WO2015198221A1 - Procédé d'obtention d'un vecteur et cellule transformée contenant celui-ci - Google Patents

Procédé d'obtention d'un vecteur et cellule transformée contenant celui-ci Download PDF

Info

Publication number
WO2015198221A1
WO2015198221A1 PCT/IB2015/054701 IB2015054701W WO2015198221A1 WO 2015198221 A1 WO2015198221 A1 WO 2015198221A1 IB 2015054701 W IB2015054701 W IB 2015054701W WO 2015198221 A1 WO2015198221 A1 WO 2015198221A1
Authority
WO
WIPO (PCT)
Prior art keywords
dna fragment
vector
cell
overhangs
transformed cell
Prior art date
Application number
PCT/IB2015/054701
Other languages
English (en)
Inventor
Muniasamy NEERATHILINGAM
Sumukh MYSORE
Original Assignee
Centre For Cellular And Molecular Platforms (C-Camp)
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 Centre For Cellular And Molecular Platforms (C-Camp) filed Critical Centre For Cellular And Molecular Platforms (C-Camp)
Publication of WO2015198221A1 publication Critical patent/WO2015198221A1/fr

Links

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
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C12N15/1027—Mutagenizing nucleic acids by DNA shuffling, e.g. RSR, STEP, RPR
    • 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

Definitions

  • the present disclosure relates to the field of Biotechnology. Particularly, it relates to recombinant DNA technology, wherein vector and enzyme free cloning (VEF-C) method is presented which results in custom plasmids harbouring desired fragments of interest in recA- null strains of host cells, such as E.coli. Moreover, the present invention is an economical and time effective alternative as against conventionally used cloning methods as well as existing ligase-independent cloning systems.
  • VEF-C vector and enzyme free cloning
  • RecA is the key enzyme in performing homologous recombination, whereas heteroduplexes are formed when one strand from each parental duplex DNA is exchanged.
  • recA activity is disadvantageous as homologous regions between plasmids can recombine and form undesired products. Therefore, a recAl mutation is generally performed in bacterial strains (example - DH5a, JM109) to reduce recombination by 10,000 fold known in the prior art.
  • the recBCD pathway is the preferred pathway for homologous recombination in E.coli.
  • three subunits namely recB, recC and recD form a heterotrimeric complex that acts as a helicase/nuclease unwinding and degrading duplex DNA.
  • the recB subunit possesses 3 '-5' helicase activity, recD acts as a 5 '-3' helicase and the nuclease function is sequestered in the recB domain.
  • the recBCD enzyme ⁇ 10 in numbers
  • recBCD This single stranded DNA is protected by SSB proteins (which is loaded by recBCD), which inhibits homologous recombination by recA (competitively) and avoids the action of helicases and nucleases on the ssDNA.
  • recA is also loaded onto the strand by recBCD itself thus displacing SSB, making it a key player in assisting the recA biochemistry in-vivo.
  • the recA protein replaces SSB from the single strand in the form of a polymer, scans for homology and performs the strand exchange reactions. Thus, recA involvement is vital functioning of the recBCD pathway.
  • recFOR pathway incorporates the function of recF, recO and recR separately.
  • the recFOR pathway plays a secondary role, occurring in the absence of recBCD.
  • recQ helicase and recJ exonuclease are two enzymes involved in generating ssDNA from dsDNA to facilitate recombination.
  • the RecO protein binds to both dsDNA and ssDNA and promotes recA- mediated renaturation of complementary strands of DNA. This reaction is enhanced when recO forms complexes with SSB. Further, recO combines with recR to make a recOR complex that loads recA onto the SSB.
  • recF protein binds to dsDNA in the presence of ATP in a stoichiometry of 1 recF per 15 nucleotides and has been proposed to direct recA to the single strand gaps in dsDNA.
  • recA displaces the SSB and performs homology search and finally completes strand exchange.
  • recF interferes with recOR function and strongly competes with recO for interacting with recR. This indicates that recF may work with recOR complex in some processes and individually in others. Thus, explaining the inconsistency of recF, recO and recR in the genomes of different bacteria.
  • recOR suppresses effects of recF overexpression, which means they have to be regulated systematically at each step in recombination for them to function together. If any of the above mentioned enzymes are missing or un-regulated, it could lead to the elimination of the pathway itself. RecA being a key player in the above pathway, its deletion leads to a recFOR and recBCD null phenotype.
  • the recE pathway is specifically only activated in recB and recC mutants by additional mutations in sbcA. These mutations lead to the activation of the recE gene, which encodes for a 96-kDa 5'-3' double strand exonuclease (Exo VIII) and recT which is a single strand DNA binding protein.
  • Exo VIII 96-kDa 5'-3' double strand exonuclease
  • recT which is a single strand DNA binding protein.
  • bacteria tend to display an adaptive characteristic when subjected to survival threatening situations. Such situations are similar to SOS response that results in the expression of various genes into the cytoplasm when triggered by external stress.
  • the present disclosure simulates such a situation in-vivo and exploits it to facilitate seamless recombination of fragments that results in custom plasmids in a recA null strain.
  • ET recombination and ligase independent recombination require pre-treatment with greater than 60 and greater than 24bp overhangs, respectively.
  • the present invention uses as less as 15bp or more of homology to get monomeric plasmids containing the gene of interest/fragments without any pre-treatment of DNA fragments ( Figure 1). Due to its flexibility, the instant disclosure demonstrates the use of this technique in generating customized plasmids of any gene or fragment desired.
  • the present disclosure involves an enzyme and vector free cloning method/strategy that results in custom plasmids harbouring desired gene of interest (GOI), wherein the DNA fragments are transformed directly into host systems without sub-cloning or restriction digestion. This facilitates cloning to expression in two days as compared to conventionally used strategies which are prolonged and error prone.
  • GOI desired gene of interest
  • the present disclosure relates to a method of obtaining vector from linear DNA fragment, said method comprising acts of - a) generating one or more linear DNA fragment with overhangs and optionally treating with Dpnl and amplifying the DNA fragment, b) transforming the said fragment into host cell to obtain transformed cell, and c) incubating said transformed cell for joining of the one or more DNA fragment and obtaining the vector.
  • the present disclosure also relates to a vector comprising one or more linear DNA fragment having overhangs, origin of replication and antibiotic marker, wherein the DNA fragments join to produce the vector.
  • the present disclosure relates to a method of obtaining transformed cell from linear DNA fragment, said method comprising acts of - a) transforming a host cell with the one or more linear DNA fragment having overhangs and b) incubating said cell for joining of the one or more DNA fragment, followed by antibiotic screening to obtain the transformed cell.
  • the present disclosure also relates to a transformed cell comprising one or more linear DNA fragment having overhangs, wherein fragments join to produce a vector within the cell.
  • FIG 1 depicts schematic representation of Vector and enzyme free Cloning (VEF-C).
  • FIG. 2 depicts customisation of plasmid with gene of interest (VEF-C).
  • Figure 3 depicts vector map for luciferase ( ⁇ 700bp) cloned into petl5b (5.3kb) in accordance with the present invention for 2 fragment joining.
  • Figure 4 depicts vector map for fragment A ( ⁇ 300bp) and vector backbone ( ⁇ 2.1Kb) in accordance with the present invention for 2 fragment joining.
  • Figure 5 depicts vector map for fragment A ( ⁇ 300bp), B(2Kb) and vector backbone ( ⁇ 2.1Kb) in accordance with the present invention for 3 fragment joining.
  • Figure 6 depicts vector Map of CKEM (product of ColEl, Kanamycin, eGFP, MBP) in accordance with the present invention for 4 fragment joining.
  • FIG. 7 depicts confirmation of SIM (template free).
  • Figure 8 depicts confirmation of SIM (Electron-microscopy).
  • the present disclosure relates to a method of obtaining vector from linear DNA fragment, said method comprising acts of:
  • the present disclosure also relates to a vector comprising one or more linear DNA fragment having overhangs, origin of replication and antibiotic marker, wherein the DNA fragments join to produce the vector.
  • the vector is produced by method as above.
  • the overhangs of the one or more DNA fragment comprise homologous sequence of atleast 15bp length.
  • the DNA fragment is generated and amplified by subjecting to PCR amplification; wherein the treating with Dpnl is carried out at temperature of about 37°C, for time duration ranging from about 1 hour to about 4 hours; and wherein the Dpnl treatment is followed by gel extraction using about 0.1 to about 3% Agarose; and PCR amplification.
  • the transformation is carried out by technique selected from group comprising heat-shock and electroporation or combination thereof.
  • the DNA fragment is linear and template-free with size ranging from about 100 bp to about 6000 Kb, and is selected from group comprising marker, antibiotic marker, origin of replication, fusion tag, affinity tag, gene of interest, promoter region, gene encoding signal sequence and regulatory region or any combinations thereof.
  • the host cell is recA-defective host cell. In yet another embodiment of the present disclosure, the host cell is E.coli.
  • the one or more DNA fragment join to result in circular and monomeric plasmid.
  • the method is applied to 2, 3 or 4 DNA fragments or combinations thereof, to result as the vector by applying external stress using bacterial antibiotic selected from group comprising kanamycin, ampicillin and chloramphenicol or any combinations thereof.
  • the transformed cell is incubated at temperature of about 37°C for time duration ranging from about 1 hour to about 1.5 hours.
  • the method is an enzyme and vector free method.
  • the DNA fragment to be generated includes selection marker or antibiotic marker and origin of replication.
  • the present disclosure relates to a method of obtaining transformed cell from linear DNA fragment, said method comprising acts of:
  • the present disclosure also relates to a transformed cell comprising one or more linear DNA fragment having overhangs, wherein fragments join to produce a vector within the cell.
  • the cell is produced by method as above.
  • the transformation is carried out by technique selected from group comprising heat-shock and electroporation or combination thereof.
  • the transformed cell is incubated at temperature of about 37°C for time duration ranging from about 1 hour to about 1.5 hours.
  • the method is an enzyme and vector free method.
  • the present disclosure involves an enzyme and vector free cloning strategy/method that results in vector/plasmids harbouring desired gene of interest (GOI).
  • the instant method gives flexibility to the user to employ desirable DNA fragments such as markers, promoters, kind of antibiotic marker, origins of replication, fusion tags, affinity tags, gene encoding signal sequence, regulatory region etc along with desired GOI, which is transformed directly into expression host systems without sub-cloning or restriction digestion (Figure 2). This facilitates cloning to expression in two days as compared to conventionally used strategies which are prolonged and error prone. Moreover, optimising protein expression is easier in such a strategy as different fragments are pooled to obtain various combinations. Upon expression of colonies, the highest expressing clone is identified and plasmid is isolated.
  • Another embodiment of the present disclosure is that inter and intra plasmid recombination is completely abolished, therefore no negative colonies are observed.
  • Yet another embodiment of the instant disclosure is the generation of vectors for various applications including other host systems like Yeast, Mammalian systems, cell-free expression systems etc.
  • recAl Upon closely observing the structure of recA and comparing it with recAl, the single strand binding ability of recAl still exists. Although it cannot bind to dsDNA like its wild type counterpart, it has been shown that complementary strand renaturation by Mg2+ mediated pathway is still possible in a recAl environment, thus explaining the 10,000-fold chance of obtaining recombination. This indicates that recombination is not completely abolished, but merely inhibited.
  • the present disclosure not only overcomes this 10,000-fold recombination event but also facilitates generation of customized plasmids based on user's choice without using any vectors or enzymes.
  • the cloning is also performed in an expression strain, shuttling of vectors or sub cloning is not required and protein expression is carried out, directly. This is time and cost effective as users can design/create a plasmid that meets their requirements.
  • the present disclosure involves a vector and enzyme free cloning (VEF-C) method/strategy that results in custom plasmids harbouring desired fragments of interest in recA-null strains of E.coli.
  • VEF-C vector and enzyme free cloning
  • This includes antibiotic resistant markers, promoters, origin of replication, fusion tags and thereof.
  • the gene of interest is transformed directly into expression host systems without sub-cloning or restriction digestion, the strategy is time and cost effective (Figure 2).
  • a survival- threatening situation is simulated in bacteria by creating a stress induced mechanism (SIM), which in turn adaptively expresses necessary proteins in its cytoplasm for facilitating 2, 3 and 4 fragment recombination. These proteins are exploited for enzyme free cloning of different fragments to result in circular monomelic and stably maintained plasmids/vectors harbouring the gene of interest.
  • SIM stress induced mechanism
  • the joining of two fragments containing homologous overhangs is performed in recA-null strain.
  • a gene of interest is PCR amplified using primers that 15-21bp overlaps with the plasmid. After amplification, both fragments are Dpnl treated and gel extracted. This product is further PCR amplified, if required and transformed into a recA-null strain of E.coli. Conventional protocol is followed for carrying out transformation, viz. heat- shock and electroporation.
  • the instant method facilitates 'cloning to expression' in two days as compared to conventionally used strategies, which are prolonged and error prone. Moreover, optimizing protein expression is easier in such a method as different fragments can be pooled to obtain various combinations.
  • the highest expressing clone is identified and plasmid isolated. Inter and intra plasmid recombination is completely abolished therefore no negative colonies are observed.
  • the plasmids/vectors generated in the instant method has application in the generation of vectors for other host systems like Other E.coli strains, Yeast, insect cells, mammalian systems, cell-free expression systems etc.
  • the instant methodology broadly employs the following acts:
  • the one or more DNA fragment with overhangs is generated in the instant method by PCR Amplification.
  • the nucleic acid molecules or DNA fragments are obtained by amplification and are isolated with gel extraction by Agarose Gel Permeation method using about 0.1 to about 3% Agarose at about 20 V to about 100V by electrophoresis.
  • the stability of vector/plasmids obtained by the instant method are observed to be consistent for more than one generation.
  • the instant method is followed to obtain vector/plasmid which has application in the generation of vectors for other host systems like E.coli strains, Yeast, insect cells, mammalian systems, cell-free expression systems etc.
  • the mechanism of recombination followed is other than that observed for recBCD, recFOR and recE pathways mediated by recA.
  • the invention involves a mechanism of recombination other than the known pathways in E.Coli (recBCD, recFOR and recE). Still further, the present invention is an economical and time effective alternative as against conventionally used cloning methods as well as existing LIC based systems.
  • Overhangs is generated by designing primers such that desired fragments are arranged as shown in figure. 2. Below illustration shows the design of the forward primer with a fragment's nucleotides (marked as X, 21-24 bp) and sequence of the flanking region (marked as N, 21-24 bp):
  • PCR reactions are performed in gradient to identify appropriate annealing temperatures.
  • a final concentration of IX PCR buffer, 200uM dNTPs, 0.5 ⁇ of forward and reverse primer each, 1-5% DMSO, 5-10 ng of template DNA, 0.25-1 unit of DNA polymerase are used and made upto 25 ⁇ with ultra-pure water. Any of these reagents are subjected to optimization, in case of non-amplified fragments. Company specific protocols are followed for generating fragments by PCR.
  • PCR clean-up is performed, which is clean up of target linear nucleic acid products and removal of non-specific products.
  • Dpnl treatment is performed by adding 1 unit in 25 ⁇ L PCR mix and samples are confirmed on a 0.2- 2% agarose gel. The Dpnl treatment is carried out at temperature of about 37°C, for time duration ranging from about 1 hour to about 4 hours.
  • Electroporation is employed with the same concentration of reagents, wherein voltage is optimized across a range of 1500-5000V, whereas capacitance is kept constant.
  • the cells are plated on LB agar containing respective antibiotic resistance (Kanamycin 25-30 ⁇ g/mL, ampicillin 40-50 ⁇ g/mL and chloramphenicol 10-15 ⁇ g/mL) and incubated at 37°C overnight (about 12-16 hours). Resulting colonies are picked, grown in LB with appropriate antibiotics and mini-prepped.
  • respective antibiotic resistance Kelamycin 25-30 ⁇ g/mL, ampicillin 40-50 ⁇ g/mL and chloramphenicol 10-15 ⁇ g/mL
  • Luciferase is cloned into pET15b such that they are not generated by conventional ligation methodology or any other means thereof.
  • the insert is cloned such that no other form of strategy other than the present invention can be employed to generate such a fragment as depicted in Figure 3.
  • Figure 4 is an illustration for a plasmid containing -300 bp gene of interest and ⁇ 2.1Kb vector backbone bearing ampicillin resistant marker is generated to demonstrate 2 fragments joining by employing present invention.
  • the combination and overhanging regions used to make the VEFC possible are novel.
  • Figure 5 is an illustration for a plasmid containing fragment A (-300 bp), fragment B (-2Kb) and ⁇ 2.1Kb vector backbone bearing ampicillin resistant marker is generated to demonstrate 3 fragments joining by employing present invention.
  • the combination and overhanging regions used to make the VEFC possible are novel.
  • a plasmid containing ColEl (origin of replication), eGFP (gene of interest), Maltose binding protein (fusion tag) and Kanamycin resistant marker is generated by the present invention.
  • ColEl oil of replication
  • eGFP gene of interest
  • Maltose binding protein fusion tag
  • Kanamycin resistant marker is generated by the present invention.
  • the combination and overhanging regions used to make the VEFC possible are non-existent but not sequence specific.

Landscapes

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

Abstract

La présente invention concerne un procédé de production de monomères circulaires de plasmides. Plus spécifiquement, la présente invention concerne la création de plasmides circulaires, sans implication d'aucune enzyme ni vecteur dans des souches à RecA délété (nulles). Ainsi, la présente invention élimine l'apparition de recombinaison inter- et intra-plasmide, qui est normalement observée dans les souches RecA1 et à une fréquence plus élevée dans les souches RecA. L'invention concerne en outre un mécanisme de recombinaison autre que les voies connues dans E. coli (recBCD, recFOR et recE). De surcroît, la présente invention représente une alternative efficace, économique en coût et en temps aux procédés de clonage habituellement utilisés, ainsi qu'aux systèmes basés sur LIC existants.
PCT/IB2015/054701 2014-06-24 2015-06-23 Procédé d'obtention d'un vecteur et cellule transformée contenant celui-ci WO2015198221A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IN6063/CHE/2013 2014-06-24
IN6063CH2013 2014-06-24

Publications (1)

Publication Number Publication Date
WO2015198221A1 true WO2015198221A1 (fr) 2015-12-30

Family

ID=54937460

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2015/054701 WO2015198221A1 (fr) 2014-06-24 2015-06-23 Procédé d'obtention d'un vecteur et cellule transformée contenant celui-ci

Country Status (1)

Country Link
WO (1) WO2015198221A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115881227A (zh) * 2022-12-28 2023-03-31 云舟生物科技(广州)股份有限公司 载体定制方法及计算机存储介质

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LI, C. ET AL.: "FastCloning: a highly simplified, purification-free, sequence-and ligation-independent PCR cloning method.", BMC BIOTECHNOLOGY, vol. 11, no. 1 : 92, 12 October 2011 (2011-10-12), XP021113264 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115881227A (zh) * 2022-12-28 2023-03-31 云舟生物科技(广州)股份有限公司 载体定制方法及计算机存储介质
CN115881227B (zh) * 2022-12-28 2024-01-26 云舟生物科技(广州)股份有限公司 载体定制方法及计算机存储介质

Similar Documents

Publication Publication Date Title
Hmelo et al. Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange
KR102271292B1 (ko) Rna-안내 게놈 편집의 특이성을 증가시키기 위한 rna-안내 foki 뉴클레아제(rfn)의 용도
US20170073663A1 (en) Targeted remodeling of prokaryotic genomes using crispr-nickases
CN106995813B (zh) 基因组大片段直接克隆和dna多分子组装新技术
WO2017043656A1 (fr) Procédé de conversion d'une séquence génomique d'une bactérie à gram positif par conversion spécifique d'une base d'acide nucléique d'une séquence d'adn ciblée, et complexe moléculaire utilisé dans celui-ci
Bidnenko et al. Termination factor Rho: from the control of pervasive transcription to cell fate determination in Bacillus subtilis
Karas et al. Cloning the Acholeplasma laidlawii PG-8A genome in Saccharomyces cerevisiae as a yeast centromeric plasmid
Blesa et al. The transjugation machinery of Thermus thermophilus: Identification of TdtA, an ATPase involved in DNA donation
Lang et al. General requirements for protein secretion by the F-like conjugation system R1
Qu et al. Group II intron inhibits conjugative relaxase expression in bacteria by mRNA targeting
Bruhn et al. Functionality of two origins of replication in Vibrio cholerae strains with a single chromosome
Grohmann et al. DNA-binding proteins regulating pIP501 transfer and replication
Park et al. Group II intron-like reverse transcriptases function in double-strand break repair
Zhang et al. A mechanism of transposon‐mediated directed mutation
CN116438302A (zh) 用于对货物核苷酸序列转位的***和方法
Agúndez et al. Site-specific integration of foreign DNA into minimal bacterial and human target sequences mediated by a conjugative relaxase
Wang et al. Strategies for gene disruptions and plasmid constructions in fission yeast
Lee et al. Linear plasmids mobilize linear but not circular chromosomes in Streptomyces: support for the ‘end first’model of conjugal transfer
WO2015198221A1 (fr) Procédé d'obtention d'un vecteur et cellule transformée contenant celui-ci
US20230183678A1 (en) In-cell continuous target-gene evolution, screening and selection
WO2004018635A2 (fr) Systeme de transformation base sur un gene d'integrase et un site de fixation pour le bacteriophage mx9 myxococcus xanthus
Whitworth et al. Genetic dissection of the light-inducible carQRS promoter region of Myxococcus xanthus
Becker et al. Relaxed specificity of the R1162 nickase: a model for evolution of a system for conjugative mobilization of plasmids
Chhakchhuak et al. Mechanism of action of bacterial transcription terminator rho
CA2492966C (fr) Methode d'expression d'adn environnemental inconnu dans des cellules hotes adaptees

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15811120

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15811120

Country of ref document: EP

Kind code of ref document: A1