escherichia coli.Motivation and strategy.Anne NY Acad Sci. 30, 1-3, from Michael Koob, University of Minnesota, USA Obtained). The strain was incubated in LB medium (tryptone 1%, yeast extract 0.5%, NaCl 0.5%). At this time, a small amount of magnesium ions were added to the medium to activate an arbitrary insertion function in the E. coli strain cells of the transposomes by the magnesium ions to be inserted into any position of the chromosome of E. coli. The E. coli mutant strain into which the transposon was inserted has resistance to chloramphenicol due to the Cm ^R gene present in the transposon, and was selected from chloramphenicol-containing medium. At this time, the exact position on the chromosome of the transposon inserted into the E. coli MG1655 λ _att :: P _tet -Km ^R chromosome was determined by sequencing primer Sac-out (5′-TGTTGTCAAAGACAGCATCCTTGAACAAGG- ￠ 3, synthesized by Genotech Co., Ltd.). No. 11)) was used to perform direct chromosome sequencing, and the results were compared with the Gene Bank DNA sequence using the BLAST program to confirm that the transposon was inserted into the E. coli strain.

Example 3

Preparation of Escherichia coli mutant strain having a deletion mutation by introducing a translocation enzyme expression vector pELTP into the strain.

A vector pELTP (see FIG. 3) expressing a translocation enzyme was introduced into E. coli mutant strains having a transposon TnRIBD prepared according to Example 2.

The vector pELTP was prepared as follows. First, translocation enzyme expression vector pXRD4043 (Gibco BRL products, Tsai, MM et al. 1987, Transposition of Tn1000: in vivo properties. J Bacteriol. 169: 5556-62), tnpA (SEQ ID NO: 7) Was digested with a restriction enzyme NaeI / ClaI (New England Biolabs Inc.) to a cleavage end, and then cleaved with a restriction enzyme BamHI (New England Biolabs Inc.) to a cleavage end, a linear temperature sensitive vector pEL3 (National Genetics). Obtained from the Institute Seiichi Yasuda, Armstrong, KA et al. 1984. A 37 X 10 ³ molecular weight plasmid-encoded protein is required for replication and copy number control in the plasmid pSC101 and its temperature-sensitive derivative pHS1.J Mol Biol. 175: 331-48) using ligase to prepare a new vector and name it pELTP. PELTP thus prepared is shown in FIG. 3.

The translocation enzyme expression vector pELTP prepared as described above was transduced into the E. coli mutant strain having the transposon TnRIBD (J. Sambrook et al. Molecular Cloning-A Laboratory Manual. 2ed. Cold Spring Harbor. 1989). Since the tnpA gene present in the expression vector pELTP is regulated by the tac promoter, IPTG (Isopropyl-beta-D-thiogalactoside) was treated at a final concentration of 1 mM and shaken at 30 ° C to express translocation enzyme. Deletion variants that occur to the right of the transposon that result in the deletion of the sacB gene (see FIG. 4A) among the deletion mutations resulting from translocation enzyme expression were selected in 5% sucrose-containing medium and occurred to the left of the transposon that induces deletion of the tetR gene. Deletion variants (see FIG. 5A) were selected in media containing kanamycin.

In Examples 2 and 3, direct genomic sequencing confirmed that the position of the inserted transposon was E. coli gene araA . After transducing the translocation enzyme expression vector pELTP to express translocation enzyme, 5% sucrose was added. Strains in which a portion of the chromosome was removed by culturing in a medium containing and a medium containing kanamycin were selected, and the results of electrophoresis by PCR amplification of these chromosomes are shown in FIGS. 4B and 5B. As can be seen in Figures 4b and 5b, it could be confirmed that a part of the chromosome of E. coli was removed.

Example 4

Partial Identification of Removed Staining and Selection of Unnecessary Genes for Growth Under Given Conditions

As can be seen in Figure 4b, the base sequence of the PCR amplification product was analyzed using primers 3C (5′-GTTCATCATGCCGTTTGTG-3 ′, SEQ ID NO: 12) and p15 (5′-CCTGCCACTGCTTCACCATCCCC-3 ′, SEQ ID NO: 13). As a result, it was confirmed that about 16 kb of the chromosome was removed. In the removed portion, 15 genes of araBC, yabI, sfuCB, tbpA, yabN, setA, leuDCBALO, and ilvI were present. Through this, it was confirmed that the 15 genes are unnecessary for growth when grown in 37 ℃, LB medium.

In addition, as can be seen in Figure 5b, the base sequence of the PCR amplification product using primers 5C (5′-CCTTAGCTCCTGAAAATCTCG-3 ′, SEQ ID NO: 14) and m10 (5′-GCTCATCGGAGATTTTCACTCCC-3 ′, SEQ ID NO: 15) As a result of analysis, it was confirmed that about 8 kb of the chromosome was removed. In the removed portion, five genes , araAD, polB, hepA, and rluA, were present. Through this, it was confirmed that the five genes are unnecessary genes for growth when grown in 37 ° C. and LB medium.

As described above, the present invention is a method for preparing E. coli mutant strains by removing any portion of the chromosome using the intramolecular potential of the transposon and the negative selection marker, so that the E. coli chromosome of various sites can be removed more efficiently, and the function is not known. The necessity of a gene under given growth conditions can be readily determined.

By removing genes necessary for growth in Escherichia coli in this way, E. coli mutants having a more genetically simple chromosome can be constructed and used for various chromosome function studies. In addition, by removing genes unnecessary for growth, E. coli mutant strains showing rapid growth can be selected and used as industrial artificial strains. In addition, by applying the present invention to not only E. coli, but also other microorganisms, there is an effect that can create a variety of microbial mutants with chromosomes selectively reduced. In addition, by introducing a new metabolism-related gene of foreign origin into the reduced microbial chromosome prepared by the method according to the present invention can be applied to make a new organism having a variety of useful functions.

Figure 1 shows the step of producing E. coli having a chromosome from which any portion of the chromosome was removed using transposon TnRIBD.

Figure 2 shows the structure of pRIBD, the parent vector of transposon TnRIBD.

Figure 3 shows the structure of pELTP, a translocation enzyme expression vector.

Figure 4 shows the removal of any part of the chromosome using the negative marker sacB gene of transposon TnRIBD, 4a using the negative marker sacB gene of transposon TnRIBD inserted into the chromosome to remove any part of the E. coli chromosome and confirm the degree of removal 4b is a photograph showing electrophoresis of PCR amplification results to confirm the degree of chromosome removal using the method of 4a.

Figure 5 shows the removal of any part of the chromosome using the negative marker tetR / P _tet -Km ^R of transposon TnRIBD, 5a shows the removal of E. coli chromosome using the negative marker tetR / P _tet -Km ^R of transposon TnRIBD inserted into the chromosome. 5B is a photograph of electrophoresis of PCR amplification results to confirm the degree of removal of chromosomes using the method of FIG. 5A.

<110> Korea Advanced Institute of Science and Technology <120> TRANSPOSON FOR BIDIRECTIONAL INTRAMOLECULAR GENOME DELETIONS, CONSTRUCTION OF NOVEL MICROORGANISM AND IDENTIFICATION OF NONESSENTIAL GENES USING THE SAME <160> 15 <170> KopatentIn 1.71 <210> 1 <211> 4206 <212> DNA <213> Artificial Sequence <220> <223> transposon TnRIBD <400> 1 ctcgagctgt ctcttataca catctcaacc ctgaagctat cttccgaagc aataaattca 60 cgtaataacg ttggcaagac tggcatgata aggccaatcc ccatggcatc gagtaacgta 120 attaccaatg cgatctttgt cgaactattc atttcacttt tctctatcac tgatagggag 180 tggtaaaata actctatcaa tgatagagtg tcaacaaaaa ttaggaatta atgatgtcta 240 gattagataa aagtaaagtg attaacagcg cattagagct gcttaatgag gtcggaatcg 300 aaggtttaac aacccgtaaa ctcgcccaga agctaggtgt agagcagcct acattgtatt 360 ggcatgtaaa aaataagcgg gctttgctcg acgccttagc cattgagatg ttagataggc 420 accatactca cttttgccct ttagaagggg aaagctggca agatttttta cgtaataacg 480 ctaaaagttt tagatgtgct ttactaagtc atcgcgatgg agcaaaagta catttaggta 540 cacggcctac agaaaaacag tatgaaactc tcgaaaatca attagccttt ttatgccaac 600 aaggtttttc actagagaat gcattatatg cactcagcgc tgtggggcat tttactttag 660 gttgcgtatt ggaagatcaa gagcatcaag tcgctaaaga agaaagggaa acacctacta 720 ctgatagtat gccgccatta ttacgacaag ctatcgaatt atttgatcac caaggtgcag 780 agccagcctt cttattcggc cttgaattga tcatatgcgg attagaaaaa caacttaaat 840 gtgaaagtgg gtcttaaaag cagcataacc tttttccgtg atggtaactt cacggtaacc 900 aagatgtcga gttaaccacc catcgatgat aagctgtcaa acatgagaat tcggtgaatc 960 ccataaattc cccggatcgg ggtttgaggg ccaatggaac gaaaacgtac gttaaggatc 1020 tctatagtgt cacctaaatc ggacgcgcgc tggtggtacc tccttagttc ctattccgaa 1080 gttcctattc tctagaaagt ataggaactt cggcgcgcct acctgtgacg gaagatcact 1140 tcgcagaata aataaatcct ggtgtccctg ttgataccgg gaagccctgg gccaactttt 1200 ggcgaaaatg agacgttgat cggcacgtaa gaggttccaa ctttcaccat aatgaaataa 1260 gatcactacc gggcgtattt tttgagttgt cgagattttc aggagctaag gaagctaaaa 1320 tggagaaaaa aatcactgga tataccaccg ttgatatatc ccaatggcat cgtaaagaac 1380 attttgaggc atttcagtca gttgctcaat gtacctataa ccagaccgtt cagctggata 1440 ttacggcctt tttaaagacc gtaaagaaaa ataagcacaa gttttatccg gcctttattc 1500 acattcttgc ccgcctgatg aatgctcatc cggaattacg tatggcaatg aaagacggtg 1560 agctggtgat atgggatagt gttcaccctt gttacaccgt tttccatgag caaactgaaa 1620 cgttttcatc gctctggagt gaataccacg acgatttccg gcagtttcta cacatatatt 1680 cgcaagatgt ggcgtgttac ggtgaaaacc tggcctattt ccctaaaggg tttattgaga 1740 atatgttttt cgtctcagcc aatccctggg tgagtttcac cagttttgat ttaaacgtgg 1800 ccaatatgga caacttcttc gcccccgttt tcaccatggg caaatattat acgcaaggcg 1860 acaaggtgct gatgccgctg gcgattcagg ttcatcatgc cgtttgtgat ggcttccatg 1920 tcggcagatg cttaatgaat acaacagtac tgcgatgagt ggcagggcgg ggcgtaaggc 1980 gcgccattta aatgaagttc ctattccgaa gttcctattc tctagaaagt ataggaactt 2040 cgaagcagct ccagcctaca gatctggccg ctaatacgac tcactatagg gaactgaccc 2100 ttaacgtacg ttttcgttcc attggccctc aaaccccaat tcgtcagact tacggttaag 2160 cagtctgaat gaattcgagc tcgccgggga tcctttttaa cccatcacat atacctgccg 2220 ttcactatta tttagtgaaa tgagatatta tgatattttc tgaattgtga ttaaaaaggc 2280 aactttatgc ccatgcaaca gaaactataa aaaatacaga gaatgaaaag aaacagatag 2340 attttttagt tctttaggcc cgtagtctgc aaatcctttt atgattttct atcaaacaaa 2400 agaggaaaat agaccagttg caatccaaac gagagtctaa tagaatgagg tcgaaaagta 2460 aatcgcgcgg gtttgttact gataaagcag gcaagaccta aaatgtgtaa agggcaaagt 2520 gtatactttg gcgtcacccc ttacatattt taggtctttt tttattgtgc gtaactaact 2580 tgccatcttc aaacaggagg gctggaagaa gcagaccgct aacacagtac ataaaaaagg 2640 agacatgaac gatgaacatc aaaaagtttg caaaacaagc aacagtatta acctttacta 2700 ccgcactgct ggcaggaggc gcaactcaag cgtttgcgaa agaaacgaac caaaagccat 2760 ataaggaaac atacggcatt tcccatatta cacgccatga tatgctgcaa atccctgaac 2820 Agcaaaaaaa tgaaaaatat caagttcctg aatttgattc gtccacaatt aaaaatatct 2880 cttctgcaaa aggcctggac gtttgggaca gctggccatt acaaaacgct gacggcactg 2940 tcgcaaacta tcacggctac cacatcgtct ttgcattagc cggagatcct aaaaatgcgg 3000 atgacacatc gatttacatg ttctatcaaa aagtcggcga aacttctatt gacagctgga 3060 aaaacgctgg ccgcgtcttt aaagacagcg acaaattcga tgcaaatgat tctatcctaa 3120 aagaccaaac acaagaatgg tcaggttcag ccacatttac atctgacgga aaaatccgtt 3180 tattctacac tgatttctcc ggtaaacatt acggcaaaca aacactgaca actgcacaag 3240 ttaacgtatc agcatcagac agctctttga acatcaacgg tgtagaggat tataaatcaa 3300 tctttgacgg tgacggaaaa acgtatcaaa atgtacagca gttcatcgat gaaggcaact 3360 acagctcagg cgacaaccat acgctgagag atcctcacta cgtagaagat aaaggccaca 3420 aatacttagt atttgaagca aacactggaa ctgaagatgg ctaccaaggc gaagaatctt 3480 tatttaacaa agcatactat ggcaaaagca catcattctt ccgtcaagaa agtcaaaaac 3540 ttctgcaaag cgataaaaaa cgcacggctg agttagcaaa cggcgctctc ggtatgattg 3600 agctaaacga tgattacaca ctgaaaaaag tgatgaaacc gctgattgca tctaacacag 3660 taacagatga aattgaacgc gcgaacgtct ttaaaatgaa cggcaaatgg tatctgttca 3720 ctgactcccg cggatcaaaa atgacgattg acggcattac gtctaacgat atttacatgc 3780 ttggttatgt ttctaattct ttaactggcc catacaagcc gctgaacaaa actggccttg 3840 tgttaaaaat ggatcttgat cctaacgatg taacctttac ttactcacac ttcgctgtac 3900 ctcaagcgaa aggaaacaat gtcgtgatta caagctatat gacaaacaga ggattctacg 3960 cagacaaaca atcaacgttt gcgccgagct tcctgctgaa catcaaaggc aagaaaacat 4020 ctgttgtcaa agacagcatc cttgaacaag gacaattaac agttaacaaa taaaaacgca 4080 aaagaaaatg ccgatatcct attggcattt tcttttattt cttatcaaca taaaggtgaa 4140 tcccataaat tccccggatc ctctagagtc gatgatggtt gagatgtgta taagagacag 4200 ctcgag 4206 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Tn5 Outer Element for tn5 trnsposase <400> 2 ctgtctctta tacacatct 19 <210> 3 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Tn-gamma delta Outer Element for Tn-gamma delta transposase <400> 3 ggggtttgag ggccaatgga acgaaaacgt acgttaagg 39 <210> 4 <211> 624 <212> DNA <213> Artificial Sequence <220> <223> tet R gene as a negative selection marker <400> 4 atgtctagat tagataaaag taaagtgatt aacagcgcat tagagctgct taatgaggtc 60 ggaatcgaag gtttaacaac ccgtaaactc gcccagaagc taggtgtaga gcagcctaca 120 ttgtattggc atgtaaaaaa taagcgggct ttgctcgacg ccttagccat tgagatgtta 180 gataggcacc atactcactt ttgcccttta gaaggggaaa gctggcaaga ttttttacgt 240 aataacgcta aaagttttag atgtgcttta ctaagtcatc gcgatggagc aaaagtacat 300 ttaggtacac ggcctacaga aaaacagtat gaaactctcg aaaatcaatt agccttttta 360 tgccaacaag gtttttcact agagaatgca ttatatgcac tcagcgctgt ggggcatttt 420 actttaggtt gcgtattgga agatcaagag catcaagtcg ctaaagaaga aagggaaaca 480 cctactactg atagtatgcc gccattatta cgacaagcta tcgaattatt tgatcaccaa 540 ggtgcagagc cagccttctt attcggcctt gaattgatca tatgcggatt agaaaaacaa 600 cttaaatgtg aaagtgggtc ttaa 624 <210> 5 <211> 658 <212> DNA <213> Artificial Sequence <220> <223> CmR gene as a positive selection marker <400> 5 atggagaaaa aaatcactgg atataccacc gttgatatat cccaatggca tcgtaaagaa 60 cattttgagg catttcagtc agttgctcaa tgtacctata accagaccgt tcagctggat 120 attacggcct ttttaaagac cgtaaagaaa aataagcaca agttttatcc ggcctttatt 180 cacattcttg cccgcctgat gaatgctcat ccggaattac gtatggcaat gaaagacggt 240 gagctggtga tatgggatag tgttcaccct tgttacaccg ttttccatga gcaaactgaa 300 acgttttcat cgctctggag tgaataccac gacgatttcc ggcagtttct acacatatat 360 tcgcaagatg tggcgtgtta cggtgaaaac ctggcctatt tccctaaagg gtttattgag 420 aatatgtttt tcgtctcagc caatccctgg gtgagtttca ccagttttga tttaaacgtg 480 gccaatatgg acaacttctt cgcccccgtt ttcaccatgg gcaaatatta tacgcaaggc 540 gacaaggtgc tgatgccgct ggcgattcag gttcatcatg ccgtttgtga tggcttccat 600 gtcggcagat gcttaatgaa tacaacagta ctgcgatgag tggcagggcg gggcgtaa 658 <210> 6 <211> 1429 <212> DNA <213> Artificial Sequence <220> <223> sacB gene as a negetive selection marker <400> 6 atgaacgatg aacatcaaaa agtttgcaaa acaagcaaca gtattaacct ttactaccgc 60 actgctggca ggaggcgcaa ctcaagcgtt tgcgaaagaa acgaaccaaa agccatataa 120 ggaaacatac ggcatttccc atattacacg ccatgatatg ctgcaaatcc ctgaacagca 180 aaaaaatgaa aaatatcaag ttcctgaatt tgattcgtcc acaattaaaa atatctcttc 240 tgcaaaaggc ctggacgttt gggacagctg gccattacaa aacgctgacg gcactgtcgc 300 aaactatcac ggctaccaca tcgtctttgc attagccgga gatcctaaaa atgcggatga 360 cacatcgatt tacatgttct atcaaaaagt cggcgaaact tctattgaca gctggaaaaa 420 cgctggccgc gtctttaaag acagcgacaa attcgatgca aatgattcta tcctaaaaga 480 ccaaacacaa gaatggtcag gttcagccac atttacatct gacggaaaaa tccgtttatt 540 ctacactgat ttctccggta aacattacgg caaacaaaca ctgacaactg cacaagttaa 600 cgtatcagca tcagacagct ctttgaacat caacggtgta gaggattata aatcaatctt 660 tgacggtgac ggaaaaacgt atcaaaatgt acagcagttc atcgatgaag gcaactacag 720 ctcaggcgac aaccatacgc tgagagatcc tcactacgta gaagataaag gccacaaata 780 cttagtattt gaagcaaaca ctggaactga agatggctac caaggcgaag aatctttatt 840 taacaaagca tactatggca aaagcacatc attcttccgt caagaaagtc aaaaacttct 900 gcaaagcgat aaaaaacgca cggctgagtt agcaaacggc gctctcggta tgattgagct 960 aaacgatgat tacacactga aaaaagtgat gaaaccgctg attgcatcta acacagtaac 1020 agatgaaatt gaacgcgcga acgtctttaa aatgaacggc aaatggtatc tgttcactga 1080 ctcccgcgga tcaaaaatga cgattgacgg cattacgtct aacgatattt acatgcttgg 1140 ttatgtttct aattctttaa ctggcccata caagccgctg aacaaaactg gccttgtgtt 1200 aaaaatggat cttgatccta acgatgtaac ctttacttac tcacacttcg ctgtacctca 1260 agcgaaagga aacaatgtcg tgattacaag ctatatgaca aacagaggat tctacgcaga 1320 caaacaatca acgtttgcgc cgagcttcct gctgaacatc aaaggcaaga aaacatctgt 1380 tgtcaaagac agcatccttg aacaaggaca attaacagtt aacaaataa 1429 <210> 7 <211> 2949 <212> DNA <213> Artificial Sequence <220> <223> tnpA gene, which is a transposase-relating gene <400> 7 atgcctgtcg actttctaac ctctgaccaa aaacagaatt acggttgtta tgctgcagaa 60 cctaatgacg tgcaactggc gcgctatttt catcttgatg aacgggatct ggccttcatt 120 aaccaacgac ggggcaaaca taataggctg ggcattgcgc ttcagctcac cacagcccgt 180 tttctgggaa catttctgac ggatttaact caggttctgc ctggtgttca acattttgtc 240 gcggtacagc ttaatatcca ccgtccagaa gttctctccc gctatgctga acgggacact 300 acccttagag aacatactgc attaattaag gaatattacg gctatcatga atttggtgat 360 tttccatggt ctttccgcct gaagcgtctg ctatataccc gggcgtggct cagtaatgag 420 cgaccgggtc tgatgtttga ttttgccact gcatggttgc ttcaaaataa ggtattactg 480 cccggagcaa ccacactagt acgtctcatc agtgaaattc gtgaaagggc aaatcagcgg 540 ctgtggaaaa agctggccgc actgccgaac aaatggcagg cagctcaagt gatggagctt 600 ctggtcattc cggaaggtca gcgtgtatca gcactggaac agttgaaaaa aggccctgtt 660 acagtcagtg gacctgcgtt taatgaagcg ctggaacgat atatccgatt acgaagtctt 720 gagttttccc gactgaactt ttccggtctg cctgccattc aactgcgtaa tctggctcgt 780 tatgctggca tggcgtcggt aaaatatatc gctcgaatgc cacagcagag aaagcttgct 840 gtacttactg cattcgttaa agcacaggaa ataacggcat tagacgatgc cgttgatgtg 900 cttgatatgc taattctgga cattatccgc gaagcaaaga aaaccgggca aaaaaaaaga 960 ctcaggacac tgaaagatct tgatcaggcc gcattgttac tggcgcgggc atgtgcattg 1020 ttgctggatg ataatacaga tgtcccagat ctcaggcagg ttatcttcaa gtgcgtaccc 1080 aaaaacagac tggcagaatc tgtaagcaag gttaatgaac ttgctcgtcc acagaacaat 1140 aatttccatg acgaaatggt tgagcagtac ggtcgggtta aacgttttct tccggcggtg 1200 ttgcgggacc tgcatttccg tgcggcaccg gcaggtgaac atgtactggc tgcgattcat 1260 tatctggcag aactgaatgg ttcgaaaaag cgcatccttg atgatgcgcc tgaacatatt 1320 atcaccggtc cctggaaacg cctcgtatac gatgcggagg gacggataca gcgtgcaggt 1380 tattcactat gtttgctgga acgccttcag gatgcactgc gccgccggga catctggctt 1440 gaaaacagtg atcgctgggg agatcctcgc gagaagttgt tgcaaggtga agagtggcag 1500 actcagcgta ttcctgtctg tcgggcactg ggacatcctg tcgatggacg taaaggtgtg 1560 caacaactgg ctattcagct ggatgagacc tggaaagccg tggcatcacg atttgaaaag 1620 aatgcggaag ttcatatctg taatgaaggt aaatatccat ccctgactat cagttgtctg 1680 gagaaacagg aagagccacc atcattgctt cgtctaaata atcggatcaa acagctactc 1740 ccaccggtag atttaacgga actgttactt gagatagatg cccagacagg atttacacat 1800 gagtttgcgc atgtcagtga atctggtgct cgagcgcaag atttgcacat cagtttatgt 1860 gcggtattga tggctgaagc ctgtaatatc ggactggaac cgctgataaa gcacaatata 1920 ccagcactga cccgccatcg gctcagttgg gtgaaacaga attaccttcg tgcagaaacg 1980 ctggtcagcg ccaatgcccg cctggttgat tttcagtcca cactggagct tgctggtcgt 2040 tggggaggtg gagaagtggc atcagctgac ggcatgcgct ttgtcacacc agtgaagacc 2100 atcaactcag gatctaacag aaaatatttt ggttctggac gaggcatcac ctggtataac 2160 ttcgtatctg atcagtactc tgggttccat ggcattgtgg tacccggtac attacgggat 2220 tcgatttttg tactggaagg acttcttgag cagcagacag ggctgaatcc agttgaaatc 2280 atgacagaca ctgcgggtag cagcgatatt attttcggtc tgttctggct actggggtat 2340 cagttttccc cccggcttgc cgatgcaggt gaggcggtgt tctggcgggt gaataaatcg 2400 gcaaactacg gagtactgga taagttggcc cgaggatatg cagatctgtc aaaagcggag 2460 agtcagtggg atgagatgat gcgaaccgct ggttcgctga agttgggtac aattcatgcg 2520 tcagaactca ttcgctcttt actgaaaagc tcgcgcccat caggactggc acaggcaatt 2580 atggaagtag ggcgtgtaaa caaaacgcta tatctcctca attatattga tgatgaagat 2640 tatcgtcggc ggatcctgac gcagcttaat cgaggagaag gccgccacgc tgtggctcgt 2700 gcgatttgtt acggacaacg cggagagatc agaaagcgtt atcgtgaagg gcaggatatg 2760 gaggaagcgt tgagctggat gcgccgtaat ggcgaagaaa ttatagatga agatatcgct 2820 cggctatctc ccctgatgca cgggcatatc aatatgttgg gccattatac attcacgttg 2880 ccagaggata ttttaaaagg ggaactgaga gctctaaatt taaatataaa caacgaatta 2940 tctccttaa 2949 <210> 8 <211> 7069 <212> DNA <213> Artificial Sequence <220> <223> Expression Vector pELPD for a transposase <400> 8 gcgctcaaag atgcaggggt aaaagctaac cgcatcttta ccgacaaggc atccggcagt 60 tcaacagatc gggaagggct ggatttgctg aggatgaagg tggaggaagg tgatgtcatt 120 ctggtgaaga agctcgaccg tcttggccgc gacaccgccg acatgatcca actgataaaa 180 gagtttgatg ctcagggtgt agcggttcgg tttattgacg acgggatcag taccgacggt 240 gatatggggc aaatggtggt caccatcctg tcggctgtgg cacaggctga acgccggagg 300 atccgataag cttactcccc atccccctgt tgacaattaa tcatcggctc gtataatgtg 360 tggaattgtg agcggataac aatttcacac aggaaacagg atcaaattat gcctgtcgac 420 tttctaacct ctgaccaaaa acagaattac ggttgttatg ctgcagaacc taatgacgtg 480 caactggcgc gctattttca tcttgatgaa cgggatctgg ccttcattaa ccaacgacgg 540 ggcaaacata ataggctggg cattgcgctt cagctcacca cagcccgttt tctgggaaca 600 tttctgacgg atttaactca ggttctgcct ggtgttcaac attttgtcgc ggtacagctt 660 aatatccacc gtccagaagt tctctcccgc tatgctgaac gggacactac ccttagagaa 720 catactgcat taattaagga atattacggc tatcatgaat ttggtgattt tccatggtct 780 ttccgcctga agcgtctgct atatacccgg gcgtggctca gtaatgagcg accgggtctg 840 atgtttgatt ttgccactgc atggttgctt caaaataagg tattactgcc cggagcaacc 900 acactagtac gtctcatcag tgaaattcgt gaaagggcaa atcagcggct gtggaaaaag 960 ctggccgcac tgccgaacaa atggcaggca gctcaagtga tggagcttct ggtcattccg 1020 gaaggtcagc gtgtatcagc actggaacag ttgaaaaaag gccctgttac agtcagtgga 1080 cctgcgttta atgaagcgct ggaacgatat atccgattac gaagtcttga gttttcccga 1140 ctgaactttt ccggtctgcc tgccattcaa ctgcgtaatc tggctcgtta tgctggcatg 1200 gcgtcggtaa aatatatcgc tcgaatgcca cagcagagaa agcttgctgt acttactgca 1260 ttcgttaaag cacaggaaat aacggcatta gacgatgccg ttgatgtgct tgatatgcta 1320 attctggaca ttatccgcga agcaaagaaa accgggcaaa aaaaaagact caggacactg 1380 aaagatcttg atcaggccgc attgttactg gcgcgggcat gtgcattgtt gctggatgat 1440 aatacagatg tcccagatct caggcaggtt atcttcaagt gcgtacccaa aaacagactg 1500 gcagaatctg taagcaaggt taatgaactt gctcgtccac agaacaataa tttccatgac 1560 gaaatggttg agcagtacgg tcgggttaaa cgttttcttc cggcggtgtt gcgggacctg 1620 catttccgtg cggcaccggc aggtgaacat gtactggctg cgattcatta tctggcagaa 1680 ctgaatggtt cgaaaaagcg catccttgat gatgcgcctg aacatattat caccggtccc 1740 tggaaacgcc tcgtatacga tgcggaggga cggatacagc gtgcaggtta ttcactatgt 1800 ttgctggaac gccttcagga tgcactgcgc cgccgggaca tctggcttga aaacagtgat 1860 cgctggggag atcctcgcga gaagttgttg caaggtgaag agtggcagac tcagcgtatt 1920 cctgtctgtc gggcactggg acatcctgtc gatggacgta aaggtgtgca acaactggct 1980 attcagctgg atgagacctg gaaagccgtg gcatcacgat ttgaaaagaa tgcggaagtt 2040 catatctgta atgaaggtaa atatccatcc ctgactatca gttgtctgga gaaacaggaa 2100 gagccaccat cattgcttcg tctaaataat cggatcaaac agctactccc accggtagat 2160 ttaacggaac tgttacttga gatagatgcc cagacaggat ttacacatga gtttgcgcat 2220 gtcagtgaat ctggtgctcg agcgcaagat ttgcacatca gtttatgtgc ggtattgatg 2280 gctgaagcct gtaatatcgg actggaaccg ctgataaagc acaatatacc agcactgacc 2340 cgccatcggc tcagttgggt gaaacagaat taccttcgtg cagaaacgct ggtcagcgcc 2400 aatgcccgcc tggttgattt tcagtccaca ctggagcttg ctggtcgttg gggaggtgga 2460 gaagtggcat cagctgacgg catgcgcttt gtcacaccag tgaagaccat caactcagga 2520 tctaacagaa aatattttgg ttctggacga ggcatcacct ggtataactt cgtatctgat 2580 cagtactctg ggttccatgg cattgtggta cccggtacat tacgggattc gatttttgta 2640 ctggaaggac ttcttgagca gcagacaggg ctgaatccag ttgaaatcat gacagacact 2700 gcgggtagca gcgatattat tttcggtctg ttctggctac tggggtatca gttttccccc 2760 cggcttgccg atgcaggtga ggcggtgttc tggcgggtga ataaatcggc aaactacgga 2820 gtactggata agttggcccg aggatatgca gatctgtcaa aagcggagag tcagtgggat 2880 gagatgatgc gaaccgctgg ttcgctgaag ttgggtacaa ttcatgcgtc agaactcatt 2940 cgctctttac tgaaaagctc gcgcccatca ggactggcac aggcaattat ggaagtaggg 3000 cgtgtaaaca aaacgctata tctcctcaat tatattgatg atgaagatta tcgtcggcgg 3060 atcctgacgc agcttaatcg aggagaaggc cgccacgctg tggctcgtgc gatttgttac 3120 ggacaacgcg gagagatcag aaagcgttat cgtgaagggc aggaagatca actgggggcg 3180 ctgggcctgg ttactaatgc agtggttctg tggaacacgc tctatatgga ggaagcgttg 3240 agctggatgc gccgtaatgg cgaagaaatt atagatgaag atatcgctcg gctatctccc 3300 ctgatgcacg ggcatatcaa tatgttgggc cattatacat tcacgttgcc agaggatatt 3360 ttaaaagggg aactgagagc tctaaattta aatataaaca acgaattatc tccttaacgt 3420 acgttttcgt tccattggaa cgccatgagc ggcctcattt cttattctga gttacaacag 3480 tccgcaccgc tgtccggtag ctccttccgg tgggcgcggg gcatgactat cgtcgccgca 3540 cttatgactg tcttctttat catgcaactc gtaggacagg tgccgatcct agagcgcacg 3600 aatgagggcc gacaggaagc aaagctgaaa ggaatcaaat ttggccgcag gcgtaccgtg 3660 gacaggaacg tcgtgctgac gcttcatcag aagggcactg gtgcaacgga aattgctcat 3720 cagctcagta ttgcccgctc cacggtttat aaaattcttg aagacgaaag ggcctcgtga 3780 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca 3840 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata 3900 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga 3960 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc 4020 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg 4080 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc 4140 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat 4200 cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact 4260 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat 4320 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga 4380 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc 4440 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga 4500 tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag 4560 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc 4620 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt 4680 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct 4740 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg 4800 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg 4860 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca 4920 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gttgatgata 4980 ccgctgcctt actgggtgca ttagccagtc tgaatgacct gtcacgggat aatccgaagt 5040 ggtcagactg gaaaatcaga gggcaggaac tgctgaacag caaaaagtca gatagcacca 5100 catagcagac ccgccataaa acgccctgag aagcccgtga cgggcttttc ttgtattatg 5160 ggtagtttcc ttgcatgaat ccataaaagg cgcctgtagt gccatttacc cccattcact 5220 gccagagccg tgagcgcagc gaactgaatg tcacgaaaaa gacagcgact caggtgcctg 5280 atggtcggag acaaaaggaa tattcagcga tttgcccgag cttgcgaggg tgctacttaa 5340 gcctttaggg ttttaaggtc tgttttgtag aggagcaaac agcgtttgcg acatcctttt 5400 gtaatactgc ggaactgact aaagtagtga gttatacaca gggctgggat ctattctttt 5460 tatctttttt tattctttct ttattctata aattataacc acttgaatat aaacaaaaaa 5520 aacacacaaa ggtctagcgg aatttacaga gggtctagca gaatttacaa gttttccagc 5580 aaaggtctag cagaatttac agatacccac aactcaaagg aaaaggacta gtaattatca 5640 ttgactagcc catctcaatt ggtatagtga ttaaaatcac ctagaccaat tgagatgtat 5700 gtctgaatta gttgttttca aagcaaatga actagcgatt agtcgctatg acttaacgga 5760 gcatgaaacc aagctaattt tatgctgtgt ggcactactc aaccccacga ttgaaaaccc 5820 tacaaggaaa gaacggacgg tatcgttcac ttataaccaa tacgctcaga tgatgaacat 5880 cagtagggaa aatgcttatg gtgtattagc taaagcaacc agagagctga tgacgagaac 5940 tgtggaaatc aggaatcctt tggttaaagg ctttgagatt ttccagtgga caaactatgc 6000 caagttctca agcgaaaaat tagaattagt ttttagtgaa gagatattgc cttatctttt 6060 ccagttaaaa aaattcataa aatataatct ggaacatgtt aagtcttttg aaaacaaata 6120 ctctatgagg atttatgagt ggttattaaa agaactaaca caaaagaaaa ctcacaaggc 6180 aaatatagag attagccttg atgaatttaa gttcatgtta atgcttgaaa ataactacca 6240 tgagtttaaa aggcttaacc aatgggtttt gaaaccaata agtaaagatt taaacactta 6300 cagcaatatg aaattggtgg ttgataagcg aggccgcccg actgatacgt tgattttcca 6360 agttgaacta gatagacaaa tggatctcgt aaccgaactt gagaacaacc agataaaaat 6420 gaatggtgac aaaataccaa caaccattac atcagattcc tacctacgta acggactaag 6480 Aaaaaacacta cacgatgctt taactgcaaa aattcagctc accagttttg aggcaaaatt 6540 tttgagtgac atgcaaagta agcatgatct caatggttcg ttctcatggc tcacgcaaaa 6600 acaacgaacc acactagaga acatactggc taaatacgga aggatctgag gttcttatgg 6660 ctcttgtatc tatcagtgaa gcatcaagac taacaaacaa aagtagaaca actgttcacc 6720 gttagatatc aaagggaaaa ctgtccatat gcacagatga aaacggtgta aaaaagatag 6780 atacatcaga gcttttacga gtttttggtg catttaaagc tgttcaccat gaacagatcg 6840 acaatgtaac agatgaacag catgtaacac ctaatagaac aggtgaaacc agtaaaacaa 6900 agcaactaga acatgaaatt gaacacctga gacaacttgt tacagctcaa cagtcacaca 6960 tagacagcct gaaacaggcg atgctgctta tcgaatcaaa gctgccgaca acacgggagc 7020 cagtgacgcc tcccgtgggg aaaaaatcat ggcaattctg gaagaaata 7069 <210> 9 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> 5 'primer for PCR of a DNA fragment comprising a Tn5 Outer Element <400> 9 gaattctcga gctgtctctt atacacatct c 31 <210> 10 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> 3 'primer for PCR of a DNA fragment comprising a Tn5 Outer Element <400> 10 acatgtctcg agctgtctct tatacacatc tc 32 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer Sac-out for a sequence analysis <400> 11 tgttgtcaaa gacagcatcc ttgaacaagg 30 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer 3C <400> 12 gttcatcatg ccgtttgtg 19 <210> 13 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer p15 <400> 13 cctgccactg cttcaccatc ccc 23 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer 5C <400> 14 ccttagctcc tgaaaatctc g 21 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer m10 <400> 15 gctcatcgga gattttcact ccc 23

Claims (10)

Tn5 translocation enzyme recognition site having the sequence of SEQ ID NO: 2, tetR gene having the sequence of SEQ ID NO: 4, Tnγδ translocation enzyme recognition site having the sequence of SEQ ID NO: 3, Cm ^R gene having the sequence of SEQ ID NO: 5, SEQ ID NO: 3 A transposon TnRIBD comprising, in sequence 5 '→ 3', a Tnγδ translocation enzyme recognition site that is reverse complementary to the sequence of, a sacB gene having the sequence of SEQ ID NO: 6, and a Tn5 translocation enzyme recognition site that is reverse complementary to the sequence of SEQ ID NO: 2. The transposon TnRIBD of claim 1, comprising the nucleotide sequence of SEQ ID NO: 1. Preparing a transposon comprising a transposon translocation enzyme recognition site and a selection marker; Inserting the transposon at an arbitrary position of a microbial chromosome and confirming the inserted position through chromosome sequencing; And Removing the left and right chromosomal portions of the inserted transposon by introducing translocation enzyme expression vectors into the chromosomes, Microbial mutant strain manufacturing method is removed any chromosomal site. The method of claim 3, wherein the transposon is a transposon TnRIBD according to claim 1 or 2. The method of claim 3, wherein the transposon, PCR amplification of translocation enzyme recognition sites of sacB and Tnγδ from pDELTA2 vector, tetR from Tn10, and Cm ^R gene from pKClox vector by PCR; Preparing a pRIBD vector by inserting OEs of sacB, tetR, Cm ^R and Tnγδ into a linear pMOD vector using ligase, The method of claim 1, wherein the pRIBD vector is digested with restriction enzyme XhoI to prepare a transposon TnRIBD according to claim 1 or 2. According to claim 3, wherein the translocation enzyme expression vector, pXRD4043 was digested with restriction enzyme NaeI / ClaI to prepare a truncated gene fragment containing a translocation enzyme related gene tnpA. Method for producing a microbial mutant strain prepared by the method comprising the step of inserting using a ligase in the linear vector pEL3 having a cutting end obtained by cutting with the restriction enzyme BamHI. The method according to any one of claims 3 to 6, by removing any chromosomal region to prepare a new microbial mutant strain, after confirming the gene of the removed part, to investigate the survival of the given mutant strain growth conditions To select non-essential microbial genes for growth. 8. The microorganism of claim 7, wherein the microorganisms are indispensable for growth, including the step of determining the cleavage size of any chromosomal site through intramolecular translocation through PCR using two primers determined at regular intervals located inside and outside the transposon. How to screen for genes. The method of claim 8, wherein the microbial genes essential for growth are selected using the primers C3 having the sequence of SEQ ID NO: 12 and primer p15 having the sequence of SEQ ID NO: 13 as the two primers. How to. The method of claim 8, wherein the microbial genes essential for growth are selected using the primers C5 having the sequence of SEQ ID NO: 14 and primer m10 having the sequence of SEQ ID NO: 15 as the two primers. How to.