WO2003089639A1 - Transposon for bidirectional intramolecular genome deletions, construction of novel microorganism and identification of nonessential genes using the same - Google Patents

Transposon for bidirectional intramolecular genome deletions, construction of novel microorganism and identification of nonessential genes using the same Download PDF

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Publication number
WO2003089639A1
WO2003089639A1 PCT/KR2003/000798 KR0300798W WO03089639A1 WO 2003089639 A1 WO2003089639 A1 WO 2003089639A1 KR 0300798 W KR0300798 W KR 0300798W WO 03089639 A1 WO03089639 A1 WO 03089639A1
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transposon
seq
nucleic acid
acid sequence
vector
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PCT/KR2003/000798
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French (fr)
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Sun-Chang Kim
Bong-Hyun Sung
Byung-Jo Yu
Jung-Min Kim
Won-Sik Lee
Choong-Hoon Lee
Jun-Hyoung Lee
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Korea Advanced Institute Of Science And Technology
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Priority to AU2003222482A priority Critical patent/AU2003222482A1/en
Publication of WO2003089639A1 publication Critical patent/WO2003089639A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1082—Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome

Definitions

  • the present invention relates to a deletion method of an optional region in genome using an intramolecular transposition of transposon and selection markers.
  • a transposon having transposase recognition sites (outer element, OE) and selection markers; and methods for identifying nonessential genes for growth under the given conditions and constructing a novel strain whose genome is partly deleted, by inserting the transposon into a genome of a microorganism and deleting an optional region bidirectionally using a transposase expression vector.
  • the techniques for studying function of a gene by inserting transposon into the chromosome and destroying the function of the gene has been well known to the art. In such techniques, however, it has not been reported to insert a transposon into chromosome of microorganism and delete optional regions around the inserted site in the chromosome. Also, even though the method of deleting the neighboring DNA segment using transposon on a vector is known, it has not been reported that nonessential genes for growth of a microorganism can be identified and a mutant microorganism with a partly deleted genome can be obtained, by inserting transposon into the chromosome of the living microorganism and removing neighboring DNA segments from the chromosome.
  • the object of the present invention is to improve the conventional method for deleting specific region of chromosome by many trials and errors in developing mutant microorganism with a partly deleted chromosome at a specific site since the gene functions of the microorganism are not fully elucidated and to provide a method and transposon for identifying and deleting the nonessential genes for growth more easily.
  • the present invention relates to a deletion of an optional region in chromosome using an intramolecular transposition of transposon and selection markers. Particularly, the present invention relates to a new method for identifying nonessential genes for growth under the given conditions and constructing novel strains with a partly deleted genome, by inserting the transposon into a genome of a microorganism and deleting an optional region bidirectionally using a transposase expression vector.
  • the present invention provides transposon with transposase recognition sites and selection markers. Further, the present invention also provides a method for developing a mutant microorganism, in which an optional chromosomal portion is deleted, comprising the steps of;
  • transposon containing transposase recognition sites and selection markers
  • transposon include Tn ⁇ and Tn5 transposase recognition sites as
  • transposase recognition site is for intramolecular transposition, and selection markers are for selection of the transposon insertion and deletion mutant.
  • transposon includes Tn5 transposase recognition site of 19 base pairs at each terminus, tetR and sacB genes as negative selection markers, Cm R gene as positive selection marker
  • transposon comprising the following elements in 5 , ⁇
  • TnRIBD 3' direction sequentially was constructed and designated as TnRIBD:
  • the length and the sequence of the transposon can vary depending on the vector used for the preparation of
  • the above transposon TnRIBD can be obtained from the pRIBD vector (see Figure 2) by treating with a restriction enzyme Xho ⁇ .
  • the processes for preparing the transposon TnRIBD from the pRIBD vector may include the following steps of:
  • the nucleic acid sequence of the transposon TnRIBD prepared by the above preparation method is as follows and is denoted SEQ ID NO:1.
  • the length and the sequence in the transposon can vary depending on the vector used for the preparation of transposon, except transposase recognition site of Tn5 and Tn ⁇ , Cm R , tetR and sacB.
  • Cm R , tetR and sacB genes are included in the transposon and preserved in the sequence, the function of the transposon does not change when more than one base of the rest sites is deleted, inserted or substituted. Also, it is
  • transposase recognition sites of Tn5 at each terminus of the TnRIBD with negative selection markers tetR and sacB on the outer left and right sides, respectively, and the positive selection marker (Cm R ) inside.
  • transposase of Tn5 is added into the above transposon TnRIBD to form transposome.
  • the transposome is transferred into microorganisms by the conventional electroporation to insert transposon into the optional site of the chromosome of the microorganism. Mutant microorganism inserted with the above transposon is selected, and then, the insertion site of the transposon in the selected mutants is identified. Since the optional inserting function of the transposase of Tn5 can be activated by Mg 2+ ion, transposome is formed in the absence of Mg 2+ ion, and the optional insertion of transposon into the microbial genome is carried out in the presence of Mg 2+ ion.
  • the above transposon includes chloramphenicol resistant gene, and thus, the strains with the transposon can be selected by cultivating them in the chloramphenicol media after chromosome insertion of the above transposon.
  • the insertion site of transposon can be identified by performing a direct genome sequencing analysis.
  • transposase gene tnpA transposase gene
  • SEQ ID NO: 7 was taken from transposase expression vector pXRD4043 on the ⁇ /ael/C/al (New England Biolabs Inc.) fragment and ligated to BamHI (New
  • the transposase expression vector constructed by the above method is called as pELTP and shown in Figure 3.
  • the base sequence is shown in SEQ ID NO: 8.
  • the degree of chromosome deletion can be approximately estimated by performing PCR using primer located on the transposon and the primer located on the genome at the distance of several kb to tens of kb from the transposon.
  • the length of chromosome deletion can be estimated accurately by an analysis of the base sequence of the PCR product using 3C (SEQ ID NO: 12) and p15 (SEQ ID NO: 13), or 5C (SEQ ID NO: 14) and m10 (SEQ ID NO: 15), respectively, as the primer located inside the transposon and the primer located at the distance of several kb to tens of kb from the transposon.
  • the sac ⁇ gene of Bacillus subtilis encodes levansucrase, which hydrolyzes sucrose into glucose and fructose and synthesizes levan by polymerizing fructose.
  • the above levan is toxic against microorganisms, and thus, the microorganisms with sacB gene cannot survive in the medium containing sucrose. Therefore, the microorganisms with sacB
  • the transposonase recognized by the transposonase to initiate intramolecular transposition and sacB gene is deleted.
  • sacB gene is deleted.
  • other genes around the sacB gene are also deleted. Therefore, if essential genes do not exist in the deleted region , the strain with the partly deleted chromosome at the sites can survive in the medium containing sucrose. Thereby, the deleted genes can be confirmed as the nonessential gene for cell survival.
  • tetracycline repressor tetR
  • the strains that have deleted tetR by intramolecular transposition can survive in the medium containing kanamycin.
  • some genes outside the tetR are deleted simultaneously with deletion of the tetR gene as above.
  • mutant microorganism with the partly deleted chromosome can be obtained.
  • the deleted genes in the chromosome can be identified as nonessential genes for growth.
  • E. coli was used as the above microorganism, and the mutants were prepared based on the above transposons Tn5 and
  • Tn5 has been reported to be inserted into an arbitrary site of the
  • the present invention related to the method of preparing the
  • the present invention relates to a method for developing mutant strains by deleting the genes on the left and right hand sides of the transposon insertion site of chromosome, and identifying nonessential genes for cell growth.
  • Figure 1 shows the steps in developing E. coli mutant strain with deletion of chromosomal segment at an arbitrary site by using transposon TnRIBD.
  • Figure 2 shows the structure of pRIBD, which is used for preparing transposon TnRIBD.
  • Figure 3 shows the structure of pELTP, which is a transposase expression vector.
  • Figure 4 shows deletion of an arbitrary portion of chromosome using negative selection maker sacB gene of transposon TnRIBD.
  • Figure 4a shows the method of confirming the degree of deletion after deleting an arbitrary portion of E. coli chromosome by using negative selection marker sacB gene.
  • Figure 4b is an electrophoresis photograph of PCR amplification result to confirm the degree of chromosome deletion by using the method in the above Figure 4a.
  • Figure 5 shows deletion of an arbitrary portion of chromosome using negative selection maker tetR/Ptet-Km R of transposon TnRIBD.
  • Figure 5a shows the method of confirming the degree of deletion after deleting an arbitrary portion of E. coli chromosome by using negative selection marker tetR/Ptet-Km R .
  • Figure 4b is an electrophoresis photograph of PCR amplification result to confirm the degree of chromosome deletion by using the method in the above Figure 5a..
  • transposon TnRIBD that can be inserted into an arbitrary site of E. coli chromosome
  • Figure 1 showing the chromosomal insertion of linear transposon
  • TnRIBD TnRIBD (SEQ ID NO:1) has sacB (SEQ ID NO:6), Cm R (SEQ ID NO:6)
  • Tn5 transposase recognition sites (Tn5 OE, SEQ ID NO:2) composed of 19 base pairs on both ends.
  • the above transposon TnRIBD can be obtained from pRIBD vector by treating with restriction enzyme Xho ⁇ as can be seen in Figure 2.
  • pRIBD was constructed by the following method: tetA and tetR genes amplified by PCR from Tn10 (Prof. Michael Koob, University of Minnesota, USA) were digested with restriction enzyme ⁇ /o-l/C/al (New England Biolabs Inc. MA, USA), cloned into a vector pKCIox (Yoon, YG, et al., 1998, Cre/loxP-mediated excision and amplification of the Escherichia coli genome, Genetic Analysis 14, 89-95), and designated as pKCtet.
  • pKCIox Yoon, YG, et al., 1998, Cre/loxP-mediated excision and amplification of the Escherichia coli genome, Genetic Analysis 14, 89-95
  • sacB gene amplified by PCR from pDELTA2 (Gibco BRL products, MD, USA) was digested with restriction enzyme SamHI (New England Biolabs Inc.), and inserted into the linearized vector pKCtet by restriction enzyme BamH ⁇ digestion and named pGtesa. After cleaving a fragment containing OE of Tn ⁇ amplified from pDELTA2 by PCR with restriction enzyme EcoRI (New
  • Cm R gene amplified from pKCIox by PCR was digested with restriction enzyme KpnUBglW (New England Biolabs Inc.), and cloned into the linearized vector pGtesa3 by Kpnl/Bglll and named pGCtesa3.
  • pMOD vector Epicentre technologies, Wl, USA
  • EcoRI/BamHI New England Biolabs Inc.
  • blunt ends were generated by T4 DNA polymerase (New England Biolabs Inc.), and self-ligated using ligase.
  • the obtained vector was named pMb2.
  • the obtained vector was named pRIBD (see Figure 2).
  • the obtained pRIBD as above was treated with restriction enzyme Xho ⁇ (New England Biolabs Inc.) and extracted on agarose gel, to be used as transposon TnRIBD.
  • transposome To inhibit the arbitrary insertion of transposome, the
  • E. coli mutant inserted with the above transposon has chloramphenicol resistance due to Cm R gene, it was selected in the media containing chloramphenicol.
  • the exact insertion position of transposon on E. coli chromosome was determined by performing a direct genome sequencing
  • Tn ⁇ transposase expression vector pELTP was introduced into E. coli
  • the above vector pELTP was prepared as follows (see Figure 3):
  • Transposase gene tnpA (SEQ ID NO: 7) was taken from transposase expression vector pXRD4043 (Gibco BRL products, Tsai, M. M. et al. 1987,
  • transposase expression vector pELTP was introduced into E. coli mutant containing transposon TnRIBD on its chromosome (J. Sambrook et al. Molecular Cloning-A Laboratory Manual. 2ed. Cold Spring Harbor. 1989). Since the transcription of the tnpA gene existing in the pELTP is controlled by tac promoter, the transposase was expressed by treating with the final concentration of 1 mM IPTG (Isopropyl-beta-D-
  • deletion mutant obtained from the result of transposase expression rightward deletion of chromosomal segment including sacB ( Figure 4a) was screened in the medium containing 5 % sucrose, and leftward deletion of chromosomal segment containing tetR at transposon insertion site ( Figure 5a) was screened in the medium containing kanamycin.
  • Example 2 the insertion position of transposon was identified to be the site of E. coli gene araA by direct genome sequencing
  • the deleted chromosomal part contains 15 genes such as araBC, yabl, sfuCB, tbpA, yabN, setA, leuDCBALO and ilvl. By this fact, it was confirmed that the above 15 genes are not essential for growth in case that the strain is cultivated
  • the deleted chromosomal part contains 5 genes such as araAD, polB, hepA and rluA. By this fact, it was confirmed that the above 5 genes are also not
  • the present invention relates to a method of developing novel E. coli strains with deletion of optional chromosomal part, using intramolecular transposition of transposon and selection markers.
  • E. coli chromosome can be deleted at various sites more efficiently, and the necessity of the genes, whose function is not known, for growth under the given conditions can be identified easily.
  • mutant E. coli with genetically simplified chromosomes can be constructed, which can be used for the research of functional genomics. Also, mutant E. coli showing a rapid growth can be used as an artificial strain for industrial use. Also, since the present invention can be applied for other microorganisms rather than E. coli, a variety of mutant microorganisms with minimized genome can be created. Further, other genes related to a specific metabolism can be collected to prepare cassettes, and inserted into the microorganism with minimized genome prepared according to the method of the present invention to create novel organisms with various useful functions.

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Abstract

Disclosed is a deletion method of an optional region in genome using an intramolecular transposition of transposon, and selection markers. Particularly, disclosed are a transposon having transposase recognition sites (outer element, OE) and selection markers; and methods for identifying nonessential genes for growth under the given conditions and constructing a novel strain, by inserting the transposon into a genome of a microorganism and deleting an optional site bidirectionally using a transposase expression vector.

Description

TRANSPOSON FOR BIDIRECTIONAL INTRAMOLECULAR GENOME
DELETIONS, CONSTRUCTION OF NOVEL MICROORGANISM AND
IDENTIFICATION OF NONESSENTIAL GENES USING THE SAME
[Technical field]
The present invention relates to a deletion method of an optional region in genome using an intramolecular transposition of transposon and selection markers. Particularly, disclosed are a transposon having transposase recognition sites (outer element, OE) and selection markers; and methods for identifying nonessential genes for growth under the given conditions and constructing a novel strain whose genome is partly deleted, by inserting the transposon into a genome of a microorganism and deleting an optional region bidirectionally using a transposase expression vector.
[Background Art]
Generally, the techniques for studying function of a gene by inserting transposon into the chromosome and destroying the function of the gene has been well known to the art. In such techniques, however, it has not been reported to insert a transposon into chromosome of microorganism and delete optional regions around the inserted site in the chromosome. Also, even though the method of deleting the neighboring DNA segment using transposon on a vector is known, it has not been reported that nonessential genes for growth of a microorganism can be identified and a mutant microorganism with a partly deleted genome can be obtained, by inserting transposon into the chromosome of the living microorganism and removing neighboring DNA segments from the chromosome.
Therefore, the object of the present invention is to improve the conventional method for deleting specific region of chromosome by many trials and errors in developing mutant microorganism with a partly deleted chromosome at a specific site since the gene functions of the microorganism are not fully elucidated and to provide a method and transposon for identifying and deleting the nonessential genes for growth more easily.
[Detailed Description of the Invention]
The present invention relates to a deletion of an optional region in chromosome using an intramolecular transposition of transposon and selection markers. Particularly, the present invention relates to a new method for identifying nonessential genes for growth under the given conditions and constructing novel strains with a partly deleted genome, by inserting the transposon into a genome of a microorganism and deleting an optional region bidirectionally using a transposase expression vector.
The present invention provides transposon with transposase recognition sites and selection markers. Further, the present invention also provides a method for developing a mutant microorganism, in which an optional chromosomal portion is deleted, comprising the steps of;
(1) constructing transposon containing transposase recognition sites and selection markers;
(2) inserting the above transposon into an optional site of microbial genome and identifying the insertion site;
(3) deleting the chromosomal portions on the left- and right-sides of the transposon insertion site by transforming the transposase expression vector into the microorganism; and (4) identifying the deleted chromosome region and selecting nonessential genes for growth.
In transposon and the above step (1) according to the present invention,
the above transposon include Tnχδ and Tn5 transposase recognition sites as
the above transposon transposase recognition sites; and negative selection markers such as sacB gene of Bacillus subtilis and tetracycline repressor gene (tetR) of Tn10, and a positive selection marker such as chloramphenicol resistant gene (CmR), as the above selection markers. Tn5 transposase
recognition site is for inserting transposon into the chromosome, Tn; £
transposase recognition site is for intramolecular transposition, and selection markers are for selection of the transposon insertion and deletion mutant.
In an embodiment of the present invention, transposon includes Tn5 transposase recognition site of 19 base pairs at each terminus, tetR and sacB genes as negative selection markers, CmR gene as positive selection marker
and Tnγδ transposase recognition site of 39 base pairs at each end of the
above CmR gene.
More particularly, transposon comprising the following elements in 5,→
3' direction sequentially was constructed and designated as TnRIBD:
- Tn5 transposase recognition site (outer element, OE) with the nucleic acid sequence of SEQ ID NO:2,
- tetR gene with the nucleic acid sequence of SEQ ID NO:4,
-
Figure imgf000006_0001
transposase recognition site with the nucleic acid sequence of
SEQ ID NO:3, - CmR gene with the nicleic acid sequence of SEQ ID NO:5,
- Tny£ transposase recognition site with the nucleic acid sequence
reverse-complementary to the sequence SEQ ID NO:3,
- sacB gene with the nucleic acid sequence of SEQ ID NO:6, and
- Tn5 transposase recognition site (outer element, OE) with the nucleic acid sequence reverse-complementary to the sequence of SEQ ID NO:2.
In the above transposon, the length and the sequence of the transposon can vary depending on the vector used for the preparation of
transposon except Tn5 transposase recognition site, Inyδ transposase
recognition site and the selection marker site, which are essential for the transposon of the present invention.
In another embodiment of the present invention, the above transposon TnRIBD can be obtained from the pRIBD vector (see Figure 2) by treating with a restriction enzyme Xho\.
The processes for preparing the transposon TnRIBD from the pRIBD vector may include the following steps of:
(i) obtaining amplified sacB and OE of Tnf£ from pDELTA2 vector
(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 U S A. 90:7874-8), tetR from Tn10, and CιmR gene from pKCIox vector (Yoon, Y G. et al., 1998. Cre/loxP-mediated excision and amplification of large segments of the Escherichia coli genome. Genet Anal. 14:89-95), by performing PCR,
(ii) constructing pRIBD vector by inserting sacB, tetR, CmR and OE of
Tn c? into linear p OD vector by using ligase; and,
(iii) preparing linear TnRIBD by cleaving the above pRIBD vector by using restriction enzyme Xhol.
The nucleic acid sequence of the transposon TnRIBD prepared by the above preparation method is as follows and is denoted SEQ ID NO:1.
TnRIBD base sequence
1 CTCGAGCTG7 CTCTTATACA CATCTCAACC CTGAAGCTAT CTTCCGAAGC AATAAATTCA II - Tnδ OE - II 61 CGTAATAACG TTGGCAAGAC TGGCATGATA AGGCCAATCC CCATGGCATC GAGTAACGTA
121 ATTACCAATG CGATCTTTGT CGAACTATTC ATTTCACTTT TCTCTATCAC TGATAGGGAG
181 TGGTAAAATA ACTCTATCAA TGATAGAGTG TCAACAAAAA TTAGGAATTA ATGΛ TGTC7>A
II - 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 CACGGCCTΛC 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 /4GCCΛGCCT7 C7T/A7TCGGC CTTGAATTGA TCATATGCGG ATTAGAAAAA CAACTTAAAT
841 GTGA A AGTGG GTCTTAAAAG CAGCATAACC TTTTTCCGTG ATGGTAACTT CACGGTAACC tetR - II 901 AAGATGTCGA GTTAACCACC CATCGATGAT AAGCTGTCAA ACATGAGAAT TCGGTGAATC 961 CCATAAATTC CCCGGATCGG GGT77GAGGG CCAATGGAAC GAAAACGTAC GTTAAGGATC
II - Tnγδ OE → II
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 261 GATCACTACC GGGCGTATTT TTTGAGTTGT CGAGATTTTC AGGAGCTAAG GAAGCTAAAA
II - 1321 TGGAGAAAAA AATCACTGGA TATACCACCG TTGATATATC CCAATGGCAT CGTAAAGAAC
CmR 1381 Λ7777GAGGC ATTTCAGTCA GTTGCTCAAT GTACCTATAA CCAGACCGTT CAGCTGGATA 1441 77ΛCGGCC777TTΛΛΛG/ACC GTAAAGAAAA ATAAGCACAA GTTTTATCCG GCCTTTATTC 1501 ΛCΛ77C77GC CCGCCTGATG AATGCTCATC CGGAΛ77ACG 7Λ7GGCAA7G AAAGACGGTG 1561 AGCTGGTGAT Λ7GGGΛ7AG7 GTTCACCCTT GTTACACCGT TTTCCATGAG CAAACTGAAA 1621 CGTTTTCATC GCTCTGGAGT GAATACCACG ACGATTTCCG GCAGTTTCTA CACATATATT 1681 CGC/AAG/A7G7 GGCG7G77ΛC GGTGAAAACC TGGCCTATTT CCCTAAAGGG TTTATTGAGA 1741 ATATGTTTTT CGTCTCAGCC AATCCCTGGG TGAGTTTCAC CAGTTTTGAT TTAAACGTGG 1801 CCA/47-47GGΛ CAACTTCTTC GCCCCCGTTT TCACCATGGG CAAATATTAT ACGCAAGGCG 1861 ACAAGGTGCT GATGCCGCTG GCGΛ7TC/4GG 77CA7CΛ7GC CGTTTGTGAT GGCTTCCATG 1921 7CGGCΛGA7G C77ΛΛ7G/A/A7ΛCAACAG7ΛC 7GCGΛ7GΛG7 GGCΛGGGCGG GGCG7AΛGGC
CmR - II
1981 GCGCCATTTA AATGAAGTTC CTATTCCGAA GTTCCTATTC TCTAGAAAGT ATAGGAACTT 2041 CGAAGCAGCT CCAGCCTACA GATCTGGCCG CTAATACGAC TCACTATAGG GAACTGACCC H -
2101 T7Λ/ACG7ACG TTTTCGTTCC ATTGGCCCTC AAACCCCAAT TCGTCAGACT TACGGTTAAG
Tnγδ OE - II
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 AGACA7G/AAC GΛ7GAAC/A7C Λ/A/AAΛG777G CAAAACAAGC AACAGTATTA ACCTTTACTA
II - sacB
2701 CCGCACTGCT GGCAGGAGGC GCAACTCAAG CGTTTGCGAA AGAAACGAAC CAAAAGCCAT 2761 /AT/A-AGG/AΛ/AC Λ7ΛCGGCΛ77 TCCCATATTA CACGCCATGA TATGCTGCAA ATCCCTGAAC
2821 AGCAAAAAAA TGAAAAATAT CAAGTTCCTG AATTTGATTC GTCCACAATT AAAAATATCT
2881 C77C7GC-AΛ/A ΛGGCC7GGΛC G777GGG/ACΛ GC7GGCCΛ77ΛCΛΛ AΛCGC7 GΛCGGC/AC7G
2941 7CGCAΛΛC7A 7CΛCGGC7ΛC CACΛ7CG7CT 77GCA77AGC CGGAGATCCT AAAAATGCGG
3001 /A7GΛCΛC.A7C G/A777ΛCA7G TTCTATCAAA AAGTCGGCGA AACTTCTATT GACAGCTGGA 3061 Λ AΛ/ACGC7GG CCGCG7C777 AΛ GΛCAGCG AC/AA/A77CGΛ TGCAAATGAT TCTATCCTAA
3121 /AΛG/ACCΛΛ AC ACAAGAATGG TCAGGTTCAG CCACATTTAC ATCTGACGGA AAAATCCGTT
3181 7Λ77C7ΛC/AC 7GA777C7CC GG7ΛA .CΛ77ΛCGGCA A ACΛ AACACTGACA ACTGCACAAG
3241 TTAACGTATC AGCATCAGAC AGCTCTTTGA ACATCAACGG TGTAGAGGAT TATAAATCAA
3301 TCTTTGACGG TGACGGAAAA ACGTATCAAA ATGTACAGCA GTTCATCGAT GAAGGCAACT 3361 ΛC/AGC7CΛGG CG/ACΛACCA7ΛCGC7GΛGAG /A7CC7C/AC7Λ CG7AG/4/AG .7ΛΛ/AGGCC/.CΛ
3421 ΛA7/AC7TΛG7Λ777GA/AGCΛ AACAC7GGΛΛ C7GA4G/A7GG C7ΛCCΛAGGC G/A/AGΛ/A7C77
3481 7Λ777Λ AC/AΛ ΛGCA7AC7Λ7 GGC/AΛΛΛGCΛ CΛ7CA77C77 CCG7CAΛGA/A AGTCAAAAAC
3541 77C7GCΛ ΛG CGATAAAAAA CGCACGGCTG AGTTAGCAAA CGGCGCTCTC GGTATGATTG
3601 AGC7ΛΛΛCGΛ TGATTACACA CTGAAAAAAG TGATGAAACC GCTGATTGCA TCTAACACAG 3661 7ΛiACΛG/A7GA AATTGAA CGC GCGAACGTCT TTAAAATGAA CGGCAAATGG TATCTGTTCA
3721 C7GΛC7CCCG CGGΛ7CAAΛA Λ7GΛCGΛ77G ACGGC/A77ΛC G7C7/A/.CGΛ7 A777ΛCΛ7GC
3781 TTGGTTATGT TTCTAATTCT TTAACTGGCC CATACAAGCC GCTGAACAAA ACTGGCCTTG
3841 7G77ΛΛΛΛ A7 GGATCTTGAT CCTAACGATG TAACCTTTAC TTACTCACAC TTCGCTGTAC
3901 C7C/AΛGCGΛ/A AGGAAACAAT GTCGTGATTA CAAGCTATAT GACAAACAGA GGATTCTACG 3961 CAGACAAACA ATCAACGTTT GCGCCGAGCT TCCTGCTGAA CATCAAAGGC AAGAAAACAT
4021 CTGTTGTCAA AGACAGCATC CTTGAACAAG GACAATTAAC AGTTAACAAA 7AΛAAACGCA sacB - II
4081 AAAGAAAATG CCGATATCCT ATTGGCATTT TCTTTTATTT CTTATCAACA TAAAGGTGAA
4141 TCCCATAAAT TCCCCGGATC CTCTAGAGTC GATGATGGTT G/AGΛTG7G7A T/AΛG/ GΛC G II - Tnδ OE - II
4201 CTCGAG As described above, the length and the sequence in the transposon can vary depending on the vector used for the preparation of transposon, except transposase recognition site of Tn5 and Tnγδ, CmR, tetR and sacB.
Thus, if all of Tn5 transposase and Tnγδ transposase recognition sites and
CmR, tetR and sacB genes are included in the transposon and preserved in the sequence, the function of the transposon does not change when more than one base of the rest sites is deleted, inserted or substituted. Also, it is
sufficient if the transposase recognition site of Tnγδ is located in between the
transposase recognition sites of Tn5 at each terminus of the TnRIBD, with negative selection markers tetR and sacB on the outer left and right sides, respectively, and the positive selection marker (CmR) inside.
In the above step (2), transposase of Tn5 is added into the above transposon TnRIBD to form transposome. The transposome is transferred into microorganisms by the conventional electroporation to insert transposon into the optional site of the chromosome of the microorganism. Mutant microorganism inserted with the above transposon is selected, and then, the insertion site of the transposon in the selected mutants is identified. Since the optional inserting function of the transposase of Tn5 can be activated by Mg2+ ion, transposome is formed in the absence of Mg2+ ion, and the optional insertion of transposon into the microbial genome is carried out in the presence of Mg2+ ion. Also, the above transposon includes chloramphenicol resistant gene, and thus, the strains with the transposon can be selected by cultivating them in the chloramphenicol media after chromosome insertion of the above transposon. The insertion site of transposon can be identified by performing a direct genome sequencing analysis.
In the above step (3), transposase of Tn^<5 can be expressed
continuously in the mutant strain with transposon in the chromosomal region to be deleted, by introducing the transposase expression vector into the above mutant strain. The chromosomal portion around the transposon is deleted
through the recognition of Tnj>£ OE by transposonase and the intramolecular
transposition thereby (see Figure 1).
In an embodiment of the present invention, transposase gene tnpA
(SEQ ID NO: 7) was taken from transposase expression vector pXRD4043 on the Λ/ael/C/al (New England Biolabs Inc.) fragment and ligated to BamHI (New
England Biolabs Inc.) fragment of temperature-sensitive vector pEL3. The transposase expression vector constructed by the above method is called as pELTP and shown in Figure 3. The base sequence is shown in SEQ ID NO: 8.
In the above step (4), the degree of chromosome deletion can be approximately estimated by performing PCR using primer located on the transposon and the primer located on the genome at the distance of several kb to tens of kb from the transposon.
In an embodiment of the present invention, the length of chromosome deletion can be estimated accurately by an analysis of the base sequence of the PCR product using 3C (SEQ ID NO: 12) and p15 (SEQ ID NO: 13), or 5C (SEQ ID NO: 14) and m10 (SEQ ID NO: 15), respectively, as the primer located inside the transposon and the primer located at the distance of several kb to tens of kb from the transposon.
The sacβ gene of Bacillus subtilis encodes levansucrase, which hydrolyzes sucrose into glucose and fructose and synthesizes levan by polymerizing fructose. The above levan, however, is toxic against microorganisms, and thus, the microorganisms with sacB gene cannot survive in the medium containing sucrose. Therefore, the microorganisms with sacB
gene can survive in the medium containing sucrose when the Tnγδ OE is
recognized by the transposonase to initiate intramolecular transposition and sacB gene is deleted. During the deletion process, other genes around the sacB gene are also deleted. Therefore, if essential genes do not exist in the deleted region , the strain with the partly deleted chromosome at the sites can survive in the medium containing sucrose. Thereby, the deleted genes can be confirmed as the nonessential gene for cell survival.
Expression of kanamycin resistant gene (KmR) under the control of
tetracycline promoter on λ attachment site of MG1655 λ att::Ptet-KmR is
inhibited by tetracycline repressor (tetR) expressed inside transposon. Therefore, the strains that have deleted tetR by intramolecular transposition can survive in the medium containing kanamycin. In this case, some genes outside the tetR are deleted simultaneously with deletion of the tetR gene as above. By using this, mutant microorganism with the partly deleted chromosome can be obtained. Also, the deleted genes in the chromosome can be identified as nonessential genes for growth.
In the present invention, E. coli was used as the above microorganism, and the mutants were prepared based on the above transposons Tn5 and
Tn^ . Tn5 has been reported to be inserted into an arbitrary site of the
chromosome (Berg, D. D. and M. M. Howe. 1989. Mobile DNA. American Society for Microbiology, Washington, D. C), and Tn^ has been reported to
be capable of intramolecular transposition (Wang, G. et al. 1993. pDUAL: A transposon-based cosmid cloning vector for generating nested deletions and DNA sequencing templates in vivo. Pro. Natl. Acad. Sci. USA 90, 7874-7878). Also, it is reported that E. coli with sacB gene of B. subtilis cannot survive in the medium with 5 % sucrose (Link, A. J. 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), and, when tetracycline repressor is expressed, kanamycin resistant gene which is under the control of tetracycline promoter cannot be expressed, and thus, E. coli cannot survive in the kanamycin medium (Koob, M. D. et al. 1994. Minimizing the genome of Escherichia coli. Motivation and strategy. Ann N Y Acad Sci. 30, 1-3).
Therefore, the present invention related to the method of preparing the
tranpson containing transposase recognition sites of Tn5 and Tnγδ and
inserting it into an arbitrary chromosomal site of a microorganism; deleting parts of the chromosome on the right and left sides of the transposon-insertion
site by Tny<5 transposase, and selecting the strain with the partial deletion of
chromosome by using negative selection markers sacB and tetR/Ptet-KmR. In other words, the present invention relates to a method for developing mutant strains by deleting the genes on the left and right hand sides of the transposon insertion site of chromosome, and identifying nonessential genes for cell growth. [Brief Description of Drawings]
Figure 1 shows the steps in developing E. coli mutant strain with deletion of chromosomal segment at an arbitrary site by using transposon TnRIBD. Figure 2 shows the structure of pRIBD, which is used for preparing transposon TnRIBD.
Figure 3 shows the structure of pELTP, which is a transposase expression vector.
Figure 4 shows deletion of an arbitrary portion of chromosome using negative selection maker sacB gene of transposon TnRIBD. Figure 4a shows the method of confirming the degree of deletion after deleting an arbitrary portion of E. coli chromosome by using negative selection marker sacB gene. Figure 4b is an electrophoresis photograph of PCR amplification result to confirm the degree of chromosome deletion by using the method in the above Figure 4a.
Figure 5 shows deletion of an arbitrary portion of chromosome using negative selection maker tetR/Ptet-KmR of transposon TnRIBD. Figure 5a shows the method of confirming the degree of deletion after deleting an arbitrary portion of E. coli chromosome by using negative selection marker tetR/Ptet-KmR. Figure 4b is an electrophoresis photograph of PCR amplification result to confirm the degree of chromosome deletion by using the method in the above Figure 5a.. EXAMPLES
The invention will be further illustrated by the following examples. It will be apparent to those having conventional knowledge in the field that these examples are given only to explain the present invention more clearly, but the invention is not limited to the examples given.
EXAMPLE 1
Construction of transposon TnRIBD that can be inserted into an arbitrary site of E. coli chromosome As Figure 1 showing the chromosomal insertion of linear transposon
TnRIBD, TnRIBD (SEQ ID NO:1) has sacB (SEQ ID NO:6), CmR (SEQ ID
NO:5), tetR (SEQ ID NO:4) and OE (SEQ ID NO:3) of Tnj , and it further has
Tn5 transposase recognition sites (Tn5 OE, SEQ ID NO:2) composed of 19 base pairs on both ends. The above transposon TnRIBD can be obtained from pRIBD vector by treating with restriction enzyme Xho\ as can be seen in Figure 2.
The above pRIBD was constructed by the following method: tetA and tetR genes amplified by PCR from Tn10 (Prof. Michael Koob, University of Minnesota, USA) were digested with restriction enzyme Λ/o-l/C/al (New England Biolabs Inc. MA, USA), cloned into a vector pKCIox (Yoon, YG, et al., 1998, Cre/loxP-mediated excision and amplification of the Escherichia coli genome, Genetic Analysis 14, 89-95), and designated as pKCtet. sacB gene amplified by PCR from pDELTA2 (Gibco BRL products, MD, USA) was digested with restriction enzyme SamHI (New England Biolabs Inc.), and inserted into the linearized vector pKCtet by restriction enzyme BamH\ digestion and named pGtesa. After cleaving a fragment containing OE of Tnγδ amplified from pDELTA2 by PCR with restriction enzyme EcoRI (New
England Biolabs Inc.), a new vector was made by inserting the cleaved fragment into the linear vector pGtesa cleaved by restriction enzyme EcoRI and named pGtesa3.
Then, CmR gene amplified from pKCIox by PCR was digested with restriction enzyme KpnUBglW (New England Biolabs Inc.), and cloned into the linearized vector pGtesa3 by Kpnl/Bglll and named pGCtesa3. After pMOD vector (Epicentre technologies, Wl, USA) was digested with restriction enzyme EcoRI/BamHI (New England Biolabs Inc.), blunt ends were generated by T4 DNA polymerase (New England Biolabs Inc.), and self-ligated using ligase. The obtained vector was named pMb2. After amplifying a DNA fragment including the sequence of Tn5 transposase recognition site of pMb2 by PCR
using 5' primer having the sequence of 5 Φ -
GAATTCTCGAGCTGTCTCTTATACACATCTC-3 <2 (SEQ ID NO: 9) and 3'
primer having the sequence of 50 -
ACATGTCTCGAGCTGTCTCTTATACACATCTC-3 <Z: (SEQ ID NO: 10), a new
vector was made by inserting the amplified DNA fragment into a linear vector pUC19 (New England Biolabs Inc.) cleaved by restriction enzyme EcoRI/Afflll (New England Biolabs Inc.) and named pMb3. Linearized pGCtesa3 by treating with restriction enzyme Hind\\\ (New England Biolabs Inc.) was cloned into the pMb3 and named pRIBDt. After cleaving vector pRIBDt with restriction enzyme EcoRV/Λ/o-1 (New England Biolabs Inc.), DNA fragment containing tetR, CmR, sacB, and OE of Tnγ δ and Tn5 was treated with T4
DNA polymerase to generate blunt ends, and self-ligated. The obtained vector was named pRIBD (see Figure 2).
The obtained pRIBD as above was treated with restriction enzyme Xho\ (New England Biolabs Inc.) and extracted on agarose gel, to be used as transposon TnRIBD.
EXAMPLE 2
Insertion of transposon TnRIBD into optional site of E. coli chromosome and identification of the insertion site
After 500 ng of transposon TnRIBD, 10 units of Tn5 transposase (Epicentre technologies), and double-distilled water were reacted for 1 h at 37
°C to form transposome. To inhibit the arbitrary insertion of transposome, the
above reaction was carried out in the absence of Mg2+ ion. Then, under the
reaction condition in the presence of magnesium ions, 1 μl of transposome
was transferred into E. coli MG1655 λ att::Ptet-KmR strain (Koob, M. D. et al.
1994. Minimizing the genome of Escherichia coli. Motivation and strategy. Ann N Y Acad Sci. 30, 1-3, obtained from Michael Koob, University of Minnesota, USA) by conventional electroporation method (Bio-Rad, Bacterial electro- transformation and pulse controller instruction manual, Cat. No 165-2098). The above strain was cultivated in LB medium (tryptone 1%, yeast extract 0.5%, NaCI 0.5%). In the above medium, a small amount of magnesium ion was added to activate the arbitrary insertion function of transposome in the cells, and thereby, transposon was inserted into an optional site of E. coli chromosome.
Since the E. coli mutant inserted with the above transposon has chloramphenicol resistance due to CmR gene, it was selected in the media containing chloramphenicol. The exact insertion position of transposon on E. coli chromosome was determined by performing a direct genome sequencing
using primer Sac-out (50 -TGTTGTCAAAGACAGCATCCTTGAACAAGG- 03,
synthesized by Genotech, Inc, SEQ ID NO: 11). It was confirmed by comparing the result of the direct genome sequencing analysis with GenBank DNA sequence using BLAST program, that transposon was inserted into the above E. coli strain.
EXAMPLE 3
Construction of E. coli mutant strain having deletion mutation of chromosomal segment by expressing transposase through introducing transposase expression vector pELTP into the above strain
Tn^ transposase expression vector pELTP was introduced into E. coli
mutant strain with transposon TnRIBD prepared according to Example 2.
The above vector pELTP was prepared as follows (see Figure 3):
Transposase gene tnpA (SEQ ID NO: 7) was taken from transposase expression vector pXRD4043 (Gibco BRL products, Tsai, M. M. et al. 1987,
Transposition of Tn1000: in vivo properties. J Bacteriol. 169: 5556-62) on the
Λ/ael/C/al (New England Biolabs Inc.) fragment and ligated to BamHI (New
England Biolabs Inc.) fragment of temperature-sensitive vector pEL3 (Obtained from Seiichi Yasuda, National Institute of Genetics, Japan, Armstrong, K. A. et al. 1984. A 37 X 103 molecular weight plasmid-encoded protein is required for replication and copy number control in the plasmid pSC101 and its temperature-sensitive derivative pHS J Mol Biol. 175: 331-48) to generate pELTP (SEQ ID NO: 8). In each cloning step, blunt ends were generated using T4 DNA polymerase (New England Biolabs Inc.). The prepared pELTP is shown in Figure 3.
The above prepared transposase expression vector pELTP was introduced into E. coli mutant containing transposon TnRIBD on its chromosome (J. Sambrook et al. Molecular Cloning-A Laboratory Manual. 2ed. Cold Spring Harbor. 1989). Since the transcription of the tnpA gene existing in the pELTP is controlled by tac promoter, the transposase was expressed by treating with the final concentration of 1 mM IPTG (Isopropyl-beta-D-
thiogalactoside) and cultivating in a shaking incubator at 30 °C. In the
deletion mutant obtained from the result of transposase expression, rightward deletion of chromosomal segment including sacB (Figure 4a) was screened in the medium containing 5 % sucrose, and leftward deletion of chromosomal segment containing tetR at transposon insertion site (Figure 5a) was screened in the medium containing kanamycin.
In Example 2 and Example 3, the insertion position of transposon was identified to be the site of E. coli gene araA by direct genome sequencing
analysis. After Tn/J transposase expression in E. coli mutant strains by
transformation of transposase expression vector pELTP, the mutant strains were cultivated in the medium containing 5 % sucrose or kanamycin to select the strains with deletion of a part of chromosome. Deletion size of chromosomal segment was assumed by PCR and electrophoretic analysis of its results was shown in Figures 4b and 5b. As can be seen in Figures 4b and 5b, it can be confirmed that parts of E. coli chromosomes are deleted.
EXAMPLE 4
Identification of the deleted chromosomal region and selection of nonessential genes for growth under given conditions
As can be seen in Figure 4b, the electrophoretic analysis of the
amplified PCR product using primer 3C (5 φ -GTTCATCATGCCGTTTGTG-3 Φ ,
SEQ ID NO: 12) and p15 (5 Φ -CCTGCCACTGCTTCACCATCCCC-3 Φ , SEQ
ID NO: 13), showed that approximately 16 kb of chromosome was deleted. The deleted chromosomal part contains 15 genes such as araBC, yabl, sfuCB, tbpA, yabN, setA, leuDCBALO and ilvl. By this fact, it was confirmed that the above 15 genes are not essential for growth in case that the strain is cultivated
in LB medium at 37 °C.
As can be seen in Figure 5b, the electrophoretic analysis of the PCR
product using primer 5C (5 φ -CCTTAGCTCCTGAAAATCTCG-3 , SEQ ID
NO: 14) and m10 (5 φ -GCTCATCGGAGATTTTCACTCCC-3 φ , SEQ ID NO:
15), showed that approximately 8 kb of the chromosome was deleted. The deleted chromosomal part contains 5 genes such as araAD, polB, hepA and rluA. By this fact, it was confirmed that the above 5 genes are also not
essential for growth in case that the strain is cultivated in LB medium at 37 °C [Industrial Applicability]
As mentioned above, the present invention relates to a method of developing novel E. coli strains with deletion of optional chromosomal part, using intramolecular transposition of transposon and selection markers. According to the present invention, E. coli chromosome can be deleted at various sites more efficiently, and the necessity of the genes, whose function is not known, for growth under the given conditions can be identified easily.
By deleting the nonessential genes for growth according to the above method, mutant E. coli with genetically simplified chromosomes can be constructed, which can be used for the research of functional genomics. Also, mutant E. coli showing a rapid growth can be used as an artificial strain for industrial use. Also, since the present invention can be applied for other microorganisms rather than E. coli, a variety of mutant microorganisms with minimized genome can be created. Further, other genes related to a specific metabolism can be collected to prepare cassettes, and inserted into the microorganism with minimized genome prepared according to the method of the present invention to create novel organisms with various useful functions.

Claims

1. A transposon comprising Tn5 transposase recognition site with the nucleic acid sequence of SEQ ID NO: 2, tetR gene with the nucleic acid
sequence of SEQ ID NO: 4, Tn^ transposase recognition site with the nucleic
acid sequence of SEQ ID NO: 3, CmR gene with the nucleic acid sequence of
SEQ ID NO: 5, Tnγδ transposase recognition site with the nucleic acid
sequence reverse-complementary to the sequence of SEQ ID NO: 3, sacB gene with the nucleic acid sequence of SEQ ID NO: 6 and Tn5 transposase recognition site with the nucleic acid sequence reverse-complementary to the
sequence of SEQ ID NO: 2, sequentially in 5'→ 3' direction.
2. The transposon (TnRIBD) according to claim 1 comprising the sequence of SEQ ID NO: 1.
3. A method for developing a mutant strain with deletion of an optional part of chromosome comprising the steps of:
- constructing transposon comprising transposase recognition sites and
selection markers;
- inserting the above transposon into an optional site of the chromosome of a microorganism and identifying the insertion site of
transposon; and
- deleting the parts of the chromosome on the left and right hand sides of the insertion site of transposon by transforming transposase expression vector into the microorganism.
4. The method for developing a mutant strain according to claim 3, wherein the above transposon is the transposon according to claim 1 or 2.
5. The method for developing a mutant strain according to claim 3,
wherein the above transposon is prepared by the steps of;
- performing PCR amplification to obtain sacB and Tnγδ transposase
recognition site from pDELTA2 vector, tetR from Tn10 and CmR gene from
pKCIox vector,
- preparing pRIBD vector by inserting sacB, tetR, CmR and OE of Tn^5
into linear pMOD vector by using ligase, and
- preparing transposon according to claim 1 or 2 by cleaving the above
pRIBD vector with restriction enzyme Xho\.
6. The method for developing a mutant strain according to claim 3, wherein the above transposase expression vector is prepared by the steps of;
- cleaving pXRD4043 with restriction enzyme Λ/ael/C/al to prepare a
gene fragment with blunt ends comprising transposase related gene tnpA, and
- inserting the above gene fragment into linear vector pEL3 with blunt
ends obtained by cleaving with restriction enzyme βamHI, by using ligase.
7. A method for identifying nonessential genes for growth of
microorganisms comprising the steps of: constructing a new mutant strain by deleting an optional part of genome according to the method in any one of claims 3 through 6; identifying the genes in the deleted part; and investigating the survival of the above mutant strain under the growth conditions.
8. The method for identifying nonessential genes for growth of microorganisms according to claim 7, comprising the additional step of determining the size of the optionally deleted chromosomal part through intramolecular transposition by performing PCR with two selected primers with certain distance between them and located in- and outside of transposon.
9. The method for identifying nonessential genes for growth of microorganisms according to claim 8, wherein primer C3 with the nucleic acid sequence of SEQ ID NO: 12 and primer p15 with the nucleic acid sequence of SEQ ID NO: 13 are used as the above two primers.
10. The method for identifying nonessential genes for growth of microorganisms according to claim 8, wherein primer C5 with the nucleic acid sequence of SEQ ID NO: 14 and primer m10 with the nucleic acid sequence of SEQ ID NO: 15 are used as the above two primers.
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