CN115369098A - Novel CRISPR (clustered regularly interspaced short palindromic repeats) related transposase - Google Patents

Novel CRISPR (clustered regularly interspaced short palindromic repeats) related transposase Download PDF

Info

Publication number
CN115369098A
CN115369098A CN202110532731.0A CN202110532731A CN115369098A CN 115369098 A CN115369098 A CN 115369098A CN 202110532731 A CN202110532731 A CN 202110532731A CN 115369098 A CN115369098 A CN 115369098A
Authority
CN
China
Prior art keywords
homology
seq
plasmid
leu
gene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202110532731.0A
Other languages
Chinese (zh)
Inventor
杨晟
杨思琪
张译文
徐佳琪
张姣
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Center for Excellence in Molecular Plant Sciences of CAS
Original Assignee
Center for Excellence in Molecular Plant Sciences of CAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Center for Excellence in Molecular Plant Sciences of CAS filed Critical Center for Excellence in Molecular Plant Sciences of CAS
Priority to CN202110532731.0A priority Critical patent/CN115369098A/en
Priority to PCT/CN2022/091102 priority patent/WO2022242464A1/en
Publication of CN115369098A publication Critical patent/CN115369098A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • 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
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • 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
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/90—Vectors containing a transposable element

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Mycology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention discloses a novel CRISPR-related transposase which is derived from a semitransparent pseudoalteromonas KMM520, can identify 16 PAMs, can insert cargo genes into 8 sites at one time, has 100 percent of efficiency and is higher than the CRISPR-related transposase derived from vibrio cholerae Tn 6677; the efficiency of transposing the 15.4kb cargo gene to the corresponding target is 100 percent, and the transposase can be used in the same escherichia coli with CRISPR related transposase derived from vibrio cholerae Tn6677, and can play a role without mutual interference, thereby having wide application prospect.

Description

Novel CRISPR (clustered regularly interspaced short palindromic repeats) related transposase
Technical Field
The invention belongs to the technical field of gene editing, and particularly relates to a novel CRISPR (clustered regularly interspaced short palindromic repeats) related transposase and application thereof in gene editing.
Background
In metabolic engineering, the coordination between different enzymes of a metabolic pathway or different metabolic pathways can be made consistent by adjusting the dose and ratio of enzyme genes to maximize the overall reaction rate. The most common way to increase the dosage of gene expression cassettes in bacteria is by plasmids, but genetic stability problems are encountered. Integrating gene expression cassettes one by one onto bacterial chromosomes is time consuming and difficult to quickly test many construction protocols. CRISPR-Cas can cleave genomic multicopy sequences or target multiple different sequences simultaneously with crRNA arrays, but is limited by the efficiency of repair of double strand breaks, making it difficult to insert more than 3 copies of a gene expression cassette at a time. Therefore, there is a need for a better method to obtain combinations of different gene doses.
In 2019, the research team of the Broad institute and the Sam Sternberg research team of the university of Columbia published two similar findings: using bacterial transposable genes, DNA sequences are precisely inserted into the genome without cutting the DNA. Wherein, the Zhang front research group extracts a transposase from blue algae, and the transposase is named as CAST, namely CRISPR-associated transposase. After a Sternberg research group discovers a unique transposable gene in Vibrio cholerae, a gene editing tool named INTEGRATE (Insertion of transposable element guiding RNA assisted targeting) is developed, and large fragment genes can be inserted into a genome without introducing DNA breakage. The novel technique, INTEGRATE, uses transposable genes to insert DNA sequences into the genome without cleaving the DNA. Both the CAST system and the INTEGRATE system can INTEGRATE DNA fragments into a preset site of an escherichia coli chromosome through transposition without depending on homologous recombination, and have no resistance marker residue and no double-strand DNA fracture.
Based on the CAST system and the INTEGRATE system, the inventor develops a Multicopy gene insertion system MUCICAT (see patent document CN 202010083919.7), which can obtain a strain with 10 copies of a Cargo gene (Cargo gene) inserted into a chromosome in 5 days, has the advantages of editing, rapidness, no marker, fixed point and the like, and at present, MUCICAT has been successfully applied to the construction of enzyme engineering strains and metabolic engineering strains (Zhang, Y., multicopy chromosomal integration using CRISPR-associated metabolic strains. Acs Synthetic Biology, 2020.). However, metabolic engineering and synthetic biology generally involve optimization of the expression levels of multiple enzymes or pathways. In one round of transposition, MUCICAT based on a single CRISPR-associated transposase can only search the optimal dose of a single cargo gene loaded with single or multiple genes, and cannot screen the optimal proportion of the gene dose of multiple genes or pathways. At present, no chromosome copy number technology for parallel amplification of multiple genes/pathways is reported. MUCICAT technology, which involves multiple CRISPR-associated transposases orthogonal to each other, promises to achieve simultaneous independent amplification of multiple genes or pathways in a single cell, creating a library of strains with different gene dose ratios to screen for optimal dose ratios of multiple genes or pathways.
Among the active CRISPR transposases, CRISPR transposases of type I-F3 derived only from Vibrio cholerae Tn6677 have high insertion and high targeting rates (Klompe, S.E., et al, transposon-encoded CRISPR-Cas systems direct RNA-guided DNA integration. Nature,2019.571 (7764): p.219-225), tilletia V-K derived from Haematococcus and Coleoptera cylindrica (Jonathan Strecker et al, RNA-guided DNA analysis with CRISPR-associated transformed space. Science, 2019), aeromonas salmonicida S44 (Miel T.Petas et al, guide RNA catalysis Enable Target Site 7-Choil-CRISPR transposons. Tn, cas and Saccharoth algae and Biocide type I-transferring, and Biocide DNA mutation, and high targeting rates (Rhync-A, S.S.1. And/or Rhync. Coli) and high targeting rates of cell A, high targeting rates (Rhync-202A, S.2021-immobilized DNA integration, S.S.S.S.S.S.1. And C. Japonica, S.1. Cholera. And/or Rhynchos. Origin.
Disclosure of Invention
According to the characteristics of the CRISPR transposase in the prior art, other novel efficient CRISPR-associated transposase or an orthogonal CRISPR-associated transposase system which can be combined with MUCICAT is ideal and applied to complicated metabolic engineering and synthetic biology design. In order to achieve the purpose, the inventor conducts extensive screening on CRISPR transposases derived from other microorganisms, and finds that a CRISPR transposition system I-F3 derived from Pseudoalteromonas translucens KMM520 (Pseudomonas transamicus KMM 520) has insertion efficiency which is no inferior to or even better than that of Vibrio cholerae Tn6677 in Escherichia coli, and can be targeted to insert between eda-purT and lacZ sites respectively without interfering with Vibrio cholerae Tn 6677. The novel CRISPR transposition system can identify all 16 PAMs without PAM dependency.
Accordingly, a first aspect of the present invention provides a CRISPR-associated transposase comprising a polypeptide selected from the group consisting of: a transposase protein tnsA derived from a bacterium of the genus pseudoalteromonas, a transposase protein tnsB derived from a bacterium of the genus pseudoalteromonas, a transposase protein tnsC derived from a bacterium of the genus pseudoalteromonas, a transposase protein tniQ derived from a bacterium of the genus pseudoalteromonas, a nuclease protein Cas5/8 derived from a bacterium of the genus pseudoalteromonas, a nuclease protein Cas6 derived from a bacterium of the genus pseudoalteromonas, and a nuclease protein Cas7 derived from a bacterium of the genus pseudoalteromonas.
Preferably, the pseudoalteromonas bacterium is a semitransparent pseudoalteromonas bacterium. More preferably, the Pseudoalteromonas translucens is Pseudoalteromonas translucens KMM520 (Pseudoalteromonas translucens KMM 520).
In a specific embodiment, the CRISPR-associated transposase described above preferably comprises a polypeptide selected from the group consisting of:
tnsA: it is a polypeptide with an amino acid sequence of SEQ ID NO. 1, or a polypeptide which has more than 95 percent of homology, preferably more than 98 percent of homology, more preferably more than 99 percent of homology and same functions with the SEQ ID NO. 1;
tnsB: it is a polypeptide with an amino acid sequence of SEQ ID NO. 2, or a polypeptide which has more than 95% homology, preferably more than 98% homology, more preferably more than 99% homology with SEQ ID NO. 2 and has the same function;
tnsC: it is a polypeptide with an amino acid sequence of SEQ ID NO. 3, or a polypeptide which has more than 95% homology, preferably more than 98% homology, more preferably more than 99% homology with SEQ ID NO. 3 and has the same function;
tniQ: it is a polypeptide having an amino acid sequence of SEQ ID NO. 4, or a polypeptide having more than 95% homology, preferably more than 98% homology, more preferably more than 99% homology with SEQ ID NO. 4 and having the same function;
cas5/8: it is a polypeptide having an amino acid sequence of SEQ ID NO. 5, or a polypeptide having 95% or more homology, preferably 98% or more homology, more preferably 99% or more homology, with SEQ ID NO. 5 and having the same function;
cas6: it is a polypeptide having an amino acid sequence of SEQ ID NO. 6, or a polypeptide having 95% or more homology, preferably 98% or more homology, more preferably 99% or more homology, with SEQ ID NO. 6 and having the same function;
cas7: it is a polypeptide having the amino acid sequence of SEQ ID NO. 7, or a polypeptide having 95% or more homology, preferably 98% or more homology, more preferably 99% or more homology, with SEQ ID NO. 7 and having the same function.
The nucleases Cas5/8, cas7 and Cas6 are Cascade complexes of a type I CRISPR system, and are linked with transposases tnsABC and tniQ to become CRISPR-associated transposases. These polypeptides are all derived from Pseudoalteromonas translucens KMM520 (Pseudomonas transamicida KMM 520).
In a second aspect, the present invention provides a gene encoding the above polypeptide.
In a specific embodiment, the gene encoding the polypeptide tnsA having the amino acid sequence of SEQ ID No. 1 is the nucleotide sequence SEQ ID No. 8 or a polynucleotide having more than 80% homology, preferably more than 85% homology, more preferably more than 90% homology, more preferably more than 95% homology to SEQ ID No. 8;
the gene encoding the polypeptide tnsB having the amino acid sequence of SEQ ID No. 2 is the nucleotide sequence SEQ ID No. 9, or a polynucleotide having more than 80% homology, preferably more than 85% homology, more preferably more than 90% homology, more preferably more than 95% homology with SEQ ID No. 9;
the gene encoding the polypeptide tnsC having the amino acid sequence of SEQ ID No. 3 is the nucleotide sequence of SEQ ID No. 10 or a polynucleotide having more than 80% homology, preferably more than 85% homology, more preferably more than 90% homology, more preferably more than 95% homology with SEQ ID No. 10;
the gene encoding the polypeptide tniQ having the amino acid sequence of SEQ ID NO. 4 is the nucleotide sequence of SEQ ID NO. 11 or a polynucleotide having more than 80% homology, preferably more than 85% homology, more preferably more than 90% homology, more preferably more than 95% homology with SEQ ID NO. 11;
the gene encoding the polypeptide Cas5/8 having the amino acid sequence of SEQ ID NO. 5 is the nucleotide sequence SEQ ID NO. 12, or a polynucleotide having more than 80% homology, preferably more than 85% homology, more preferably more than 90% homology, more preferably more than 95% homology with SEQ ID NO. 12;
the gene encoding the polypeptide Cas6 having the amino acid sequence of SEQ ID NO. 6 is the nucleotide sequence SEQ ID NO. 13, or a polynucleotide having more than 80% homology, preferably more than 85% homology, more preferably more than 90% homology, more preferably more than 95% homology with SEQ ID NO. 13;
the gene encoding the polypeptide Cas7 having the amino acid sequence of SEQ ID NO. 7 is the nucleotide sequence SEQ ID NO. 14 or a polynucleotide having more than 80% homology, preferably more than 85% homology, more preferably more than 90% homology, more preferably more than 95% homology with SEQ ID NO. 14.
A third aspect of the invention is to provide a CRISPR transposome system. Like the transposition system INTEGRATE system or CAST system, the CRISPR transposable system of the present invention also includes plasmid pQCascade, helper plasmid pTns, and helper plasmid pDonor carrying the cargo gene. Any two of the three plasmids may even be combined into one plasmid, or even the three plasmids may be combined together to form one plasmid. In particular, the method comprises the steps of,
a plasmid, pqqacade, for CRISPR transposon systems comprising a gene fragment selected from the group consisting of: the above-mentioned Cas 5/8-encoding gene; the above-mentioned Cas 6-encoding gene; the Cas 7-encoding gene described above; the tniQ-encoding gene described above.
The polypeptides are all derived from pseudoalteromonas translucency KMM520 (C)Pseudoalteromonas transiduda KMM 520), which plasmid, pqcacide, is designated herein as pqcacadeptr. Similarly, the plasmids pTns and pDonor of the present invention may be hereinafter denoted as pTnsPtr and pDonorPtr, respectively.
Preferably, the plasmid pQCascadePtr may further include the following genes: crRNA sequences targeting the genomic target site, cloDF13 replicons, promoters such as anhydrotetracycline inducible promoter, streptomycin resistance gene.
The crRNA in the CRISPR transposase described above can function in the form of array.
In one embodiment, the spacer of the crRNA series may be a spacer that targets a single site in the genome of the cell to be treated.
In another embodiment, the crRNA array can be a crRNA array (also called crisprraray, crRNA array, or array) that targets multiple sites in the genome of the cells to be treated.
Preferably, the crRNA sequence is a crRNA array targeting multiple sites in the genome, wherein the repeat region repeat comprises one or more sequences selected from the group consisting of: the nucleotide sequence is repeat1 of SEQ ID NO. 15, the nucleotide sequence is repeat2 of SEQ ID NO. 16, the nucleotide sequence is repeat3 of SEQ ID NO. 17, the nucleotide sequence is repeat4 of SEQ ID NO. 18, and 32N (namely [ N32 ]) in the nucleotide sequences SEQ ID NOs:15-18 are any base A, T, G or C.
A helper plasmid pTnsPtr for CRISPR transposon systems, for use with the plasmid pqcracadeptr described above, comprising a gene fragment selected from the group consisting of: the above-mentioned tnsA-encoding gene, the above-mentioned tnsB-encoding gene, and the above-mentioned tnsC-encoding gene.
When the CRISPR transposase gene sequence is used for CRISPR transposition, it is required to function together with a promoter, left End (LE) -cargo-Right End (RE), which is available in a host (gram-negative bacteria such as Escherichia coli, vibrio natriensis and Tatamibacterium citreum; gram-positive bacteria such as Corynebacterium glutamicum), whether in a plasmid form or an integrated form.
Preferably, the helper plasmid pTnsPtr described above further includes the following genes: colA replicons, promoters such as anhydrotetracycline inducible promoter, kanamycin resistance gene.
As an embodiment of combining two plasmids into one plasmid, the present invention provides a plasmid pqcrastnsptr for CRISPR transposon system, which is combined with the plasmid pqcracadeptr and helper plasmid pTnsPtr described above, comprising: the above-mentioned Cas5/8, cas6, cas7, tniQ, tnsA, tnsB and tnsC genes, crRNA sequences targeting a genomic target site, colA replicons, promoters such as anhydrotetracycline inducible promoter, kanamycin resistance gene.
A helper plasmid pDonorPtr for CRISPR transposon systems for use in conjunction with the above plasmid pqqascadeptr and the above helper plasmid ptr, comprising gene fragments selected from the group consisting of: a Left End (LE) sequence having a nucleotide sequence of SEQ ID NO. 19 or a sequence having more than 80% homology, preferably more than 85% homology, more preferably more than 90% homology, more preferably more than 95% homology with SEQ ID NO. 19 and comprising 33bp of the 3' end of SEQ ID NO. 19; the sequence Right End (RE) having the nucleotide sequence of SEQ ID NO:20 or a sequence 27bp more than 80%, preferably more than 85%, more preferably more than 90%, more preferably more than 95% homologous to SEQ ID NO:20 and comprising the 5' end of SEQ ID NO: 20; the Cargo gene of interest (Cargo gene).
Because LE with the nucleotide sequence of SEQ ID NO. 19 and RE with the nucleotide sequence of SEQ ID NO. 20 are from the pseudoalteromonas translucency KMM520 (Pseudoalteromonas transiduda KMM 520), the plasmid pDonor being named pDonorPtr herein.
Preferably, the helper plasmid pDonorPtr described above may further include the following genes: pMB1 replicon, ampicillin resistance gene, target Cargo gene (Cargo gene); or the following genes: p15A replicon, chloramphenicol resistance gene, cargo gene of interest (Cargo gene).
As an embodiment of the above three plasmids combined into one plasmid, the present invention provides a plasmid pEffectorPtr for CRISPR transposon system, which is combined by the above plasmid pqqascadepptr, the above helper plasmid pTnsPtr and the above helper plasmid pDonorPtr, comprising: the above-mentioned Cas5/8, cas6, cas7, tniQ, tnsA, tnsB and tnsC genes, left End (LE) and Right End (RE) sequences, target cargo genes, crRNA sequences targeting a genomic target site, colA replicons, promoters such as anhydrotetracycline inducible promoter, kanamycin resistance gene.
Based on the above plasmid, a fourth aspect of the present invention provides a CRISPR transposon system, comprising: plasmids pQCascadePtr, pTnsPtr and pDionorPtr; or plasmids pQCasTnsPtr and pDonorPtr; or plasmid pEffectorPtr.
A fifth aspect of the present invention provides a combination of the above CRISPR transposon system derived from Pseudoalteromonas translucida KMM520 and the CRISPR transposon system derived from Vibrio cholerae (Vibrio cholerae) Tn6677 of the prior art, in particular,
a CRISPR transposon system comprising Vibrio cholerae (Vibrio cholerae) in addition to the CRISPR transposon system (comprising plasmids pQCascadePtr, pTnsPtr and pDONOrPtr, or plasmids pQCasTnsPtr and pDONOrPtr, or plasmid pEffectorPtr) described aboveVibrio cholerae) Tn 6677-derived CRISPR transposase-associated plasmid.
The gene sequence of the I-F3 type CRISPR transposase of Vibrio cholerae Tn6677 is NCBI: NZ _ ALED01000027.1.
The CRISPR transposase associated plasmid derived from Vibrio cholerae Tn6677 comprises a plasmid pQCastnsVch and an auxiliary plasmid pDONOrVch, wherein
Plasmid pqcrastnsvch comprises: the genes Cas5/8, cas6, cas7, tniQ, tnsA, tnsB and tnsC from vibrio cholerae Tn6677, the CloDF13 replicon, promoters such as anhydrotetracycline inducible promoter, streptomycin resistance gene;
the plasmid pDOnorVch comprises a CRISPR array from vibrio cholerae Tn6677, left End (LE) and Right End (RE), a pMB1 replicon, an ampicillin resistance gene and a target Cargo gene (Cargo gene).
In one embodiment, the plasmids pQCastnsVch and pDOnORVch described above may be combined into the plasmid pEffectiorVch, similar to the combination of two or three plasmids of the present invention into one plasmid pQCastnsPtr and pEffectiorPtr.
In the case of using anhydrotetracycline inducible promoter as the above promoter, when the CRISPR transposon system derived from Pseudomonas semitransparent KMM520 as described above is used to transform E.coli BL21 (DE 3), BL21Star TM In the case of strains such as (DE 3) and W3110 (DE 3), the strain can be induced by anhydrotetracycline. That is, willPlasmids pQCastnsPtr and pDenorPtr were used to transform E.coli BL21 (DE 3), BL21Star TM In the case of strains such as (DE 3) and W3110 (DE 3), the induction is carried out using anhydrotetracycline.
Similarly, in the case where the above-mentioned promoter employs a anhydrotetracycline inducible promoter, when the above-mentioned CRISPR transposon system derived from Pseudomonas semitransparent KMM520 is used in combination with the CRISPR transposon system derived from Vibrio cholerae Tn6677, the induction can also be performed using anhydrotetracycline. That is, the plasmids pEffecterptr and pEffecterpVH, or pQCastnsVch, pDONOrVch, pQCastnsPtr and pDONOrPtr were used to transform E.coli BL21 (DE 3), BL21Star TM In the case of strains such as (DE 3) and W3110 (DE 3), the induction is carried out using anhydrotetracycline.
The CRISPR transposable systems derived from the two microorganisms can be used in the same Escherichia coli, and can play a role without mutual interference, and the two functions play a role in an orthogonal mode.
The sixth aspect of the present invention provides the above-mentioned CRISPR-associated transposase, the above-mentioned gene encoding a polypeptide, the above-mentioned plasmid pQCascadePtr, the above-mentioned plasmid pTnsPtr, the plasmid pqcasttnsvtr, the plasmid pEffectorPtr, and the use of the above-mentioned CRISPR transposon system in gene editing.
The gene editing may be any intracellular gene editing, in particular in microbial (including fungal and bacterial) cells, in particular industrial microbial cells.
Preferably, the bacteria for gene editing include gram-negative bacteria such as Escherichia coli, vibrio natriegens, tatma citrobacter, gram-positive bacteria such as Corynebacterium glutamicum, and the like, but are not limited thereto.
Experiments prove that the novel CRISPR-related transposase system developed by the invention can insert cargo genes into 8 sites at one time, the efficiency is 100 percent, and the efficiency is higher than that of the CRISPR-related transposase from vibrio cholerae Tn 6677; the efficiency of transposing 15.4kb cargo gene to the corresponding target was 100%; the novel CRISPR-associated transposase can recognize 16 PAMs. The invention uses two CRISPR related transposase systems in the same escherichia coli for the first time, and the functions can be exerted without mutual interference, thereby providing a choice for accelerating the construction of metabolic engineering strains.
Drawings
FIG. 1 shows a photograph of an electrophoretogram of the targeting crRNA3 (lacZ) site in example 1.
FIG. 2 shows the gel electrophoresis of the insertion of the cargo gene GFP at 8 sites in the genome of the strain of example 2. Wherein NC is a Negative Control (Negative Control).
FIG. 3 is a histogram of the colony PCR and nucleic acid gel electrophoresis in example 2 to verify the insertion of 8 sites in each clone's genome. Wherein the abscissa is the copy number of the cargo gene GFP, and the ordinate is the cargo gene GFP insertion rate.
FIG. 4 shows gel electrophoresis images of the insertion of cargo gene GFP carried by Ptr and cargo gene "terminator sequence" carried by Vch at 6 sites in the genome of the strain in example 3. Where NC is a negative control.
FIG. 5 is a schematic diagram of the structure of plasmid pQCascadePtr.
FIG. 6 is a schematic diagram of the structure of plasmid pDONOrPtr.
FIG. 7 is a schematic structural diagram of the plasmid pTnsPtr.
FIG. 8 is a schematic diagram of the structure of plasmid pQCastnsPtr.
FIG. 9 is a schematic diagram of the structure of plasmid pQCastnsVch.
FIG. 10 is a schematic diagram of the structure of plasmid pEffectorPtr.
FIG. 11 is a structural diagram of plasmid pEffectorVch.
FIG. 12 is a schematic diagram of the structure of plasmid pVnQCastnsPtr.
FIG. 13 is a schematic diagram of the structure of plasmid pCgQCastnsVch.
FIG. 14 is a schematic diagram of the structure of plasmid pCgDonorPtr.
FIG. 15 shows a photograph of a gel electrophoresis image of the crtYf gene locus targeting Corynebacterium glutamicum ATCC13032 strain in example 5.
Detailed Description
The novel CRISPR related transposase system is derived from the semitransparent pseudoalteromonas KMM520, is further developed and perfected for a gene editing tool CAST system, an INTEGRATE system and a MUCICAT system, and especially improves the gene copy quantity and the cargo gene insertion efficiency.
Herein, for the sake of convenience of description, the term "CRISPR-associated transposase system" is sometimes abbreviated as "CRISPR-associated transposase", "CRISPR transposase", or "CRISPR transposase system" or the like, which represent the same meanings and may be used interchangeably.
Herein, for the sake of simplicity of description, a certain protein such as Cas6 is sometimes mixed with the name of its encoding gene (DNA), and those skilled in the art will understand that they represent different substances at different description occasions. Those skilled in the art will readily understand their meanings based on the context and context. For example, for tnsA, when used to describe transposase function or class, refers to a protein; when described as a gene, refers to a gene encoding the transposase tnsA protein.
Similarly, for ease of description, sometimes RNAs such as crRNA are mixed with the names of the genes encoding them, and those skilled in the art will understand that they represent different substances in different description situations. Those skilled in the art will readily understand their meanings based on the context and context.
Each plasmid pqcas captopr, pTnsPtr, pDonorPtr, pqcasttnsvtr and pEffectorPtr provided by the present invention respectively comprises a plurality of gene elements, for example, the plasmid pqcas captopr comprises Cas5/8 gene, cas6 gene, cas7 gene, tniQ encoding gene, crRNA gene, cloDF13 replicon, promoter such as anhydrotetracycline inducible promoter, streptomycin resistance gene, and the arrangement sequence of these gene elements can be arbitrary, and those skilled in the art can arrange according to the custom and easily prepare plasmids.
It is understood that for the genes encoding the polypeptides Cas5/8, cas7, cas6, tnsABC (i.e., tnsA, tnsB and tnsC) and tniQ of the present invention, one skilled in the art can perform codon optimization depending on the particular species of the cell to be treated, such as E.coli, and is not limited only to the nucleotide sequences SEQ ID NOs:8-14 described above.
The purpose of codon optimization is to enable optimal expression of these polypeptides in the cells to be treated. Codon optimization is one technique that can be used to maximize protein expression in an organism by increasing the translation efficiency of a gene of interest. Different organisms often show a particular preference for one of several codons encoding the same amino acid due to mutagenesis tendencies and natural selection. For example, in rapidly growing microorganisms such as E.coli, the optimized codons reflect the composition of their respective pools of genomic tRNA's. Thus, in a fast growing microorganism, low frequency codons for an amino acid can be replaced by codons for the same amino acid but with a high frequency. Thus, expression of optimized DNA sequences is improved in fast growing microorganisms.
The invention will be further illustrated with reference to the following specific examples. It is to be understood that these examples are for illustrative purposes only and are not limiting upon the present invention. Further, it should be understood that various changes and modifications may be made by one skilled in the art after reading the concept of the present invention and those equivalents may also fall within the scope of the invention as defined by the appended claims.
The addition amount, content and concentration of various substances are referred to herein, wherein the percentage refers to the mass percentage unless otherwise specified.
In the examples herein, if no specific description is made about the reaction temperature or the operation temperature, the temperature is usually referred to as room temperature (15 to 30 ℃).
Examples
Materials and methods
The whole gene synthesis in the examples was performed by Nanjing Kingsrei Biotech, inc., the primer synthesis was performed by PitanCurrent Biotechnology (Shanghai), and the sequencing was performed by Oncki Biotechnology, inc.
The molecular biological experiments in the examples include plasmid construction, digestion, ligation, competent cell preparation, transformation, culture medium preparation, and the like, and are mainly performed with reference to "molecular cloning experimental manual" (third edition), sambrook, d.w. rasel (american), translation of huang peitang et al, scientific press, beijing, 2002). The specific experimental conditions can be determined by simple experiments if necessary.
PCR amplification experiments were performed according to the reaction conditions or kit instructions provided by the supplier of the plasmid or DNA template. If necessary, it can be adjusted by simple experiments.
LB culture medium: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, pH7.2, and high temperature and high pressure sterilizing at 121 deg.C for 20min. 20g/L agar powder is additionally added into the solid culture medium.
LBv2 medium: v2 salt (21.7204 g mmol/L NaCl, 0.3.2 g mmol/L KCl,4.723.14g mmol/L MgCl) was added to LB medium 2 )。
BHIS medium: 37g/L BHI,91g/L sorbitol. 20g/L agar powder is additionally added into the solid culture medium.
In the examples, plasmids pQCascadePtr, pTnsPtr and pCgQCasTnsPtr were constructed and synthesized by Soujin Siry Biotechnology Co., ltd, and plasmids pDOnorPtr, pQCasTnsPtr, pEffectorPtr, pCgDonor Ptr, pQCasTnsVch, pDOnorVch and pEffectorVch were constructed by Poplar subject group of molecular plant science innovation center of Chinese academy of sciences. Wherein
Plasmid pQCascadePtr comprises the genes tniQ (SEQ ID NO: 11), cas5/8 (SEQ ID NO: 12), cas7 (SEQ ID NO: 14) and Cas6 (SEQ ID NO: 13) derived from Pseudomonas transcucida KMM520, a crRNA sequence targeting a genomic target site, a CloDF13 replicon, a promoter such as a anhydrotetracycline inducible promoter, a streptomycin resistance gene, and is manufactured by Nanjing Kingsry Biotech Co., ltd, and has the nucleotide sequence of SEQ ID NO:21 and the structure shown in FIG. 5.
The plasmid pDronPtr comprises the sequences of the genes LE (SEQ ID NO: 19) and RE (SEQ ID NO: 20) derived from Pseudomonas translucida KMM520, the pMB1 replicon, the ampicillin resistance gene, the Cargo gene of interest Cargo, for example a promoterless chloramphenicol resistant CmR gene fragment, the structure of which is shown in FIG. 6.
The plasmid pTnsPtr comprises the genes tnSA (SEQ ID NO: 8), tnSB (SEQ ID NO: 9) and tnSC (SEQ ID NO: 10) derived from Pseudomonas transcucida KMM520, a ColA replicon, a promoter such as a anhydrotetracycline inducible promoter, a kanamycin resistance gene, which was assigned to the synthesis by Nanjing Kingsry Biotech Co., ltd., the nucleotide sequence of which is SEQ ID NO:22, and the structure of which is shown in FIG. 7.
Plasmid pQCastNSPtr comprises the genes tnSA (SEQ ID NO: 8), tnSB (SEQ ID NO: 9), tnSC (SEQ ID NO: 10), tnIQ (SEQ ID NO: 11), cas5/8 (SEQ ID NO: 12), cas7 (SEQ ID NO: 14) and Cas6 (SEQ ID NO: 13) derived from Pseudomonas transcucida KMM520, a crRNA sequence targeting a genomic target site, a ColA replicon, a promoter such as anhydrotetracycline inducible promoter, a kanamycin resistance gene, the structure of which is shown in FIG. 8.
Plasmid pQCastnsVch contains genes Cas5/8, cas7, cas6, tniQ, tnSA, tnSB, tnSC and CRISPR array derived from Vibrio cholerae Tn6677, cloDF13 replicon, promoters such as anhydrotetracycline inducible promoter and streptomycin resistance gene, and the structure of the plasmid is shown in FIG. 9 and is constructed by Yangtze topic group of molecular plant science and innovation center of China academy of sciences.
Plasmid pEffectoPtr comprises genes tnSA (SEQ ID NO: 8), tnSB (SEQ ID NO: 9), tnSC (SEQ ID NO: 10), tniQ (SEQ ID NO: 11), cas5/8 (SEQ ID NO: 12), cas7 (SEQ ID NO: 14) and Cas6 (SEQ ID NO: 13) derived from Pseudomonas transcuciida KMM520, a crRNA sequence targeting a genomic target site, LE (SEQ ID NO: 19) and RE (SEQ ID NO: 20) sequences, a cargo gene of interest, a ColA replicon, a promoter such as a tetracycline-inducible promoter, a kanamycin-resistant gene, the structure of which is shown in FIG. 10.
Plasmid pEffectorVch contains genes Cas5/8, cas7, cas6, tniQ, tnSA, tnSB, tnSC and CRISPR array, LE and RE sequences derived from Vibrio cholerae Tn6677, target cargo genes, cloDF13 replicons, promoters such as anhydrotetracycline inducible promoter, streptomycin resistance gene, the structure of which is shown in FIG. 11 and is constructed by the Yangxi topic group of molecular plant science and innovation center of China academy of sciences.
Example 1 verification of transposition activity of CRISPR-associated transposase
By targeting E.coli BL21Star TM (DE 3) genomic lacZ and T7RNA polymerase pre-lacZ, confirming that the CRISPR-associated enzyme derived from pseudoalteromonas translucens KMM520 has programmable transposition activity.
The plasmid pQCascadePtr-cr3 targeting genomic lacZ and pre-lacZ for T7RNA polymerase and validation primers were constructed as shown in Table 1. Wherein the suffix "-cr3" in the plasmid name pQCascadePtr-cr3 represents the construction of the targeted genomic lacZ.
Table 1: primer sequences
Figure BDA0003068539600000111
Figure BDA0003068539600000121
Note: in the table the primer name suffix F represents the forward primer and R represents the reverse primer.
1.1 obtaining pQCascadePtr framework fragment by enzyme digestion
The pQCascadePtr plasmid was digested with NcoI and BamHI to obtain pQCascadePtr backbone fragments.
Enzyme digestion reaction system (50 mu L)
Figure BDA0003068539600000122
The enzyme digestion reaction conditions are as follows: 37 ℃ for 1h. And separating the plasmid fragment after enzyme digestion by gel electrophoresis and recovering the gel.
Restriction enzyme kits were purchased from Thermofisiher, and DNA gel recovery kits were purchased from Shanghai Tulong Biotech, inc.
1.2 primer annealing self-Assembly
The primer pairs PtrDR-F and PtrDR-R in Table 1 were annealed and self-assembled to obtain a DR fragment containing 4bp cohesive ends complementary to the cohesive ends of pQCascadePtr plasmid backbone NcoI and BamHI after digestion.
Annealing self-lap system (50 mu L)
Figure BDA0003068539600000123
Maintaining at 95 deg.C for 5min, reducing the temperature by 5-10 deg.C per minute, maintaining at 16 deg.C for 10min, and adding ddH 2 Diluting with O20 times for later use.
1.3 ligation construction of plasmid pQCascadePtr-DR
T4 connection system (10. Mu.L)
Figure BDA0003068539600000124
Figure BDA0003068539600000131
Ligation was performed at 16 ℃ for 1h. T4 ligase kit was purchased from TAKARA.
The ligation products were all transformed into DH 5. Alpha. Competent cells (purchased from Shenzhen Kangsheng Life technologies, ltd.), and screened on LB solid plate containing streptomycin to obtain plasmid pQCascadePtr-DR. The correct sequence was verified by sequencing with primer 8.
1.4 obtaining pQCascadePtr-DR framework fragment by enzyme digestion
BsaI enzyme cuts pQCascadePtr-DR plasmid to obtain pQCascadePtr-DR skeleton segment.
Enzyme digestion reaction system (50 mu L)
Figure BDA0003068539600000132
The enzyme digestion reaction conditions are as follows: 37 ℃ for 1h. And separating the plasmid fragment after enzyme digestion by gel electrophoresis and recovering the gel.
Restriction enzyme kits were purchased from Thermofisiher, and DNA gel recovery kits were purchased from Toulungo.
1.5 primer annealing self-overlap
The primer pairs Ptrcr3-F and Ptrcr3-R in Table 1 annealed to self-assemble to obtain a DR fragment containing 4bp cohesive ends complementary to the cohesive ends of pQCascadePtr-DR plasmid backbone BsaI after digestion.
Annealing self-lap system (50 mu L)
Figure BDA0003068539600000133
Maintaining at 95 deg.C for 5min, reducing the temperature by 5-10 deg.C per minute, maintaining at 16 deg.C for 10min, and adding ddH 2 Diluting with O20 times for later use.
1.6 ligation construction of plasmid pQCascadePtr-cr3
T4 connection system (10. Mu.L)
Figure BDA0003068539600000134
Figure BDA0003068539600000141
Ligation was performed at 16 ℃ for 1h. T4 ligase kit was purchased from TAKARA.
The ligation products were all transformed into DH 5. Alpha. Competent cells (purchased from Shenzhen Kangsheng Life technologies, ltd.), and screened on an LB solid plate containing streptomycin to obtain plasmid pQCascadePtr-cr3. The correct sequencing was verified with primer 8.
1.7 transformation of transposable tool plasmid and Induction of transposition
Electrotransformation of pDionorptr and pTnsPtr into E.coli BL21Star TM (DE 3), screening was carried out on LB solid plates containing ampicillin and kanamycin at 37 ℃. Selecting positive clone, preparing into electroconceptive cell, electrically transforming pQCascadPtr-cr 3 into the above-mentioned bacteria, and making LB solid plate containing ampicillin, kanamycin and streptomycin under the condition of 37 deg.CThe above-mentioned screening was carried out to obtain E.coli BL21Star containing pDionorptr, pTnsPtr and pQCascadePtr-cr3 TM (DE 3) Strain. A portion of the colonies on the plate was scraped and resuspended in liquid LB medium and replated on LB solid plates containing 100ng/ml of anhydrotetracycline, ampicillin, kanamycin and streptomycin, which were responsible for inducing the expression of the transposition-associated enzymes. A mycoderm may be formed by culturing at 37 ℃ for 16h, which is normal condition. The above-mentioned colonies on plates containing 100ng/ml anhydrotetracycline were scraped off, a portion was resuspended in liquid LB medium, and OD was adjusted 600 After reaching about 0.5, the cells were diluted 50-fold with liquid LB medium, 100. Mu.L of the diluted cells were pipetted and spread on LB solid plates to which was added anhydrotetracycline, ampicillin, kanamycin and streptomycin at a final concentration of 1000ng/ml, and cultured at 37 ℃ for 24 hours.
1.8 colony PCR identification efficiency of targeting crRNA3
The efficiency of targeting two sites of crRNA3 was verified by colony PCR using the primer pairs crRNA3-F/crRNA3-R and crRNA3-R/T7lacZcr3-R in Table 1, located upstream and downstream of the insertion site.
Colony PCR reaction (10 μ L):
Figure BDA0003068539600000142
PCRMix was purchased from Novodka.
And (3) PCR reaction conditions:
Figure BDA0003068539600000151
the donor insert on plasmid pDenorPtr comprises LE (Left end), RE (Right end) and cargo gene CmR fragment (promoterless chloramphenicol resistance gene fragment) with 1433bp total, 1601/1759bp positive band and 168/326bp negative band. Statistical analysis showed that 16 clones had insertions at two sites. The gel electrophoresis pattern is shown in FIG. 1.
FIG. 1 clearly shows the bands of the CmR fragment of the cargo gene by targeting this CmR fragmentTo the plasmid cloned in Escherichia coli BL21Star TM (DE 3) genomic lacZ and T7RNA polymerase pre-lacZ, demonstrating that CRISPR-associated transposase from Pseudomonas semitransparent KMM520 has programmable transposition activity.
Example 2 targeting of 8 different sites of the genome with array to achieve multicopy integration
2.1 construction of plasmid pQCastnsPtr-array8
With reference to the method of example 2 in patent document CN202010083919.7, escherichia coli BL21Star is targeted TM (DE 3) the genome has 8 different sites of crRNA combined to form an array sequence consisting of 9 fixed forward repeats and 8 spacers targeting different sites, inserted between the NcoI and BamHI sites of pQCastnsPtr plasmid to construct plasmid pQCastnsPtr-array8. The array sequence synthesis and the plasmid construction are completed by Nanjing Kinshire Biotechnology Ltd.
2.2 transformation of transposable tool plasmid and Induction of transposition
Electrotransformation of plasmids pDionorptr, pQCastnsptr-array8 to E.coli BL21Star TM (DE 3), the transformation was carried out as in step 1.7. Transposition is induced by the plate colonies and the passage operation is the same as the step 1.7. The cargo gene in plasmid pDonorPtr is the green fluorescent protein GFP gene (about 1.29 kb).
2.3 colony PCR identification of genomic 8 site insertions
Forward and reverse primers, namely primers required for verification, are designed at the upstream and downstream of 8 sites in the genome respectively, and the sequences are shown in Table 2.
Table 2, verification of the sequences of primers required for insertion of 8 sites in the genome
Figure BDA0003068539600000152
Figure BDA0003068539600000161
Note: in the table the primer name suffix F represents the forward primer and R represents the reverse primer.
The PCR system and reaction conditions are the same as those in step 1.8.
The insertion condition of 8 sites of each cloned genome is verified through colony PCR and nucleic acid gel electrophoresis, as shown in figure 2, the whole insertion of the cargo genes of 8 sites can be completed in a target colony at one time, and the specific distribution of the copy number of the cargo genes is shown in figure 3. The result shows that the insertion efficiency of the cargo gene is 100% by using the novel CRISPR related transposase, which is higher than that of the CRISPR related transposase derived from vibrio cholerae Tn 6677.
Example 3 orthogonal experiments of two CRISPR-associated transposases
The orthogonality of the novel CRISPR-associated transposase derived from Pseudomonas transaucida KMM520 with the CRISPR-associated transposase derived from Vibrio cholerae Tn6677 was investigated.
3.1 plasmid construction of pQCasTnsPtr-nagman and pQCasTnsVch-cr3
Targeting Escherichia coli BL21Star TM (DE 3) the crRNA of the genomes nagB, nagE and manX, forming array sequence consisting of 5 fixed direct repeats and 4 spacers spaced to target different sites, was inserted between the NcoI and BamHI sites of pQCastnsPtr plasmid to construct plasmid pQCastnsPtr-nagman. The array sequence synthesis and the plasmid construction are completed by Nanjing Kinshire Biotechnology Ltd.
The construction method of pQCastnsVch-cr3 plasmid is the same as steps 1.4, 1.5 and 1.6, and the plasmid pQCastnsVch comes from the laboratory.
The primers used for plasmid construction and identification are listed in Table 3.
Table 3, construction and identification of the primers used for plasmids pQCastnsPtr-nagman and pQCastnsVch-cr3
Serial number Primer and method for producing the same Sequence (5 '→ 3')
1 Vchcr3-F ATAACTATCCCATTACGGTCAATCCGCCGTTTGTTCCG
2 Vchcr3-R TTCACGGAACAAACGGCGGATTGACCGTAATGGGATAG
3 tetR-seq AGTGAGTATGGTGCCTATCT
4 crRNA3-F CACCACAGATGAAACGCCG
5 crRNA3-R CATCTACACCAACGTGACCTATCC
6 T7lacZcr3-R CACCCATCTCGTAAGACTCATG
7 nagEF AACCTCGCATTAATCTTCGC
8 nagER GCAGTAACAGCAACAGAAAGCA
9 nagBF TGCAATATGACCGTCGTTACC
10 nagBR TGAGACTGATCCCCCTGACT
11 manXF GAAATGCTGTTAGGCGAGCA
12 manXR TGAATCAGACGGTCGTCGAT
13 manXF2 GATGAAGTGGCTGCGGATAC
14 manXR2 CCTTACGGACTTCCAGCTCA
3.2 transformation of transposable tool plasmid and Induction of transposition
Electrotransformation of plasmids pDOnORPtr, pQCastnsPtr-nagman, pDOnORVch and pQCastnsVch-cr3 to E.coli BL21Star TM (DE 3), the transformation was performed as in step 1.7. Transposition induction of the plate colonies and passage operation were performed in the same manner as in step 1.7. Wherein the cargo gene carried by pDroPtr is the green fluorescent protein GFP gene (about 1.29 kb) and the cargo gene carried by pDroVch is the terminator sequence (about 0.64 kb).
3.3 colony PCR identification of genomic insertions
The genomic lacZ and the pre-lacZ of T7RNA polymerase were targeted with CRISPR-associated transposase derived from Vibrio cholerae Tn6677, the cargo gene size being 0.64kb. The crRNA array of pqqastnsptr was designed to target genomes nagB, nagE and manX, the cargo gene green fluorescent protein GFP gene (approximately 1.29 kb). After an escherichia coli transposition experiment is carried out, positive bands are detected by using primers on a target site on lacZ and upstream and downstream, the sizes of the positive bands are 1.0kb and 1.17kb, and the sizes of the negative bands are 0.17kb and 0.33kb; the positive bands were approximately 2.47kb, 2.70kb, 2.49kb and 2.36kb negative bands of 0.43kb, 0.66kb, 0.46kb and 0.33kb as determined by PCR using primers upstream and downstream of the target sites on nagB, nagE and manX. As a result, as shown in fig. 4, CRISPR-associated transposase derived from vibrio cholerae Tn6677 and pqgastnsvtr both target to corresponding sites, respectively, and insert corresponding cargo genes (the cargo gene of pDonorPtr is a green fluorescent protein GFP gene, and the cargo gene carried by pdonorvh is a terminator sequence), so as to obtain a strain with 2 × 4 copies, with an efficiency of about 100%, and the result is verified by sequencing, as shown in fig. 4.
Fig. 4 shows the insertion situation of cargo gene GFP carried by Ptr and cargo gene "terminator sequence" carried by Vch at 6 sites in the genome of the strain, which indicates that two CRISPR-associated transposases can be used in the same escherichia coli and can function without interfering with each other, thereby providing a selection scheme for accelerating the construction of metabolic engineering strains.
Example 4 verification of transposition activity of CRISPR-associated transposase in Vibrio natriegens
The CRISPR-associated enzyme derived from pseudoalteromonas translucens KMM520 was demonstrated to have programmable transposition activity in vibrio natriegens by targeting the vibrio natriegens ATCC14048 genome wbfF gene.
Plasmid pVnQCastNSPtr comprises the genes tnSA (SEQ ID NO: 8), tnSB (SEQ ID NO: 9), tnSC (SEQ ID NO: 10), tniQ (SEQ ID NO: 11), cas5/8 (SEQ ID NO: 12), cas7 (SEQ ID NO: 14) and Cas6 (SEQ ID NO: 13) derived from Pseudomonas transcucida KMM520, a crRNA sequence targeting a genomic target site, a p15A replicon, a promoter such as a anhydrotetracycline inducible promoter, a chloramphenicol resistance gene, and the plasmid structure is shown in FIG. 12.
The plasmid pDOnorPtr-GFP comprises the sequences of the genes LE (SEQ ID NO: 19) and RE (SEQ ID NO: 20) derived from Pseudomonas translucida KMM520, a pMB1 replicon, an ampicillin resistance gene, a promoter-free green fluorescent protein gene fragment of the Cargo gene of interest (Cargo).
The construction of the genome-targeted wbfF pVnQCastnsPtr-wbfF plasmid and validation primers are listed in Table 4. Wherein the suffix "-wbfF" in the plasmid name pVnQCastnsPtr-wbfF represents the construction of the targeted genome wbfF. The construction method of plasmid pVnQCastnsPtr-wbfF is the same as that in steps 1.4, 1.5 and 1.6.
Table 4: primer sequences
Numbering Primer and method for producing the same Sequence (5 '→ 3')
1 wbfF-F GAAATCTCTCTTTGGTATCTCTATCAGTCTACTCTAAG
2 wbfF-R TTCACTTAGAGTAGACTGATAGAGATACCAAAGAGAGA
3 wbfF-test-F TCAGCAGAACAACGACACTCAG
4 wbfF-test-R TATTGCCTTGGTTATCTACCGTGAC
5 tetR-seq AGTGAGTATGGTGCCTATCT
Note: in the table the primer name suffix F represents the forward primer and R represents the reverse primer.
4.1 transformation of transposable tool plasmid and Induction of transposition
pDONOrPtr-GFP was electrotransformed into Vibrio natriegens ATCC14048 and screened on LBv2 solid plates containing ampicillin at 30 ℃. After selecting positive clones and preparing into electrotransformation competent cells, the cells were electroporated with pVnQCastnsPtr-wbfF and screened at 30 ℃ on LBv2 solid plate containing ampicillin and chloramphenicol to obtain the Vibrio natriegens ATCC14048 strain containing pDenorpPtr-GFP and pVnQCastnsPtr-wbfF. A portion of the colonies on the plate was scraped and resuspended in liquid LBv2 medium and replated on LBv2 solid plates containing 100ng/ml anhydrotetracycline, ampicillin and chloramphenicol responsible for inducing expression of the transposition-associated enzyme. When the culture is carried out for 12 hours at the temperature of 30 ℃, a mycoderm can be formed, and the normal condition is met. The above containing 100ng/ml anhydrotetracycline plate clones scraped a portion of the heavy suspension in liquid LBv2 medium, OD adjusted 600 After about 0.5, the cells were diluted 50-fold with liquid LBv2 medium, 100. Mu.L of the diluted cells were pipetted and plated on LBv2 solid plates supplemented with 1000ng/ml of anhydrotetracycline, ampicillin and chloramphenicol at a final concentration, and the plates were incubated at 30 ℃ for 12 hours.
4.2 colony PCR identification efficiency of targeting wbfF Gene
The efficiency of targeting two sites of wbfF was verified by colony PCR using the primer pair wbfF-test-F/wbfF-test-R in Table 4, located upstream and downstream of the insertion site. The colony PCR method is the same as that in step 1.8.
The donor insert on the plasmid pDionorPtr-GFP comprises LE (Left end), RE (Right end) and a cargo gene GFP fragment (green fluorescent protein gene fragment without promoter), which are 720bp in total, a positive band is 1220bp, and a negative band is 500bp. Statistical analysis showed that 16 clones had insertions. It was confirmed that the CRISPR-associated transposase derived from pseudoalteromonas translucens KMM520 also has programmable transposition activity in vibrio natriegens.
Example 5 verification of transposition activity of CRISPR-associated transposase in Corynebacterium glutamicum
The plasmid pCgQCastnsPtr used in the examples comprises the genes tnSA (SEQ ID NO: 8), tnSB (SEQ ID NO: 9), tnsC (SEQ ID NO: 10), tniQ (SEQ ID NO: 11), cas5/8 (SEQ ID NO: 12), cas7 (SEQ ID NO: 14) and Cas6 (SEQ ID NO: 13) derived from Pseudomonas transcucida KMM520, all of which are codon optimized for Corynebacterium glutamicum, a crRNA sequence targeting a target site of the genome, a ColA replicon, a pBL1ts replicon, promoters such as anhydrotetracycline inducible promoter, kanamycin resistant gene, the structure of which is shown in FIG. 13.
The plasmid pCgDonorPtr comprises the sequences of the genes LE (SEQ ID NO: 19) and RE (SEQ ID NO: 20) derived from Pseudomonas translucida KMM520, the pMB1 replicon, the pGA1 replicon, the spectinomycin resistance gene, the Cargo gene of interest Cargo, e.g., a promoterless chloramphenicol resistance CmR gene fragment, the structure of which is shown in FIG. 14.
Transposition activity of CRISPR-associated enzymes derived from pseudoalteromonas translucens KMM520 in corynebacterium glutamicum was confirmed by targeting the corynebacterium glutamicum ATCC13032 genomic crtYf gene.
The crRNA sequence for constructing the targeted genomic crtYf gene is as follows:
AGGCAACCATAGGGCAGGAATCAGAAGTACTG。
5.1 transformation of transposable tool plasmid and Induction of transposition
The pCgDonorPtr and pCgQCastnsPtr were electrotransformed into Corynebacterium glutamicum ATCC13032, and selection was performed at 30 ℃ on BHIS solid plates containing spectinomycin and kanamycin to obtain Corynebacterium glutamicum ATCC13032 strain containing pCgDonorPtr and pCgQCastnsPtr. A part of the clones on the plate is scraped and resuspended in a liquid BHIS medium, and the liquid BHIS medium is replated on a BHIS solid plate containing 100ng/ml of anhydrotetracycline, spectinomycin and kanamycin, wherein the anhydrotetracycline is responsible for inducing the expression of the transposition-associated enzyme. When the culture is carried out for 24 hours at the temperature of 30 ℃, a mycoderm can be formed, and the normal condition is met. The above-mentioned material containing 100nCloning on g/ml anhydrotetracycline plates A portion was scraped and resuspended in liquid BHIS medium, OD adjusted 600 After about 0.5, the cells were diluted 50-fold with a liquid BHIS medium, 100. Mu.L of the diluted cells were pipetted and spread on a BHIS solid plate to which was added anhydrotetracycline, spectinomycin and kanamycin at a final concentration of 1000ng/ml, and cultured at 30 ℃ for 48 hours.
5.2 colony PCR identification efficiency of targeting crtYf
The efficiency of targeting crtYf was verified by colony PCR using the primer pair F-crtYf (TGCTGTGGGAACTTTTCGGT) and R-crtYf (ACTACCATCCCGAGGTTGA) located upstream and downstream of the insertion site.
The donor inserts on plasmid pDenorPtr included LE (Left end), RE (Right end) and the cargo gene CmR fragment (promoterless chloramphenicol resistance gene fragment) of 1433bp total, 2110bp positive band and 677bp negative band. Statistically, 6 clones were successfully inserted. Gel electrophoresis pattern as shown in fig. 15, the transposable activity of the CRISPR-associated transposase derived from pseudoalteromonas translucens KMM520 in corynebacterium glutamicum was confirmed.
Sequence listing
<110> prominent innovation center of molecular plant science of Chinese academy of sciences
<120> a novel CRISPR-associated transposase
<130> SHPI2110093
<160> 22
<170> SIPOSequenceListing 1.0
<210> 1
<211> 209
<212> PRT
<213> Pseudoalteromonas translucida KMM520
<400> 1
Met Tyr Arg Arg Lys Leu Lys Tyr Ser Arg Val Lys Asn Leu His Lys
1 5 10 15
Phe Ala Ser Gln Lys Asn Lys Ser Thr Cys Leu Val Glu Ser Ser Leu
20 25 30
Glu Phe Asp Ala Cys Phe His Phe Glu Phe Ser Pro Pro Ile Ala Ala
35 40 45
Phe Glu Ala Gln Pro Leu Gly Tyr Glu Tyr Glu Phe Asp Asn Arg Ile
50 55 60
Cys Arg Tyr Thr Pro Asp Phe Leu Leu Thr His Thr Asp Gly Thr Gln
65 70 75 80
Lys Phe Ile Glu Val Lys Pro Gln Ser Lys Ile Ala Asp Glu Asp Phe
85 90 95
Arg Ala Arg Phe Ile Glu Lys Gln Ala Ile Ala Lys Gln Asp Gly Arg
100 105 110
Asp Leu Ile Leu Val Thr Asp Lys Gln Ile Arg Val Tyr Pro Thr Leu
115 120 125
Asn Asn Leu Lys Leu Leu His Arg Tyr Ser Gly Phe Gln Ser Leu Thr
130 135 140
Glu Leu Gln Ala Ser Val Leu Glu Leu Val Lys Gln Tyr Gly Ser Ile
145 150 155 160
Lys Val Gly Gln Leu Ile Arg Tyr Leu Lys Val Thr Ala Gly Glu Leu
165 170 175
Leu Ala Thr Val Leu Arg Leu Leu Ser Leu Gly Gln Leu Phe Ala Asp
180 185 190
Leu Thr Thr Asn Glu Ile Ser Ile Glu Thr Ala Ile Trp Ser Asn Asn
195 200 205
Val
<210> 2
<211> 607
<212> PRT
<213> Pseudoalteromonas translucida KMM520
<400> 2
Met Phe Asn Asn Asp Leu Phe Asp Asp Glu Phe Asn Gln Pro Leu Pro
1 5 10 15
Lys Ala Glu Thr Lys Leu Pro Gln Asn Tyr Thr Lys Asp Leu Gln Ala
20 25 30
Leu Pro Glu Lys Ile Lys Thr Thr Thr Phe Ala Lys Leu Lys Tyr Ile
35 40 45
Gln Trp Leu Glu Ala Asn Ile Gln Gly Gly Trp Thr Gln Lys Asn Leu
50 55 60
Glu Pro Leu Leu Lys Leu Met Pro Asp Val Glu Gly Glu Lys Lys Pro
65 70 75 80
Ser Trp Arg Thr Ala Ala Arg Trp Tyr Ser Ala Tyr Thr Asn Ala Asp
85 90 95
Lys Asn Ile Met Ala Leu Ile Pro Ser His Gln Lys Lys Gly Asn Arg
100 105 110
Glu Arg Asp Thr Thr Thr Asp Lys Phe Phe Glu Lys Ala Leu Glu Arg
115 120 125
Tyr Leu Val Lys Glu Lys Pro Ser Val Ala Ser Ala Tyr Lys Phe Tyr
130 135 140
Lys Asp Leu Val Ile Ile Glu Asn Asp Ser Val Val Asp Ser Val Leu
145 150 155 160
Lys Pro Leu Thr Tyr Lys Ala Phe Lys Asn Arg Ile Asp Asn Leu Pro
165 170 175
Gln Tyr Glu Val Met Ile Ala Arg Tyr Gly Lys Arg Leu Ala Asp Ile
180 185 190
Ala Tyr Asn Lys Val Glu Gly His Lys Arg Pro Ile Arg Val Leu Glu
195 200 205
Lys Val Glu Ile Asp His Thr Pro Leu Asp Leu Ile Leu Leu Asp Asp
210 215 220
Glu Leu His Ile Pro Leu Gly Arg Pro Thr Leu Thr Met Leu Val Asp
225 230 235 240
Val Tyr Ser His Cys Ile Val Gly Tyr Tyr Phe Ser Phe Ser Glu Pro
245 250 255
Ser Tyr Asp Ala Val Arg Arg Ala Met Leu Asn Ala Met Lys Pro Lys
260 265 270
Ser Glu Val Ala Lys Leu Tyr Pro Asp Thr Ile Asn Glu Trp Lys Cys
275 280 285
Ala Gly Lys Ile Glu Thr Leu Val Val Asp Asn Gly Ala Glu Phe Trp
290 295 300
Ser Asn Ser Leu Glu Leu Ala Cys Glu Glu Ile Gly Ile Asn Thr Gln
305 310 315 320
Tyr Asn Pro Val Ala Lys Pro Trp Leu Lys Pro Phe Val Glu Arg Met
325 330 335
Phe Gly Thr Ile Asn Thr Glu Leu Leu Asp Pro Val Pro Gly Lys Thr
340 345 350
Phe Ser Asn Ile Leu Gln Lys His Glu Tyr Asn Pro Lys Lys Asp Ala
355 360 365
Ile Met Arg Phe Thr Thr Phe Met Gln Leu Phe His Lys Trp Val Val
370 375 380
Asp Val Tyr His Gln Asp Ala Asp Ser Arg Phe Lys Tyr Ile Pro Ser
385 390 395 400
Gln Leu Trp Asp Gln Gly Phe Asn Thr Leu Pro Pro Thr Met Leu Ser
405 410 415
Asp Ala Asp Leu Gln Gln Leu Asp Val Val Leu Ser Ile Ser Asn His
420 425 430
Arg Val Leu Arg Lys Gly Gly Ile Arg Leu Glu Asn Leu Ser Tyr Asp
435 440 445
Ser Thr Glu Leu Ala Asn Tyr Arg Lys Gln Phe Ser His Lys Val Ser
450 455 460
Gln Glu Val Leu Ile Lys Leu Asn Pro Asp Asp Ile Ser Tyr Ile Tyr
465 470 475 480
Val Tyr Leu Asp Lys Leu Glu His Tyr Ile Lys Val Pro Cys Ile Asp
485 490 495
Pro Asn Gly Tyr Thr Gln Asn Leu Ser Leu Asn Gln His Lys Ile Asn
500 505 510
Ile Arg Ile His Arg Asp Phe Ile Ser Gly Ser Ile Asp Asn Val Gly
515 520 525
Leu Ala Lys Ala Arg Met Phe Ile His Asn Lys Ile Gln Asn Glu Phe
530 535 540
Glu Glu Leu Lys Asn Ala Pro Lys His Ser Lys Val Lys Gly Gly Lys
545 550 555 560
Ala Leu Ala Lys His Gln Asn Ile Ser Ser Asp Ser Gln Lys Ser Ile
565 570 575
Thr His Ser Lys Pro Val Glu Ala Lys Lys Val Thr Pro Lys Glu Gln
580 585 590
Pro Thr Asp Ser Trp Asp Asp Phe Ile Ser Asp Leu Asp Gly Phe
595 600 605
<210> 3
<211> 333
<212> PRT
<213> Pseudoalteromonas translucida KMM520
<400> 3
Met Leu Thr Asp Lys Gln Lys Glu Lys Leu Asn Glu Phe Arg Asp Val
1 5 10 15
Phe Ile Glu Tyr Pro Ile Ile Thr Thr Ile Phe Asn Asp Phe Asp Arg
20 25 30
Leu Arg Leu Gly Lys Gly Leu Thr Gly Glu Lys Pro Cys Met Leu Leu
35 40 45
Asn Gly Asp Thr Gly Thr Gly Lys Thr Ala Leu Ile Lys Gln Tyr Lys
50 55 60
Glu Arg His Leu Pro Gln Phe Ile Asn Gly Val Met Asn His Pro Val
65 70 75 80
Leu Val Ser Arg Ile Pro Ser Asn Pro Thr Leu Glu Ser Thr Leu Ala
85 90 95
Glu Leu Leu Lys Asp Leu Gly Gln Val Gly Ser Thr Glu Arg Lys Leu
100 105 110
Arg Ile Asn Gly Thr Arg Leu Thr Thr Ser Leu Ile Lys Cys Leu Lys
115 120 125
Thr Cys Gly Thr Glu Leu Ile Ile Ile Asp Glu Phe Gln Glu Leu Ile
130 135 140
Glu His Asn Gln Gly Lys Lys Arg Arg Glu Ile Ala Asn Arg Leu Lys
145 150 155 160
Tyr Ile Asn Asp Glu Ala Gly Val Ser Ile Val Leu Val Gly Met Pro
165 170 175
Trp Ala Glu Lys Ile Ala Asp Glu Pro Gln Trp Ser Ser Arg Leu Leu
180 185 190
Ile Arg Arg Gln Leu Pro Tyr Phe Lys Leu Ser Glu Asn Pro Lys His
195 200 205
Phe Val Gln Leu Ile Ile Gly Leu Ala Asn Arg Met Pro Phe Ala Glu
210 215 220
Lys Pro Asn Leu Ser Glu Gln Ala Thr Val Phe Thr Leu Phe Ser Leu
225 230 235 240
Ser Lys Gly Cys Phe Arg Thr Leu Lys Tyr Phe Leu Asp Asp Ala Val
245 250 255
Leu Tyr Ala Leu Met Asp Asn Ala Lys Thr Leu Thr Thr Lys His Leu
260 265 270
Val Lys Ala Phe Glu Val Leu Phe Pro Asp Val Pro Asn Leu Phe Thr
275 280 285
Leu Pro Val Ala Glu Ile Thr Ala Ser Glu Val Glu Arg Tyr Ser Leu
290 295 300
Tyr Lys Pro Glu Ser Ser Gln Asp Glu Asp Pro Phe Ile Ala Thr Lys
305 310 315 320
Phe Thr Asp Arg Met Pro Ile Ser Gln Leu Leu Arg Lys
325 330
<210> 4
<211> 391
<212> PRT
<213> Pseudoalteromonas translucida KMM520
<400> 4
Met His Phe Leu Val Gln Thr Lys Ser Tyr Pro Asp Glu Ala Leu Glu
1 5 10 15
Ser Tyr Leu Leu Arg Leu Ala Arg Asp Asn Ser Tyr Asn Gly Tyr Ser
20 25 30
Glu Leu Ala Asp Ile Leu Trp Gln Trp Leu Ala Glu Gln Asp Asn Glu
35 40 45
Leu Glu Gly Ala Leu Pro Leu Ala Leu Ser Lys Val Asp Val Tyr His
50 55 60
Ala Arg Gln Ala Ser Ser Phe Arg Ile Arg Ala Leu Lys Leu Val Ala
65 70 75 80
Gln Leu Ala Asp Val Asn Ala Gly Asp Ile Leu Ala Leu Ala Trp Arg
85 90 95
Arg Ser Asn Phe Lys Phe Gly Asn Leu Ala Ala Val Ser Arg Asn Glu
100 105 110
Leu Ala Ile Pro Leu Glu Leu Leu Arg Thr Asp Asn Ile Pro Val Cys
115 120 125
Ile Lys Cys Leu Ser Glu Ser Ser His Ile Pro Phe Tyr Trp His Leu
130 135 140
Lys Pro Tyr Lys Ala Cys His Lys His Lys Ser Gln Leu Ile Thr Arg
145 150 155 160
Cys Lys Glu Cys Tyr Asp Leu Ile Asp Tyr Arg Ala Ser Glu Ala Phe
165 170 175
Leu Glu Cys Val Cys Gly Cys Lys Ile Thr Asn Ser Glu Gln Leu Asn
180 185 190
Asp Ala Asp Phe Lys Ile Ala Ile Ala Leu Ala Ser Ser Asn Ser Gln
195 200 205
Lys Ile Val Gly Leu Ile Ser Trp Phe Ala Lys Val Lys Gln Leu Asp
210 215 220
Val Ser Asp Ala Asp Phe Asn Cys Ala Phe Val Asp Tyr Phe Asn Thr
225 230 235 240
Trp Pro Glu Ser Leu Thr Thr Glu Leu Asp Leu Leu Thr Asn Asn Ala
245 250 255
Arg Leu Lys Gln Leu Asn Pro Phe Asn Lys Thr Lys Phe Ser Ser Val
260 265 270
Tyr Gly Asp Leu Ile Arg Asp Gly Gln Ile Ala Ala Thr Ser Asn Arg
275 280 285
Lys Asn Lys Val Ile Asp Glu Ile Ile Ser Tyr Phe Val Glu Leu Val
290 295 300
Asp Ser Asn Pro Lys Ala Lys His Pro Asn Ile Gly Asp Leu Leu Leu
305 310 315 320
Cys Thr Phe Asp Ala Ala Val Leu Leu Asn Thr Thr Thr Glu Gln Val
325 330 335
Tyr Arg Leu His Gln Glu Ala Phe Leu Asn Cys Ala Tyr Ser Gln Lys
340 345 350
Lys His Glu Gln Leu Arg Ala Asp Ser His Val Phe Tyr Leu Arg Gln
355 360 365
Val Ile Glu Leu Gln Gln Ala Phe Ala Ala Glu Lys Pro Leu Thr Lys
370 375 380
Lys Gln Phe Ile Ala Pro Trp
385 390
<210> 5
<211> 683
<212> PRT
<213> Pseudoalteromonas translucida KMM520
<400> 5
Met Asn Leu Gln Asp Ala Leu Ala Ile Glu Pro Leu Lys Glu Lys Thr
1 5 10 15
Thr Ala Leu Arg Lys Leu Phe Val Pro Tyr Thr Ser His Val Glu Val
20 25 30
Asp Gly Phe Glu Glu Leu Ala Leu Thr Val Leu Ile Asn Leu Val Tyr
35 40 45
Lys Arg Ser Glu Ile Asp Asp Leu Thr Ser Ala Arg Thr Ala Lys Ser
50 55 60
Val Leu Arg Asp Glu Val Leu Leu Ser Lys Cys Ile Asn Glu Val Lys
65 70 75 80
Trp Phe His Thr His Asn Leu Lys Tyr Pro Asp Ile Arg Val Ser His
85 90 95
Gln Arg Leu Ile Ser Glu Val Val Ser Glu Asp Ile Ala Gly Ile Cys
100 105 110
Ser Arg Ser Leu Pro Leu Ser Phe Gly Trp Ser His Asn Ser Ala Glu
115 120 125
Ile Asn His Ala Lys Leu Phe Leu Thr Ser Phe Asn Trp Gln Gly Glu
130 135 140
Val Thr Cys Leu Ala Arg Leu Leu Ile Asn Glu Glu Pro Val Trp Ile
145 150 155 160
Asn Leu Ile Arg Ala Tyr Gly Phe Thr Lys Lys Ala Val Leu Glu Ile
165 170 175
Ser Gly Lys Ile Lys Gln Gln Leu Pro Val Ala Glu Phe Pro Leu Glu
180 185 190
Val Ser Ser Phe Ser Pro Gln Leu Gln Met Pro Phe Gln Gln Ser Tyr
195 200 205
Leu Val Val Thr Pro Val Val Ser His Ala Met Leu Ala Lys Ile Gln
210 215 220
Gln Leu Thr Thr Asp Arg Lys Leu Asn Phe Ala Leu Val Glu His Ser
225 230 235 240
Arg Pro Ala Asn Val Gly Asp Leu Ala Ser Ser Val Gly Gly Asn Ile
245 250 255
Arg Val Leu Arg Tyr Phe Pro Lys Thr Tyr Ser Lys Ala Val Asn Arg
260 265 270
Ser Lys Val Ala Asn Asn Asp Ile Glu Lys Ala Phe Lys Ile Arg Ala
275 280 285
Leu Leu Ser Ser Gln Phe Gln Gln Ala Leu Leu Val Leu Val Gly Ile
290 295 300
Lys Gln Phe Asn Thr Leu Arg Gln Lys Arg Leu Ala Arg Val Ala Ala
305 310 315 320
Ile Arg Gln Val Arg Val Ser Leu Gln Leu Trp Leu Asp Asn Ile Leu
325 330 335
Glu Ala Lys Asn Asn Ala Gln Asn Gln Val Tyr Pro Glu Trp Val Arg
340 345 350
His Tyr Leu Asp Gln Ser Ile Thr Asn Cys Ile Ser Gln Phe Ser Asn
355 360 365
Val Leu Asn Glu Ser Leu Gly Asn Leu Ser Lys Leu Lys Arg Phe Ala
370 375 380
Tyr His Pro Asn Leu Met Gly Leu Phe Lys Ala Gln Leu Asn Tyr Val
385 390 395 400
Phe Thr His Cys Ala Ala Glu Gln Glu Ile Leu Asn Asp Glu Gln Ile
405 410 415
Val Tyr Val His Cys Gln Asp Met Arg Val Phe Asp Ala Glu Ala Met
420 425 430
Ala Asn Pro Tyr Ile Gln Gly Met Pro Ser Leu Thr Ala Leu Asn Gly
435 440 445
Leu Ala His Asn Phe Glu Arg Lys Leu Lys Asn Phe Ile Asp Pro Ser
450 455 460
Ile Lys Cys Ile Gly Ser Ala Ile Tyr Ile Glu Asn Tyr Gln Leu His
465 470 475 480
Thr Gly Lys Pro Leu Pro Glu Pro Ser Lys Leu Lys Gln Val Ala Gly
485 490 495
Arg Ser His Val Ile Arg Ser Gly Ile Ile Asp Lys Pro Lys Cys Asp
500 505 510
Ile Thr Leu Asp Leu Val Phe Arg Leu Phe Val Pro Asn Thr Glu Leu
515 520 525
Leu Asp Lys Leu Asn Ser Gln Leu Ile Lys Pro Ala Leu Pro Ser Ser
530 535 540
Phe Ala Gly Gly Thr Met His Pro Pro Ser Leu Tyr Gln Asn Ile Asp
545 550 555 560
Trp Cys His Val His Thr Lys Pro Ser Glu Leu Phe Lys Lys Leu Lys
565 570 575
Ala Lys Ser Ser Asn Gly Ser Trp Leu Tyr Pro Ser Lys Lys Val Val
580 585 590
Lys Ser Phe Glu Gln Leu Ile Asp Ala Leu Asn Ser Asn Phe Asn Leu
595 600 605
Arg Pro Ala Ala Ile Gly Leu Ala Ala Leu Glu Glu Pro Val Lys Arg
610 615 620
Asp Ala Ala Leu His Glu Tyr His Cys Tyr Ala Glu Pro Val Ile Gly
625 630 635 640
Leu Leu Glu Cys Val Ser Asn Thr Ser Val Lys Tyr Ala Gly Ala Lys
645 650 655
Gln Phe Phe His Asp Ala Phe Trp Val Met Asp Val Gln Lys Glu Ser
660 665 670
Met Leu Met Lys Lys Ser Lys Phe Glu Tyr Glu
675 680
<210> 6
<211> 200
<212> PRT
<213> Pseudoalteromonas translucida KMM520
<400> 6
Leu Lys Arg Tyr Tyr Phe Thr Ile Thr Tyr Leu Pro Gln Ser Cys Asp
1 5 10 15
Val Ser Leu Leu Ala Gly Arg Cys Ile Gly Ile Leu His Gly Phe Met
20 25 30
Ser Ser Arg Glu Ile Ser Asn Ile Gly Val Cys Phe Pro Lys Trp Asn
35 40 45
Glu Gln Thr Ile Gly Asn Glu Leu Ala Phe Val Ser Thr Asn Lys Lys
50 55 60
Gln Leu Thr Asn Leu Ser Gln Gln Ser Tyr Phe Glu Met Met Ala His
65 70 75 80
Asp Lys Leu Phe Gly Leu Ser Lys Ile Leu Glu Val Pro Val Asn Gln
85 90 95
Ser Glu Val Met Phe Val Arg Asn Gln Ser Val Ala Lys Ala Phe Val
100 105 110
Gly Glu Lys Gln Arg Arg Leu Lys Arg Ala Lys Lys Arg Ala Glu Ala
115 120 125
Arg Gly Glu Val Tyr Asn Pro Glu Tyr Lys Phe Glu Ala Lys Asp Ile
130 135 140
Gly His Phe His Ser Ile Pro Val Ser Ser Lys Gly Asn Gly Gln Ser
145 150 155 160
Tyr Val Leu His Ile Gln Lys Asn Glu Asn Ala Glu Ser Ile Lys Asn
165 170 175
Gln Phe Asn Asn Tyr Gly Phe Ala Thr Asn Gln Ile Phe Leu Gly Thr
180 185 190
Val Pro Ser Leu Asn Thr Leu Leu
195 200
<210> 7
<211> 342
<212> PRT
<213> Pseudoalteromonas translucida KMM520
<400> 7
Met Gln Leu Pro Arg His Leu Ser Tyr Thr Arg Ser Leu Ser Pro Ser
1 5 10 15
Lys Ala Val Phe Phe Tyr Lys Thr Pro Glu Ser Asp Phe Glu Pro Leu
20 25 30
Gln Ile Glu Gln Asn Lys Leu Val Gly Gln Lys Ser Gly Phe Gly Asp
35 40 45
Ala Tyr Gln Lys Gln Asn Val Ala Lys Asn Leu Ala Pro Gln Asp Leu
50 55 60
Ala Phe Gly Asn Pro Gln Thr Ile Asp Val Cys Tyr Val Pro Pro Thr
65 70 75 80
Val Asn Glu Leu Phe Cys Arg Phe Ser Leu Arg Val Glu Ala Asn Cys
85 90 95
Ile Glu Pro His Val Cys Asp Asp Pro Lys Val Ile Tyr Trp Leu Lys
100 105 110
Arg Phe Phe Glu Thr Tyr Lys Lys His Asn Gly Leu Asn Glu Val Ala
115 120 125
Thr Arg Tyr Ala Lys Asn Ile Leu Met Gly Asn Trp Leu Trp Arg Asn
130 135 140
Arg Gln Ser Pro Asn Val Asp Ile Glu Ile Leu Thr Glu His Ala Ala
145 150 155 160
Pro Ile Val Val Glu Gly Ala Gln Lys Leu Lys Trp Gln Gly Asn Trp
165 170 175
Gln Asn Asn Gln Thr Ala Leu Leu Thr Leu Ser Glu Ser Ile Gln Glu
180 185 190
Gly Leu Ser Asn Pro Gln Asn Tyr Cys Tyr Leu Asp Ile Thr Ala Lys
195 200 205
Ile Lys Asn Ala Phe Ser Gln Glu Val His Pro Ser Gln Lys Phe Val
210 215 220
Asp Asn Val Glu Gln Gly Met Ser Ser Lys Gln Leu Ala Tyr Thr Gln
225 230 235 240
Val Gly Asp Lys Lys Ala Ala Ser Leu Asn Ser Gln Lys Val Gly Ala
245 250 255
Ala Ile Gln Thr Ile Asp Asp Trp Tyr Glu Glu Gly Tyr Lys Pro Leu
260 265 270
Arg Thr His Glu Tyr Gly Ala Asp Lys Gln Ile Leu Val Ala His Arg
275 280 285
Thr Pro Lys Ser His Ser Asp Phe Tyr Ser Leu Leu Pro Arg Ile Ala
290 295 300
Leu His Ile Lys His Met Glu Lys His Gly Leu Glu Gln Ser Glu Gln
305 310 315 320
Ser Asn Ser Ile His Phe Ile Ala Ala Val Leu Ile Lys Gly Gly Leu
325 330 335
Phe Gln Arg Ser Lys Gly
340
<210> 8
<211> 630
<212> DNA
<213> Pseudoalteromonas translucida KMM520
<400> 8
atgtacagaa gaaaactaaa atactcccgt gtaaaaaatc ttcataaatt tgctagtcaa 60
aaaaataaat ctacttgttt agtcgaatcc tctttagagt ttgatgcgtg tttccatttt 120
gaattttcac caccaatagc cgcatttgaa gcacaacctc taggttacga atatgagttc 180
gataaccgta tttgccgtta cacacctgac tttttactta cccacacaga cggcacgcaa 240
aaatttatag aagtaaaacc gcaaagcaaa attgctgacg aagactttcg tgcacgtttt 300
attgaaaagc aagccatagc taagcaagat ggacgcgact taatactggt tactgataaa 360
caaatccgtg tatacccaac actcaataac ttaaagcttt tgcatcgcta ctctggtttt 420
cagtctttaa cagaattgca agcatcggta ctagaacttg ttaagcagta cggctctatc 480
aaagtgggcc agttaatcag atatttaaaa gtaactgccg gtgagctact tgctacggtg 540
cttcgcttac tatcactagg gcagttattt gccgacttaa ctacaaatga aatatcaata 600
gaaacagcaa tttggtctaa caatgtttaa 630
<210> 9
<211> 1824
<212> DNA
<213> Pseudoalteromonas translucida KMM520
<400> 9
atgtttaata acgatttgtt tgatgatgag tttaaccagc cattaccaaa agctgaaacc 60
aaactacctc aaaattacac taaagactta caagcccttc ctgaaaaaat aaaaacaaca 120
acatttgcta agcttaaata tattcaatgg cttgaggcta atattcaagg tggttggaca 180
caaaaaaatc ttgaaccttt attaaaatta atgcctgatg ttgagggtga aaaaaagcca 240
agttggagaa cagccgcacg atggtatagc gcttacacca atgcggataa aaatattatg 300
gcgctaatac caagccacca aaaaaagggt aatagggagc gcgatacaac cactgataag 360
ttttttgaaa aagcacttga gcgttactta gtaaaagaaa aaccatcagt ggcttcggct 420
tacaagttct ataaagactt agttattatc gaaaacgaca gtgttgttga cagtgtttta 480
aagcctttaa catacaaagc gtttaaaaac agaatagata acttaccgca atacgaagta 540
atgattgctc gttatggtaa gcgccttgct gatattgctt ataataaggt tgaagggcat 600
aaacggccta tccgagtact tgaaaaagtt gaaattgacc atacgccact tgatcttatt 660
ttattagatg atgagctaca tattccacta ggtaggccta cactcaccat gttggtagat 720
gtgtatagcc attgtattgt tggctattac tttagcttca gtgagcctag ctatgatgca 780
gtaaggcgag caatgctaaa tgcgatgaaa cctaaaagtg aagtggcaaa actataccct 840
gatacgatta atgagtggaa gtgtgctggc aaaattgaaa cactcgttgt tgataatggc 900
gctgaatttt ggagcaacag ccttgaactt gcttgtgaag aaataggcat taatactcaa 960
tataacccag tcgcaaagcc ttggttaaaa ccatttgtag aacgtatgtt tggaacaata 1020
aatactgagt tattagatcc tgttcccggt aaaacctttt ctaacatttt acaaaagcat 1080
gaatacaatc caaaaaaaga tgcaatcatg cgctttacga cctttatgca gttatttcat 1140
aaatgggtag tagacgttta tcatcaagat gccgacagtc gctttaagta cataccgagt 1200
caactgtggg atcaaggttt taatacgtta ccaccaacaa tgctaagtga tgctgatctt 1260
caacaactag atgttgtgct cagtatttca aatcatcggg tacttcgtaa aggtgggata 1320
cggctagaaa acttaagcta cgacagtact gaactggcca attatagaaa gcaatttagc 1380
cataaagtat ctcaagaagt tttaattaaa ttaaatcccg atgatatttc ttatatatat 1440
gtttaccttg ataagctaga gcattacata aaagtgccat gcatagatcc aaacggttac 1500
acccaaaatt taagtttgaa tcagcataaa ataaatatac gcatccaccg cgactttatt 1560
tcgggctcta tcgataatgt aggcttagca aaagcgcgca tgtttattca taacaaaatt 1620
caaaacgagt ttgaagagtt aaaaaatgcg ccaaaacact caaaagtaaa gggtggtaaa 1680
gcgttagcta aacatcaaaa tatcagtagt gactcacaaa agtcaataac gcatagcaaa 1740
cccgtagagg ccaaaaaggt tacacctaaa gagcaaccaa ctgatagctg ggatgatttt 1800
atctcagact tagatggatt ttaa 1824
<210> 10
<211> 1002
<212> DNA
<213> Pseudoalteromonas translucida KMM520
<400> 10
atgctgaccg ataaacaaaa agaaaagctg aatgaatttc gtgatgtatt tattgaatac 60
ccaataataa ccaccatatt taacgacttc gatagattaa gacttggtaa agggctaaca 120
ggtgaaaagc cttgcatgct cttaaatggc gatacaggca caggtaaaac agcactgatc 180
aagcaatata aagaacgaca tttaccgcaa tttattaatg gtgttatgaa ccaccctgta 240
ttggtaagcc gcatacctag taacccgaca ttagaatcta ctttagcaga gcttcttaaa 300
gatttagggc aagtaggcag cacagagcgt aagctacgaa taaacggcac tcgcttaacg 360
acatcattaa taaaatgcct aaaaacatgt ggcacagagc ttataattat tgatgagttc 420
caagagctaa ttgagcacaa ccaaggtaaa aagcgccgcg agattgctaa tcgattaaaa 480
tatattaacg acgaagcggg tgtatcaatt gtattggtag gtatgccgtg ggcagaaaaa 540
atagcagacg agccccagtg gtcatctcgt ttattaataa ggcggcagtt gccttatttt 600
aagttgtcag aaaacccaaa gcattttgta caactaataa ttggtctagc caaccgtatg 660
ccatttgccg aaaagccaaa cttaagtgag caagcaacag tgtttacttt gttctcatta 720
tcaaaaggtt gctttagaac attaaaatac tttttagatg atgccgtact ttatgcatta 780
atggacaacg cgaaaactct cacaaccaag catttagtta aagcatttga ggtactcttt 840
ccggatgttc ctaatttatt taccttgcct gtagcagaaa taacagcaag cgaagtcgag 900
cgctattcac tttataagcc tgaaagctct caagatgaag acccgtttat agcgaccaag 960
tttactgacc ggatgccgat tagtcagttg ttaaggaaat aa 1002
<210> 11
<211> 1176
<212> DNA
<213> Pseudoalteromonas translucida KMM520
<400> 11
atgcattttt tagttcaaac aaaatcttac ccagatgagg cgcttgaaag ctatttgctg 60
aggcttgcaa gggataactc atacaatggc tatagtgagc ttgctgatat tttgtggcaa 120
tggcttgcag agcaagataa tgagcttgaa ggtgcgctgc cgttagcgct gagtaaagtt 180
gatgtttatc atgctaggca agcgagcagc tttagaataa gagcgcttaa gttggttgct 240
caattagcag atgtaaacgc tggtgacatt cttgcacttg cttggaggcg cagtaatttt 300
aaatttggca accttgccgc agtaagtcga aatgaactgg ctattcccct tgagctactt 360
cgtactgata acatacctgt ttgcattaaa tgcttgtctg aatcttccca tattcccttt 420
tattggcatt taaagcccta taaggcgtgt cataagcata agtcacaatt aattacacgt 480
tgtaaggagt gctatgactt aattgattac agagcctctg aggcgttttt agagtgtgtt 540
tgcggttgta aaataaccaa tagtgaacag ttaaacgatg cagactttaa aattgcaatt 600
gcgcttgcaa gtagtaacag ccaaaaaata gtagggttga tatcgtggtt tgcgaaggtt 660
aagcaacttg atgtaagtga tgcagacttt aactgcgctt ttgttgatta ctttaatact 720
tggcctgaaa gccttaccac tgaattagat ttactcacaa ataatgcgcg actcaagcaa 780
cttaaccctt ttaataaaac taagttcagc tctgtttatg gcgatttaat ccgtgatggt 840
caaatagctg caacaagtaa ccggaaaaac aaagtaattg atgagattat tagttatttt 900
gtcgaattag ttgatagtaa ccctaaagct aaacatccaa atattggtga cttactgctt 960
tgtacttttg atgccgcagt attgttaaac actactacag agcaagttta caggcttcat 1020
caagaagcct ttttaaactg tgcttattca caaaaaaagc acgaacagct cagagctgat 1080
agccatgtat tttatttacg ccaagtgatt gaactacaac aagcattcgc agctgaaaag 1140
cctctaacaa aaaaacaatt tatagcgccg tggtaa 1176
<210> 12
<211> 2052
<212> DNA
<213> Pseudoalteromonas translucida KMM520
<400> 12
atgaacttac aagatgcact tgcaattgaa ccactaaaag aaaaaaccac agcacttaga 60
aaattgttcg ttccatacac gtctcatgtc gaggtagatg gctttgaaga actagcgctg 120
actgtgctca ttaatcttgt ttataagcga agtgagattg atgatttaac aagtgcaaga 180
actgctaaaa gtgtactacg cgatgaagtg ttactgagta agtgcattaa cgaagtgaaa 240
tggtttcata ctcataattt aaaatacccc gatatacgag taagccatca acgtttaatt 300
agtgaagttg taagtgaaga tattgcgggc atttgcagcc ggtcattacc tttaagtttt 360
ggctggtcgc acaacagtgc tgaaattaat catgcaaagc tatttttaac ctcgtttaat 420
tggcaaggtg aagtgacttg tttagcaagg ctgttaatta atgaagagcc tgtttggatt 480
aatttaataa gagcatacgg gtttactaaa aaggcggttt tagaaatctc gggtaaaata 540
aaacagcagt tgccagtggc agagttccca ttagaagtaa gctctttttc accacaatta 600
caaatgccat ttcagcaaag ctaccttgtg gttacgcctg tagtaagcca cgcaatgctg 660
gctaaaattc agcaattaac aacagatcgt aagttaaatt ttgctttagt tgagcactca 720
agacctgcca atgttggcga tttagcaagc tcagtaggcg gcaatataag agtgctgcgt 780
tactttccta aaacatattc aaaggctgtt aaccgctcta aagtagccaa taatgatatt 840
gagaaagcat ttaaaattcg tgcgctatta agtagtcaat ttcaacaggc gcttttggtg 900
ttggtaggca ttaaacagtt taatacgtta aggcaaaaac gattagcgcg agtagcggct 960
attaggcaag tacgtgttag cttgcagtta tggcttgata atattcttga agctaaaaat 1020
aacgcgcaaa accaagttta ccctgagtgg gtaaggcatt acttagatca gagtattact 1080
aactgtatta gccaatttag taacgtacta aatgagagcc ttggtaattt aagtaagctc 1140
aaacgctttg cgtatcaccc taatttaatg ggactgttta aagcgcagtt aaactatgta 1200
tttactcact gtgcagctga acaagaaata ttaaatgatg agcagatagt gtatgtacat 1260
tgccaagata tgcgagtgtt tgatgctgag gcaatggcta atccgtatat tcaaggcatg 1320
ccgtcactta ctgctttaaa tgggcttgct cataactttg agcgtaagct aaaaaacttt 1380
atagaccctt caattaagtg tattggcagt gctatttaca ttgaaaacta tcaattacat 1440
acaggtaaac cattacctga gccaagcaag ttaaaacaag ttgcagggcg tagtcatgta 1500
ataagatctg gaattatcga taaaccaaaa tgtgacataa cactcgattt agtatttaga 1560
ctttttgtac caaatactga gctgttagat aagttaaata gtcagcttat aaagcccgca 1620
ctaccgtctt catttgcagg cgggactatg catccacctt cgttatatca aaatattgac 1680
tggtgccatg tacataccaa accgagcgag ctgtttaaaa aacttaaagc aaaatcgtca 1740
aatggcagtt ggttatatcc ttcaaaaaaa gtagttaaaa gttttgaaca attaattgat 1800
gcccttaaca gtaactttaa tttaagaccc gctgcaattg gcttggctgc gcttgaagaa 1860
cccgtaaagc gagatgcagc attacatgaa taccattgtt atgcagagcc cgtaattggg 1920
ctgttagagt gtgttagcaa tacatcagta aagtacgcag gggctaagca gttctttcat 1980
gacgcatttt gggttatgga tgttcaaaaa gagtctatgc ttatgaaaaa gtctaagttt 2040
gagtatgaat aa 2052
<210> 13
<211> 603
<212> DNA
<213> Pseudoalteromonas translucida KMM520
<400> 13
ttgaagcgct attattttac cattacttat ttaccccaaa gttgtgatgt aagccttctt 60
gctgggcgtt gtatcggtat tttgcatggg tttatgagct cacgtgaaat aagtaatatt 120
ggtgtgtgct ttcctaaatg gaatgagcaa acaataggta atgaattagc gtttgtatca 180
acaaataaaa agcaattaac caatctatct cagcaaagct attttgagat gatggctcat 240
gacaagttat ttggcttatc aaaaatactt gaagtaccag taaaccaaag cgaagtcatg 300
tttgttcgca accaatcggt agcaaaagca tttgttggcg aaaagcaaag gcgattaaag 360
cgagctaaaa aacgagctga agccagaggc gaagtttaca accctgaata taaatttgag 420
gcaaaggaca taggccattt tcattcaata cccgtatcaa gcaaaggcaa tggtcaaagt 480
tatgttttgc atatacaaaa aaatgaaaat gctgaatcca taaaaaatca gtttaacaat 540
tatggctttg ctacaaatca aatatttcta ggtacggttc cttctttaaa taccctttta 600
taa 603
<210> 14
<211> 1029
<212> DNA
<213> Pseudoalteromonas translucida KMM520
<400> 14
atgcaattac ctcggcactt aagttacacg cgttcgctct cacccagtaa agcggtgttt 60
ttttataaaa caccagagtc tgactttgaa ccgctacaaa tagagcaaaa taaattagtt 120
gggcagaagt cagggtttgg cgatgcgtat caaaagcaaa atgtggctaa aaatttagcg 180
ccacaagatc tcgcgtttgg aaaccctcaa acaattgatg tgtgttatgt acctccaacg 240
gtaaatgagc tattttgtcg tttttcactc agggttgagg ctaattgtat tgagccacat 300
gtatgtgatg accctaaagt tatttattgg ttaaaacggt ttttcgaaac ctataaaaaa 360
cacaatggcc ttaatgaagt tgcaacgcgc tatgctaaaa atatactgat gggcaactgg 420
ctttggcgta accgccaatc accaaatgtt gatattgaaa tccttactga gcacgcagcc 480
ccgattgttg ttgaaggtgc acaaaaacta aaatggcaag gcaactggca aaataatcaa 540
acggcattat taacgttgtc agaatctatt caagaagggc taagcaatcc tcaaaattat 600
tgttatttag atataaccgc aaaaattaaa aatgcattta gccaagaggt tcatcctagt 660
caaaagtttg tagataatgt tgaacaaggt atgtcatcta aacaacttgc atatactcaa 720
gtaggcgata aaaaagcagc aagtttgaat tcacaaaaag taggggctgc tatccaaact 780
attgatgatt ggtatgagga aggttacaaa cctttacgca ctcacgagta tggcgcagat 840
aagcaaatat tagttgcaca cagaacacct aagagccatt cagactttta ttcattactc 900
ccgcgcattg ctttgcatat taaacacatg gaaaagcatg gtttagagca aagtgaacaa 960
tcaaactcaa ttcactttat tgcggcagtg ctgatcaaag gtggcttgtt tcaaaggagt 1020
aaaggttga 1029
<210> 15
<211> 88
<212> DNA
<213> Pseudoalteromonas translucida KMM520
<220>
<221> misc_feature
<222> (29)..(60)
<223> n is a, c, g, or t
<400> 15
gtgaactgcc gagtaggcag ctggaaatnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60
gtgaactgcc gagtaggcag ctgaagtt 88
<210> 16
<211> 88
<212> DNA
<213> Pseudoalteromonas translucida KMM520
<220>
<221> misc_feature
<222> (29)..(60)
<223> n is a, c, g, or t
<400> 16
gtgaactgcc gagtaggcag ctggaaatnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60
gtgaactgcc gagtaggcag ctggaaat 88
<210> 17
<211> 88
<212> DNA
<213> Pseudoalteromonas translucida KMM520
<220>
<221> misc_feature
<222> (29)..(60)
<223> n is a, c, g, or t
<400> 17
gtgaactgcc gagtaggcag ctgaagttnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60
gtgaactgcc gagtaggcag ctgaagtt 88
<210> 18
<211> 88
<212> DNA
<213> Pseudoalteromonas translucida KMM520
<220>
<221> misc_feature
<222> (29)..(60)
<223> n is a, c, g, or t
<400> 18
gtgaactgcc gagtaggcag ctgaagttnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60
gtgaactgcc gagtaggcag ctggaaat 88
<210> 19
<211> 632
<212> DNA
<213> Pseudoalteromonas translucida KMM520
<400> 19
ttaattttcc ttaattattt ttaaagttag actgatttag acttggaaaa gcttaatgat 60
tggagagcta aattgactaa tagtattcag tcaagtttaa ttagttttaa gcgatccatg 120
cataactatt tctgtacaat gctatatttt caccaattaa taatttttaa tcaagcctac 180
attatgaaat atactatacc catttgaact cttctatttg taccattgtc ggtagcaaaa 240
acttatgggg ttttgaacgt cacttaaatt gtaagcattt gcgatggagg cgcgtttaga 300
gtcaaccttg attctgatat gctccgaatt tttggtaaga atataagtgt gagagtagct 360
aatgtggata cgcctgagtt aagggaaaaa tgtgaaaatg aaataactcg ttatcatgca 420
aagtgactaa ggttataatc ttccgtttat ggcacatagc agccaactaa acttgacagt 480
atttttatgt ggttggcttt ataaaaccag catttggtaa cattatgcca atttttactt 540
caatattatg ccaacataca ctacactaac ggagctgtag cacaataagc tcgtttgtac 600
ttatgccaac ttatacttca aacaacattg gg 632
<210> 20
<211> 113
<212> DNA
<213> Pseudoalteromonas translucida KMM520
<400> 20
ttgggtgttg tttgaagtat aagttgacat atctgtacta aaagatggca taaattggaa 60
gtgtaaggtg gcatagtcta gtatttaacc aaatggttaa atggttgact cac 113
<210> 21
<211> 8138
<212> DNA
<213> Artificial sequence ()
<400> 21
cattaattcc taatttttgt tgacactcta tcattgatag agttatttta ccactcccta 60
tcagtgatag agaaaagtga actctagaaa taattttgtt taactttaaa aggagatata 120
ccatgggtga actgccgagt aggcagctgg aaatgagacc tctggtctcg tgaactgccg 180
agtaggcagc tggaaatgga tccgaattcg agctcggcgc gcctgcaggt cgacaagctt 240
gcggccgctc taatctagac atcattaatt cctaattttt gttgacactc tatcattgat 300
agagttattt taccactccc tatcagtgat agagaaaagt gaactctaga aataattttg 360
tttaacttta aaggagatat acatatgttt ttgcaaagac ctaaataacc aacaagaagg 420
agatatacat atgcactttc tggtgcagac caagagctac ccggacgagg cgctggaaag 480
ctatctgctg cgtctggcgc gtgataacag ctacaacggt tatagcgagc tggcggacat 540
cctgtggcag tggctggcgg aacaagataa cgagctggaa ggtgcgctgc cgctggcgct 600
gagcaaggtg gacgtttacc acgcgcgtca ggcgagcagc ttccgtatcc gtgcgctgaa 660
actggtggcg caactggcgg acgttaacgc gggtgatatt ctggcgctgg cgtggcgtcg 720
tagcaacttc aagtttggca acctggcggc ggtgagccgt aacgagctgg cgatcccgct 780
ggaactgctg cgtaccgata acatcccggt ttgcattaaa tgcctgagcg agagcagcca 840
cattccgttt tactggcacc tgaagccgta taaagcgtgc cacaagcaca aaagccagct 900
gatcacccgt tgcaaggagt gctacgacct gattgattat cgtgcgagcg aggcgtttct 960
ggaatgcgtt tgcggttgca aaatcaccaa cagcgaacaa ctgaacgacg cggatttcaa 1020
gatcgcgatt gcgctggcga gcagcaacag ccagaaaatc gtgggcctga ttagctggtt 1080
cgcgaaggtg aaacaactgg acgttagcga cgcggatttc aactgcgcgt ttgttgatta 1140
cttcaacacc tggccggaga gcctgaccac cgaactggac ctgctgacca acaacgcgcg 1200
tctgaagcag ctgaacccgt ttaacaagac caaattcagc agcgtgtacg gtgacctgat 1260
ccgtgatggc caaattgcgg cgaccagcaa ccgtaagaac aaagttatcg acgagatcat 1320
tagctatttt gtggaactgg ttgatagcaa cccgaaggcg aaacacccga acattggtga 1380
cctgctgctg tgcaccttcg atgcggcggt gctgctgaac accaccaccg agcaggttta 1440
ccgtctgcac caagaagcgt ttctgaactg cgcgtatagc cagaagaaac acgaacaact 1500
gcgtgcggat agccacgtgt tctatctgcg tcaggttatc gagctgcagc aagcgtttgc 1560
ggcggaaaaa ccgctgacca agaaacaatt cattgcgccg tggtaactta tgaacctgca 1620
ggatgcgctg gcgattgagc cgctgaagga aaaaaccacc gcgctgcgta agctgttcgt 1680
gccgtacacc agccacgttg aggtggatgg ttttgaggaa ctggcgctga ccgtgctgat 1740
caacctggtt tataagcgta gcgaaattga cgatctgacc agcgcgcgta ccgcgaaaag 1800
cgtgctgcgt gacgaggttc tgctgagcaa gtgcatcaac gaagtgaaat ggttccacac 1860
ccacaacctg aagtacccgg acatccgtgt tagccaccaa cgtctgatta gcgaggtggt 1920
tagcgaagat atcgcgggta tttgcagccg tagcctgccg ctgagctttg gctggagcca 1980
caacagcgcg gagatcaacc acgcgaaact gttcctgacc agctttaact ggcagggtga 2040
agtgacctgc ctggcgcgtc tgctgattaa cgaggaaccg gtttggatca acctgattcg 2100
tgcgtacggt ttcaccaaga aagcggttct ggagatcagc ggcaagatta aacagcaact 2160
gccggtggcg gagttcccgc tggaagttag cagctttagc ccgcagctgc aaatgccgtt 2220
tcagcaaagc tatctggtgg ttaccccggt ggttagccac gcgatgctgg cgaagatcca 2280
gcaactgacc accgaccgta aactgaactt cgcgctggtt gagcacagcc gtccggcgaa 2340
cgttggtgat ctggcgagca gcgtgggtgg caacattcgt gttctgcgtt actttccgaa 2400
gacctatagc aaagcggtga accgtagcaa agttgcgaac aacgatatcg aaaaggcgtt 2460
caaaattcgt gcgctgctga gcagccagtt tcagcaagcg ctgctggtgc tggttggcat 2520
caagcagttc aacaccctgc gtcaaaaacg tctggcgcgt gtggcggcga tccgtcaagt 2580
gcgtgttagc ctgcaactgt ggctggacaa cattctggag gcgaagaaca acgcgcagaa 2640
ccaagtgtac ccggaatggg ttcgtcacta tctggatcaa agcatcacca actgcattag 2700
ccagttcagc aacgttctga acgaaagcct gggtaacctg agcaagctga aacgttttgc 2760
gtaccacccg aacctgatgg gcctgttcaa agcgcaactg aactatgtgt ttacccactg 2820
cgcggcggag caggaaatcc tgaacgacga gcaaattgtg tacgttcact gccaggacat 2880
gcgtgttttc gatgcggaag cgatggcgaa cccgtatatc cagggtatgc cgagcctgac 2940
cgcgctgaac ggcctggcgc acaacttcga gcgtaagctg aaaaacttta ttgatccgag 3000
catcaagtgc attggtagcg cgatctacat tgagaactat caactgcaca ccggcaaacc 3060
gctgccggaa ccgagcaagc tgaaacaggt ggcgggtcgt agccacgtta tccgtagcgg 3120
catcattgac aagccgaaat gcgacattac cctggatctg gtgttccgtc tgtttgttcc 3180
gaacaccgaa ctgctggata agctgaacag ccaactgatt aagccggcgc tgccgagcag 3240
ctttgcgggt ggcaccatgc acccgccgag cctgtaccag aacattgact ggtgccacgt 3300
gcacaccaag ccgagcgagc tgtttaagaa actgaaggcg aaaagcagca acggtagctg 3360
gctgtatccg agcaagaaag tggttaaaag cttcgaacag ctgatcgacg cgctgaacag 3420
caactttaac ctgcgtccgg cggcgattgg cctggcggcg ctggaggaac cggtgaagcg 3480
tgatgcggcg ctgcacgagt accactgcta tgcggaaccg gttatcggtc tgctggagtg 3540
cgtgagcaac accagcgtta agtacgcggg cgcgaaacaa ttctttcacg acgcgttctg 3600
ggtgatggat gttcagaagg aaagcatgct gatgaagaaa agcaaatttg agtatgaata 3660
atgcagctgc cgcgtcacct gagctacacc cgtagcctga gcccgagcaa ggcggtgttc 3720
ttttataaaa ccccggagag cgacttcgaa ccgctgcaga tcgagcaaaa caaactggtg 3780
ggtcagaaga gcggttttgg cgatgcgtac cagaagcaaa acgttgcgaa aaacctggcg 3840
ccgcaggacc tggcgtttgg taacccgcaa accattgatg tgtgctatgt tccgccgacc 3900
gtgaacgaac tgttctgccg ttttagcctg cgtgttgagg cgaactgcat cgaaccgcac 3960
gtgtgcgacg atccgaaggt tatttactgg ctgaaacgtt tctttgaaac ctataagaaa 4020
cacaacggtc tgaacgaagt ggcgacccgt tacgcgaaga acatcctgat gggcaactgg 4080
ctgtggcgta accgtcagag cccgaacgtt gacatcgaga ttctgaccga acacgcggcg 4140
ccgattgtgg ttgagggtgc gcagaagctg aaatggcaag gcaactggca gaacaaccaa 4200
accgcgctgc tgaccctgag cgagagcatc caggaaggtc tgagcaaccc gcaaaactac 4260
tgctatctgg atatcaccgc gaagattaaa aacgcgttca gccaggaagt gcacccgagc 4320
caaaagtttg tggacaacgt tgaacagggt atgagcagca aacagctggc gtatacccaa 4380
gtgggcgata agaaagcggc gagcctgaac agccagaagg ttggcgcggc gatccaaacc 4440
attgacgatt ggtacgagga aggttataaa ccgctgcgta cccatgagta tggtgcggac 4500
aagcaaatcc tggtggcgca ccgtaccccg aaaagccaca gcgattttta tagcctgctg 4560
ccgcgtatcg cgctgcacat taagcacatg gaaaaacacg gtctggagca gagcgaacaa 4620
agcaacagca tccacttcat tgcggcggtt ctgattaagg gtggcctgtt tcagcgtagc 4680
aaaggatgaa gcgttactat ttcaccatca cctacctgcc gcaaagctgc gatgtgagcc 4740
tgctggcggg tcgttgcatc ggcattctgc acggtttcat gagcagccgt gagatcagca 4800
acattggcgt gtgctttccg aaatggaacg agcagaccat cggtaacgaa ctggcgtttg 4860
ttagcaccaa caagaaacaa ctgaccaacc tgagccagca aagctatttc gagatgatgg 4920
cgcacgacaa gctgtttggc ctgagcaaaa ttctggaagt gccggttaac cagagcgaag 4980
tgatgttcgt tcgtaaccaa agcgtggcga aggcgtttgt tggtgaaaag caacgtcgtc 5040
tgaaacgtgc gaagaaacgt gcggaggcgc gtggcgaagt gtacaacccg gagtataagt 5100
tcgaagcgaa agatatcggt cactttcaca gcattccggt gagcagcaag ggtaacggcc 5160
agagctacgt tctgcacatc caaaagaacg agaacgcgga aagcattaaa aaccagttca 5220
acaactatgg ctttgcgacc aaccaaattt tcctgggcac cgtgccgagc ctgaacaccc 5280
tgctgtaagg taccaccctt aatctgacct aggctgctgc caccgctgag caataactag 5340
cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa cctcaggcat 5400
ttgagaagca cacggtcaca ctgcttccgg tagtcaataa accggtaaac cagcaataga 5460
cataagcggc tatttaacga ccctgccctg aaccgacgac cgggtcatcg tggccggatc 5520
ttgcggcccc tcggcttgaa cgaattgtta gacattattt gccgactacc ttggtgatct 5580
cgcctttcac gtagtggaca aattcttcca actgatctgc gcgcgaggcc aagcgatctt 5640
cttcttgtcc aagataagcc tgtctagctt caagtatgac gggctgatac tgggccggca 5700
ggcgctccat tgcccagtcg gcagcgacat ccttcggcgc gattttgccg gttactgcgc 5760
tgtaccaaat gcgggacaac gtaagcacta catttcgctc atcgccagcc cagtcgggcg 5820
gcgagttcca tagcgttaag gtttcattta gcgcctcaaa tagatcctgt tcaggaaccg 5880
gatcaaagag ttcctccgcc gctggaccta ccaaggcaac gctatgttct cttgcttttg 5940
tcagcaagat agccagatca atgtcgatcg tggctggctc gaagatacct gcaagaatgt 6000
cattgcgctg ccattctcca aattgcagtt cgcgcttagc tggataacgc cacggaatga 6060
tgtcgtcgtg cacaacaatg gtgacttcta cagcgcggag aatctcgctc tctccagggg 6120
aagccgaagt ttccaaaagg tcgttgatca aagctcgccg cgttgtttca tcaagcctta 6180
cggtcaccgt aaccagcaaa tcaatatcac tgtgtggctt caggccgcca tccactgcgg 6240
agccgtacaa atgtacggcc agcaacgtcg gttcgagatg gcgctcgatg acgccaacta 6300
cctctgatag ttgagtcgat acttcggcga tcaccgcttc cctcatactc ttcctttttc 6360
aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta 6420
tttagaaaaa taaacaaata gctagctcac tcggtcgcta cgctccgggc gtgagactgc 6480
ggcgggcgct gcggacacat acaaagttac ccacagattc cgtggataag caggggacta 6540
acatgtgagg caaaacagca gggccgcgcc ggtggcgttt ttccataggc tccgccctcc 6600
tgccagagtt cacataaaca gacgcttttc cggtgcatct gtgggagccg tgaggctcaa 6660
ccatgaatct gacagtacgg gcgaaacccg acaggactta aagatcccca ccgtttccgg 6720
cgggtcgctc cctcttgcgc tctcctgttc cgaccctgcc gtttaccgga tacctgttcc 6780
gcctttctcc cttacgggaa gtgtggcgct ttctcatagc tcacacactg gtatctcggc 6840
tcggtgtagg tcgttcgctc caagctgggc tgtaagcaag aactccccgt tcagcccgac 6900
tgctgcgcct tatccggtaa ctgttcactt gagtccaacc cggaaaagca cggtaaaacg 6960
ccactggcag cagccattgg taactgggag ttcgcagagg atttgtttag ctaaacacgc 7020
ggttgctctt gaagtgtgcg ccaaagtccg gctacactgg aaggacagat ttggttgctg 7080
tgctctgcga aagccagtta ccacggttaa gcagttcccc aactgactta accttcgatc 7140
aaaccacctc cccaggtggt tttttcgttt acagggcaaa agattacgcg cagaaaaaaa 7200
ggatctcaag aagatccttt gatcttttct actgaaccgc tctagatttc agtgcaattt 7260
atctcttcaa atgtagcacc tgaagtcagc cccatacgat ataagttgta attctcatgt 7320
tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg 7380
gtcgagatcc cggtgcctaa tgagtgagct aacttaccgt tgtaaaacga cggccagtga 7440
attcctgatg aatcccctaa tgatttttat caaaatcatt aaggttacca tcacggaaaa 7500
aggttatgct gcttttaaga cccactttca catttaagtt gtttttctaa tccgcatatg 7560
atcaattcaa ggccgaataa gaaggctggc tctgcacctt ggtgatcaaa taattcgata 7620
gcttgtcgta ataatggcgg catactatca gtagtaggtg tttccctttc ttctttagcg 7680
acttgatgct cttgatcttc caatacgcaa cctaaagtaa aatgccccac agcgctgagt 7740
gcatataatg cattctctag tgaaaaacct tgttggcata aaaaggctaa ttgattttcg 7800
agagtttcat actgtttttc tgtaggccgt gtacctaaat gtacttttgc tccatcgcga 7860
tgacttagta aagcacatct aaaactttta gcgttattac gtaaaaaatc ttgccagctt 7920
tccccttcta aagggcaaaa gtgagtatgg tgcctatcta acatctcaat ggctaaggcg 7980
tcgagcaaag cccgcttatt ttttacatgc caatacaatg taggctgctc tacacctagc 8040
ttctgggcga gtttacgggt tgttaaacct tcgattccga cctcattaag cagctctaat 8100
gcgctgttaa tcactttact tttatctaat ctagacat 8138
<210> 22
<211> 6320
<212> DNA
<213> Artificial sequence ()
<400> 22
acgatcgtaa aaggatctca agaagatcct ttacggattc ccgacaccat cactctagat 60
ttcagtgcaa tttatctctt caaatgtagc acctgaagtc agccccatac gatataagtt 120
gtaattctca tgttagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct 180
ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg 240
ttgcgctgat gaatccccta atgattttta tcaaaatcat taaggttacc atcacggaaa 300
aaggttatgc tgcttttaag acccactttc acatttaagt tgtttttcta atccgcatat 360
gatcaattca aggccgaata agaaggctgg ctctgcacct tggtgatcaa ataattcgat 420
agcttgtcgt aataatggcg gcatactatc agtagtaggt gtttcccttt cttctttagc 480
gacttgatgc tcttgatctt ccaatacgca acctaaagta aaatgcccca cagcgctgag 540
tgcatataat gcattctcta gtgaaaaacc ttgttggcat aaaaaggcta attgattttc 600
gagagtttca tactgttttt ctgtaggccg tgtacctaaa tgtacttttg ctccatcgcg 660
atgacttagt aaagcacatc taaaactttt agcgttatta cgtaaaaaat cttgccagct 720
ttccccttct aaagggcaaa agtgagtatg gtgcctatct aacatctcaa tggctaaggc 780
gtcgagcaaa gcccgcttat tttttacatg ccaatacaat gtaggctgct ctacacctag 840
cttctgggcg agtttacggg ttgttaaacc ttcgattccg acctcattaa gcagctctaa 900
tgcgctgtta atcactttac ttttatctaa tctagacatc attaattcct aatttttgtt 960
gacactctat cattgataga gttattttac cactccctat cagtgataga gaaaagtgaa 1020
ctctagaaat aattttgttt aactttaaaa ggagatatac catgtaccgt cgtaagctga 1080
aatatagccg tgttaagaac ctgcacaaat ttgcgagcca gaagaacaaa agcacctgcc 1140
tggtggagag cagcctggaa ttcgacgcgt gcttccactt tgagttcagc ccgccgatcg 1200
cggcgtttga agcgcaaccg ctgggttacg agtatgaatt cgataaccgt atttgccgtt 1260
acaccccgga ctttctgctg acccacaccg atggcaccca gaagttcatc gaggttaagc 1320
cgcaaagcaa aattgcggac gaggattttc gtgcgcgttt catcgaaaag caggcgattg 1380
cgaaacaaga cggtcgtgat ctgatcctgg tgaccgacaa gcagattcgt gtttacccga 1440
ccctgaacaa cctgaaactg ctgcaccgtt atagcggctt tcagagcctg accgagctgc 1500
aagcgagcgt gctggaactg gttaagcagt acggtagcat caaagtgggc caactgattc 1560
gttatctgaa agttaccgcg ggtgaactgc tggcgaccgt gctgcgtctg ctgagcctgg 1620
gccaactgtt cgcggatctg accaccaacg agatcagcat tgaaaccgcg atctggagca 1680
acaatgttta ataacgacct gttcgacgat gagtttaacc agccgctgcc gaaggcggaa 1740
accaaactgc cgcagaacta taccaaggat ctgcaagcgc tgccggagaa gatcaaaacc 1800
accaccttcg cgaagctgaa atacattcaa tggctggagg cgaacatcca gggtggctgg 1860
acccaaaaga acctggaacc gctgctgaaa ctgatgccgg acgttgaggg tgaaaagaaa 1920
ccgagctggc gtaccgcggc gcgttggtat agcgcgtaca ccaacgcgga taagaacatt 1980
atggcgctga tcccgagcca ccagaagaaa ggcaaccgtg aacgtgacac caccaccgat 2040
aagttctttg agaaagcgct ggaacgttac ctggtgaagg agaaaccgag cgttgcgagc 2100
gcgtataagt tctacaaaga cctggtgatc attgaaaacg acagcgtggt tgatagcgtt 2160
ctgaaaccgc tgacctataa ggcgtttaaa aaccgtattg acaacctgcc gcagtatgag 2220
gttatgatcg cgcgttacgg caagcgtctg gcggatattg cgtacaacaa ggtggaaggc 2280
cacaaacgtc cgattcgtgt gctggagaaa gttgaaatcg accacacccc gctggatctg 2340
attctgctgg acgatgagct gcacatcccg ctgggtcgtc cgaccctgac catgctggtt 2400
gacgtttata gccactgcat cgtgggctac tatttcagct ttagcgagcc gagctacgat 2460
gcggttcgtc gtgcgatgct gaacgcgatg aagccgaaaa gcgaagtggc gaaactgtac 2520
ccggacacca ttaacgagtg gaagtgcgcg ggtaaaatcg aaaccctggt ggttgataac 2580
ggcgcggagt tctggagcaa cagcctggaa ctggcgtgcg aggaaatcgg tattaacacc 2640
cagtataacc cggtggcgaa gccgtggctg aaaccgttcg ttgagcgtat gtttggcacc 2700
atcaacaccg aactgctgga cccggttccg ggcaagacct tcagcaacat cctgcaaaaa 2760
cacgaataca acccgaagaa agacgcgatt atgcgtttca ccacctttat gcagctgttt 2820
cacaagtggg tggttgatgt gtatcaccaa gacgcggata gccgtttcaa atacattccg 2880
agccagctgt gggaccaagg ctttaacacc ctgccgccga ccatgctgag cgatgcggat 2940
ctgcagcaac tggatgtggt tctgagcatc agcaaccacc gtgtgctgcg taagggtggc 3000
attcgtctgg agaacctgag ctatgacagc accgaactgg cgaactaccg taagcagttc 3060
agccacaaag tgagccaaga ggttctgatc aaactgaacc cggacgatat tagctacatc 3120
tatgtgtacc tggacaagct ggaacactat attaaagttc cgtgcatcga tccgaacggt 3180
tacacccaga acctgagcct gaaccaacac aagatcaaca ttcgtatcca ccgtgacttt 3240
attagcggta gcatcgataa cgttggcctg gcgaaggcgc gtatgttcat tcacaacaaa 3300
atccagaacg agtttgagga actgaagaac gcgccgaaac acagcaaggt gaaaggtggc 3360
aaggcgctgg cgaaacacca gaacattagc agcgacagcc aaaagagcat cacccacagc 3420
aaaccggtgg aggcgaagaa agttaccccg aaagaacaac cgaccgatag ctgggacgat 3480
ttcatcagcg acctggatgg tttttaatta tgctgaccga caagcagaaa gaaaagctga 3540
acgagttccg tgatgttttt attgaatacc cgatcattac caccatcttc aacgactttg 3600
atcgtctgcg tctgggtaaa ggcctgaccg gcgagaagcc gtgcatgctg ctgaacggtg 3660
acaccggcac cggtaaaacc gcgctgatta aacagtataa ggaacgtcac ctgccgcaat 3720
tcatcaacgg tgttatgaac cacccggtgc tggttagccg tattccgagc aacccgaccc 3780
tggaaagcac cctggcggag ctgctgaaag acctgggtca agtgggcagc accgagcgta 3840
agctgcgtat taacggcacc cgtctgacca ccagcctgat caaatgcctg aagacctgcg 3900
gcaccgaact gatcattatc gatgagtttc aggaactgat tgagcacaac caaggcaaga 3960
aacgtcgtga aattgcgaac cgtctgaaat acatcaacga cgaggcgggt gttagcattg 4020
tgctggttgg catgccgtgg gcggaaaaga tcgcggatga gccgcagtgg agcagccgtc 4080
tgctgatccg tcgtcaactg ccgtatttca aactgagcga gaacccgaag cactttgtgc 4140
agctgattat cggtctggcg aaccgtatgc cgttcgcgga aaaaccgaac ctgagcgagc 4200
aagcgaccgt tttcaccctg tttagcctga gcaaaggctg cttccgtacc ctgaagtact 4260
ttctggacga tgcggtgctg tatgcgctga tggacaacgc gaagaccctg accaccaaac 4320
acctggtgaa ggcgttcgaa gttctgtttc cggatgtgcc gaacctgttt accctgccgg 4380
ttgcggagat caccgcgagc gaggtggaac gttacagcct gtataagccg gaaagcagcc 4440
aggacgagga cccgttcatt gcgaccaaat ttaccgatcg tatgccgatc agccaactgc 4500
tgcgtaagta actcgagccg ctgagcaata actagcataa ccccttgggg cctctaaacg 4560
ggtcttgagg ggttttttgc tgaaacctca ggcatttgag aagcacacgg tcacactgct 4620
tccggtagtc aataaaccgg taaaccagca atagacataa gcggctattt aacgaccctg 4680
ccctgaaccg acgacaagct gacgaccggg tctccgcaag tggcactttt cggggaaatg 4740
tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga 4800
attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga 4860
ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg 4920
cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca 4980
atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga 5040
gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca 5100
acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt 5160
cgtgattgcg cctgagcgag acgaaatacg cggtcgctgt taaaaggaca attacaaaca 5220
ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa 5280
tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac 5340
catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc 5400
agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt 5460
ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat 5520
tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt 5580
aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcatactctt cctttttcaa 5640
tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt 5700
tagaaaaata aacaaatagg catgctagcg cagaaacgtc ctagaagatg ccaggaggat 5760
acttagcaga gagacaataa ggccggagcg aagccgtttt tccataggct ccgcccccct 5820
gacgaacatc acgaaatctg acgctcaaat cagtggtggc gaaacccgac aggactataa 5880
agataccagg cgtttccccc tgatggctcc ctcttgcgct ctcctgttcc cgtcctgcgg 5940
cgtccgtgtt gtggtggagg ctttacccaa atcaccacgt cccgttccgt gtagacagtt 6000
cgctccaagc tgggctgtgt gcaagaaccc cccgttcagc ccgactgctg cgccttatcc 6060
ggtaactatc atcttgagtc caacccggaa agacacgaca aaacgccact ggcagcagcc 6120
attggtaact gagaattagt ggatttagat atcgagagtc ttgaagtggt ggcctaacag 6180
aggctacact gaaaggacag tatttggtat ctgcgctcca ctaaagccag ttaccaggtt 6240
aagcagttcc ccaactgact taaccttcga tcaaaccgcc tccccaggcg gttttttcgt 6300
ttacagagca ggagattacg 6320

Claims (16)

1. A CRISPR-associated transposase comprising a polypeptide selected from the group consisting of: a transposase protein tnsA derived from a bacterium of the genus pseudoalteromonas, a transposase protein tnsB derived from a bacterium of the genus pseudoalteromonas, a transposase protein tnsC derived from a bacterium of the genus pseudoalteromonas, a transposase protein tniQ derived from a bacterium of the genus pseudoalteromonas, a nuclease protein Cas5/8 derived from a bacterium of the genus pseudoalteromonas, a nuclease protein Cas6 derived from a bacterium of the genus pseudoalteromonas, and a nuclease protein Cas7 derived from a bacterium of the genus pseudoalteromonas.
2. The CRISPR-associated transposase of claim 1, wherein the Pseudoalteromonas bacterium is Pseudoalteromonas translucens, preferably wherein the Pseudoalteromonas translucens is Pseudoalteromonas translucens KMM520 (Pseudoalteromonas translucens transfucida KMM 520).
3. The CRISPR-associated transposase of claim 2,
the tnsA is a polypeptide with an amino acid sequence of SEQ ID NO. 1, or a polypeptide which has more than 95% homology, preferably more than 98% homology, more preferably more than 99% homology and has the same function with the SEQ ID NO. 1;
the tnSB is a polypeptide with an amino acid sequence of SEQ ID NO. 2, or a polypeptide which has more than 95% homology, preferably more than 98% homology, more preferably more than 99% homology and has the same function with the SEQ ID NO. 2;
the tnsC is a polypeptide with an amino acid sequence of SEQ ID NO. 3, or a polypeptide which has more than 95% homology, preferably more than 98% homology, more preferably more than 99% homology with SEQ ID NO. 3 and has the same function;
the tniQ is a polypeptide having an amino acid sequence of SEQ ID NO. 4, or a polypeptide having more than 95% homology, preferably more than 98% homology, more preferably more than 99% homology, with SEQ ID NO. 4 and having the same function;
the Cas5/8 is a polypeptide with an amino acid sequence of SEQ ID NO. 5, or a polypeptide which has more than 95% homology, preferably more than 98% homology, more preferably more than 99% homology and has the same function with the SEQ ID NO. 5;
the Cas6 is a polypeptide with an amino acid sequence of SEQ ID NO. 6, or a polypeptide which has more than 95% homology, preferably more than 98% homology, more preferably more than 99% homology with the SEQ ID NO. 6 and has the same function;
the Cas7 is a polypeptide having an amino acid sequence of SEQ ID NO. 7, or a polypeptide having 95% or more homology, preferably 98% or more homology, more preferably 99% or more homology, with SEQ ID NO. 7 and having the same function.
4. A gene encoding the polypeptide as claimed in any one of claims 1 to 3.
5. The gene according to claim 4,
the gene encoding the polypeptide tnsA having the amino acid sequence of SEQ ID No. 1 is the nucleotide sequence of SEQ ID No. 8 or a polynucleotide having more than 80% homology, preferably more than 85% homology, more preferably more than 90% homology, more preferably more than 95% homology with SEQ ID No. 8;
the gene encoding the polypeptide tnsB having the amino acid sequence of SEQ ID NO. 2 is the nucleotide sequence SEQ ID NO. 9, or a polynucleotide having more than 80% homology, preferably more than 85% homology, more preferably more than 90% homology, more preferably more than 95% homology with SEQ ID NO. 9;
the gene encoding the polypeptide tnsC having the amino acid sequence of SEQ ID No. 3 is the nucleotide sequence of SEQ ID No. 10 or a polynucleotide having more than 80% homology, preferably more than 85% homology, more preferably more than 90% homology, more preferably more than 95% homology with SEQ ID No. 10;
the gene encoding the polypeptide tniQ having the amino acid sequence of SEQ ID No. 4 is the nucleotide sequence of SEQ ID No. 11, or a polynucleotide having more than 80% homology, preferably more than 85% homology, more preferably more than 90% homology, more preferably more than 95% homology with SEQ ID No. 11;
the gene encoding the polypeptide Cas5/8 with the amino acid sequence of SEQ ID No. 5 is the nucleotide sequence SEQ ID No. 12, or a polynucleotide having more than 80% homology, preferably more than 85% homology, more preferably more than 90% homology, more preferably more than 95% homology with SEQ ID No. 12;
the gene encoding the polypeptide Cas6 having the amino acid sequence of SEQ ID No. 6 is the nucleotide sequence SEQ ID No. 13 or a polynucleotide having more than 80% homology, preferably more than 85% homology, more preferably more than 90% homology, more preferably more than 95% homology with SEQ ID No. 13;
the gene encoding the polypeptide Cas7 having the amino acid sequence of SEQ ID NO. 7 is the nucleotide sequence SEQ ID NO. 14 or a polynucleotide having more than 80% homology, preferably more than 85% homology, more preferably more than 90% homology, more preferably more than 95% homology with SEQ ID NO. 14.
6. A plasmid, pqqacade or pqqacadeptr, for use in CRISPR transposon systems comprising a gene fragment selected from the group consisting of: a Cas 5/8-encoding gene as claimed in claim 4 or 5; a Cas 6-encoding gene as set forth in claim 4 or 5; a Cas 7-encoding gene as set forth in claim 4 or 5; a tniQ encoding gene as claimed in claim 4 or 5.
7. The plasmid pQCascadePtr of claim 6, wherein the spacer of the crRNA sequence, spacer, is a spacer that targets a single site in the genome or is an array of crRNAs that targets multiple sites in the genome.
8. The plasmid pQCascadePtr of claim 7, wherein the crRNA sequence is a genomic multisite targeted crRNA array, and wherein the repeat region repeat comprises one or more sequences selected from the group consisting of: the nucleotide sequence is repeat1 of SEQ ID NO. 15, the nucleotide sequence is repeat2 of SEQ ID NO. 16, the nucleotide sequence is repeat3 of SEQ ID NO. 17 and the nucleotide sequence is repeat4 of SEQ ID NO. 18, wherein 32N (N32) in the nucleotide sequences of SEQ ID NOs:15-18 are any base A, T, G or C.
9. A helper plasmid pTns or pTnsPtr for CRISPR transposon systems, for use in conjunction with the plasmid pqracadeptr of any of claims 6-8, comprising gene segments selected from the group consisting of: the gene encoding tnsA as claimed in claim 4 or 5; the tnsB-encoding gene as set forth in claim 4 or 5; the tnsC-encoding gene as set forth in claim 4 or 5.
10. A plasmid pqcrastnsptr for use in CRISPR transposon systems, which is the combination of the plasmid pqcratdtr of any one of claims 6 to 8 and the helper plasmid pTnsPtr of claim 9, comprising: the above Cas5/8, cas6, cas7, tniQ, tnSA, tnSB and tnSC genes, crRNA sequence targeting genome target site, colA replicon, promoter, kanamycin resistance gene.
11. A helper plasmid pDonorPtr for CRISPR transposon systems for use with the plasmid pqqascadeptr of any one of claims 6 to 8 and the helper plasmid pTnsPtr of claim 9, comprising gene segments selected from the group consisting of: a Left End (LE) having the nucleotide sequence of SEQ ID NO. 19 or a sequence having more than 80% homology, preferably more than 85% homology, more preferably more than 90% homology, more preferably more than 95% homology with SEQ ID NO. 19 and comprising 33bp of the 3' end of SEQ ID NO. 19; the sequence Right End (RE) having the nucleotide sequence of SEQ ID NO:20 or a sequence 27bp more than 80%, preferably more than 85%, more preferably more than 90%, more preferably more than 95% homologous to SEQ ID NO:20 and comprising the 5' end of SEQ ID NO: 20; the Cargo gene of interest (Cargo gene).
12. A plasmid pEffectorPtr for use in a CRISPR transposon system, formed by combining the plasmid pqqascadeptr of any one of claims 6-8, the helper plasmid pTnsPtr of claim 9, and the helper plasmid pDonorPtr of claim 11, comprising: the above Cas5/8, cas6, cas7, tniQ, tnsA, tnsB and tnsC genes, left End (LE) and Right End (RE) sequences, crRNA sequences targeting the genomic target site, colA replicons, promoters, kanamycin resistance genes.
13. A CRISPR transposon system, comprising: the plasmid pqqcacadepptr of any one of claims 6 to 8, the helper plasmid pTnsPtr of claim 9, the helper plasmid pDonorPtr of claim 11; or the plasmid pQCasTnsPtr of claim 10, the helper plasmid pDonorPtr of claim 11; or the plasmid pEffectorPtr of claim 12.
14. The CRISPR transposase system of claim 13, further comprising a Vibrio cholerae (Vibrio cholerae) Tn6677 derived CRISPR transposase-associated plasmid comprising plasmid pQCastnsVch and helper plasmid pDenoroVch,
wherein plasmid pQCastnsVch comprises: cas5/8, cas6, cas7, tniQ, tnSA, tnSB and tnsC genes from vibrio cholerae Tn6677, cloDF13 replicons, a promoter and a streptomycin resistance gene;
the plasmid pDronVch comprises Left End (LE) and Right End (RE) from Vibrio cholerae Tn6677, and a target Cargo gene (Cargo gene).
15. Use of a CRISPR-associated transposase of any of claims 1 to 3, a gene of claim 4 or 5, a plasmid pQCascadePtr of any of claims 6 to 8, a plasmid pTnsPtr of claim 9, a plasmid pqcastnptr of claim 10, a plasmid pDonorPtr of claim 11, a plasmid peffecterptr of claim 12, a CRISPR transposon system of claim 13 or 14 for gene editing.
16. Use of the CRISPR transposon system of claim 13 or 14 in gene editing, for gene editing of gram negative bacteria such as e.
CN202110532731.0A 2021-05-17 2021-05-17 Novel CRISPR (clustered regularly interspaced short palindromic repeats) related transposase Pending CN115369098A (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN202110532731.0A CN115369098A (en) 2021-05-17 2021-05-17 Novel CRISPR (clustered regularly interspaced short palindromic repeats) related transposase
PCT/CN2022/091102 WO2022242464A1 (en) 2021-05-17 2022-05-06 Novel crispr-associated transposase

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110532731.0A CN115369098A (en) 2021-05-17 2021-05-17 Novel CRISPR (clustered regularly interspaced short palindromic repeats) related transposase

Publications (1)

Publication Number Publication Date
CN115369098A true CN115369098A (en) 2022-11-22

Family

ID=84058343

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110532731.0A Pending CN115369098A (en) 2021-05-17 2021-05-17 Novel CRISPR (clustered regularly interspaced short palindromic repeats) related transposase

Country Status (2)

Country Link
CN (1) CN115369098A (en)
WO (1) WO2022242464A1 (en)

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2019284926C1 (en) * 2018-06-13 2023-04-27 Caribou Biosciences, Inc. Engineered cascade components and cascade complexes
EP3935179A4 (en) * 2019-03-07 2022-11-23 The Trustees of Columbia University in the City of New York Rna-guided dna integration using tn7-like transposons
WO2020186262A1 (en) * 2019-03-14 2020-09-17 Cornell University Compositions and methods for gene targeting using crispr-cas and transposons
WO2021016135A1 (en) * 2019-07-19 2021-01-28 Synthego Corporation Stabilized crispr complexes

Also Published As

Publication number Publication date
WO2022242464A1 (en) 2022-11-24

Similar Documents

Publication Publication Date Title
US20230383290A1 (en) High-throughput precision genome editing
US10612043B2 (en) Methods of in vivo engineering of large sequences using multiple CRISPR/cas selections of recombineering events
US10287590B2 (en) Methods for generating libraries with co-varying regions of polynuleotides for genome modification
EP1034260B1 (en) Novel dna cloning method relying on the e. coli rece/rect recombination system
JP4264703B2 (en) Synthetic genes and bacterial plasmids lacking CpG
US20200255830A1 (en) Novel crispr-associated transposon systems and components
WO2016205623A1 (en) Methods and compositions for genome editing in bacteria using crispr-cas9 systems
CN107142272A (en) A kind of method for controlling plasmid replication in Escherichia coli
US9567590B2 (en) Method of introducing a mitochondrial genome into a eukaryotic cell mitochondrion
CN102703424B (en) A kind of method of genome of E.coli point mutation of recombined engineering mediation
CN103097537A (en) Self-deleting plasmid
Bost et al. Application of the endogenous CRISPR-Cas type ID system for genetic engineering in the thermoacidophilic archaeon Sulfolobus acidocaldarius
CN111850025B (en) Gene editing system and method applied to mycobacterium tuberculosis
KR102151064B1 (en) Gene editing composition comprising sgRNAs with matched 5&#39; nucleotide and gene editing method using the same
CN115369098A (en) Novel CRISPR (clustered regularly interspaced short palindromic repeats) related transposase
Dhundale et al. Mutations that affect production of branched RNA-linked msDNA in Myxococcus xanthus
WO2020036181A1 (en) Method for isolating or identifying cell, and cell mass
CN100494384C (en) T vector and its construction method
Kim et al. Directed evolution and identification of control regions of ColE1 plasmid replication origins using only nucleotide deletions
WO2022267843A1 (en) Library construction method based on long overhang sequence ligation
CN102533741B (en) Swine pseudo attp site and use of swine pseudo attp site
Cartman et al. Clostridial gene tools
WO2023183627A1 (en) Production of reverse transcribed dna (rt-dna) using a retron reverse transcriptase from exogenous rna
Domenech Corts Efficient and Precise Genome Editing in Shewanella with Recombineering and CRISPR/Cas9-mediated Counter-selection
JP2022513642A (en) A DNA-cutting means based on the Cas9 protein from Clostridium cellulolyticum, a biotechnically important bacterium.

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination