WO2009106668A1 - Vecteurs et utilisations du transposon mboumar - Google Patents

Vecteurs et utilisations du transposon mboumar Download PDF

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WO2009106668A1
WO2009106668A1 PCT/ES2009/070044 ES2009070044W WO2009106668A1 WO 2009106668 A1 WO2009106668 A1 WO 2009106668A1 ES 2009070044 W ES2009070044 W ES 2009070044W WO 2009106668 A1 WO2009106668 A1 WO 2009106668A1
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recombinant peptide
seq
amino acid
acid sequence
polynucleotide
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PCT/ES2009/070044
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Spanish (es)
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Pedro LORITE MARTÍNEZ
Teresa Amalia PALOMEQUE MESSÍA
José Antonio CARRILLO ÁVILA
Martín MUÑOZ LÓPEZ
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Universidad De Jaén
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • 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
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome

Definitions

  • the present invention is included in the area of molecular biology, specifically within molecular genetics, and more specifically in the field of study of mobile genetic elements or transposons. It refers to the vectors necessary for the construction of genetic tools based on transposons, and their uses.
  • Transposons are DNA sequences that can move to different positions within the genome of a cell, a process called transposition. In general they are classified into two groups:
  • RNA is copied into the DNA by a reverse transcriptase (often also encoded by the transposon) and inserted from New in the genome.
  • transposons whose transposition mechanism does not imply RNA formation. They are generally transposed by a "cut and paste” process, using a transposase enzyme.
  • Transposases are the enzymes responsible for their mobility and are synthesized by the transposon itself. In vivo they can cause transposition of genetic material within the same chromosome, between chromosomes, and even between chromosomes and non-chromosomal genetic material, such as mitochondrial DNA. This movement of genetic material can cause serious disturbances in the organism in which it occurs, although it is also known that it has been of great importance throughout the evolution. At first the transposons were considered as "junk DNA". However, it is currently thought that they can influence the host genome in different ways. Thus, it is considered that they can modulate the gene expression by acting as promoters, activators, silencers, alternative processing sites or sites indicative of epigenetic modifications.
  • transposons can be active in somatic cells, opening the possibility that they can play an important role generating diversity between cells with the same genome. Specifically, it has been considered that this diversity could be generated by changes in the regulation of the expression of the transposase which, in turn, could affect the regulation of the gene expression of certain genes in certain cells.
  • the effects could be beneficial (as probably occurs in cases of differentiation of neuronal cells, or in the activity of RAG proteins, important in the recombination processes that occur in the genome of lymphocytes) or deleterious (carcinogenic processes) depending on the moment of development and cell type (Collier and Largaespada. Transposable elements and the dynamic somatic genome. Rewied 2007. Genome Biology 2007, 8 (Suppl 1: S5).
  • Maritime transposons are sequences capable of moving around the genome that are flanked by inverted terminal repeated sequences (inverted terminal repeats or ITRs). They encode a single protein, the transposase, which, joining these ITRs, cleaves the mariner and integrates it elsewhere in the genome.
  • the Mos1 transposase catalyzes the movement of the Mos1 transposon found in Drosophila maur ⁇ tania, which is not only the first isolated mariner element, but also the first naturally active mariner element isolated.
  • the mariner elements constitute a large family of transposons widely spread in the animal kingdom. They encode the enzyme transposase that is the only requirement for its transposition.
  • This enzyme has two domains: the N-terminal domain or DNA binding domain and the C-terminal domain or catalytic domain, which retains the DD (34) D motif.
  • DD DD
  • In vivo transposition reactions are carried out independently of the cellular or host conditions: Generally they are carried out with purified transposases, and plasmids, gene fragments, or DNA can be used as targets. genomic isolated. In successive reactions, plasmids containing transposons can be transferred to host cells.
  • transposases Since most of the transposases found are inactive, and given the large number of applications and interest that MLEs have in biotechnology, it is necessary to construct artificial transposases that are active, from the natural defects, for use as genetic tools.
  • the most commonly used procedure consists of, from defective copies of the transposases found in various organisms in a natural way, correct the mutations that inactivate the enzyme by means of directed mutagenesis. Therefore, finding a naturally active transposase, which facilitates its production as a recombinant protein, without the need to introduce modifications in the amino acid sequence, would be a notable advance in this sector of the technique.
  • the joint use of this transposase and the constructions made with the ITRs can constitute an in vitro transposition system that can be used in various experiments, such as the generation of random mutations.
  • Another possible application of this system is the transgenesis and mutagenesis randomly in the genome of prokaryotic or eukaryotic cells, by insertion between the Mboumar ITRs of the desired sequence.
  • the advantage of the use of vectors based on this type of transposable elements is their versatility, since they are not host-specific, since their only requirement is the presence of the transposase that they themselves produce.
  • a recombinant peptide hereinafter peptide of the invention, consisting essentially of the amino acid sequence SEQ ID NO: 1
  • SEQ ID NO: 1 collects the amino acid sequence of the transposase enzyme isolated from the Messorbouvier ⁇ species.
  • the global identity of the transposases homologous to Mboumar, at the amino acid level, and more specifically at the level of the amino acid sequence that is collected in SEQ ID NO: 1, is 70% or more, and more preferably 80% or more and more preferably 90, or 95% or more.
  • the correspondence between the amino acid sequence of the putative transposase (s) and the sequence of other transposases can be determined by methods known in the art. For example, those can be determined by a direct comparison of the amino acid sequence information from the putative homologous transposase, and the amino acid sequence that is collected in SEQ ID NO: 1 of this specification.
  • the amino acid sequence of the recombinant peptide has an identity of at least 70% with SEQ ID NO: 1, preferably at least 80%, more preferably at least 90% and even more preferably at least 95%.
  • the transposase activity includes its binding activity to the IRT and / or RDs elements ("Deleted regions"), the cleavage of the nucleic acid sequences between these IRT and / or RDs elements and its sequence insertion activity in A specific target
  • these peptides homologous to the Mboumar transposase have been modified to enhance the biological activity of said enzyme.
  • amino acids can be substituted in SEQ ID NO: 1 to enhance said activity.
  • the methods for producing such related derived sequences for example, by site-directed mutagenesis, random mutagenesis, or enzymatic cleavage and / or nucleic acid ligation, are well known in the art, as are the methods for determine if the nucleic acid thus modified has a significant homology with the sequence that is being considered, for example, by hybridization.
  • the recombinant peptide is homologous to the amino acid sequence of SEQ ID NO: 1.
  • the recombinant peptide of the invention is used as a transposase. In another aspect of the invention, the recombinant peptide of the invention is used as a genetic tool.
  • polynucleotide of the invention which is translated to the peptide encoded by SEQ ID NO: 1, and which is selected from the list comprising: a) nucleic acid molecules that encode a peptide comprising the amino acid sequence of SEQ ID NO: 1, b) nucleic acid molecules whose complementary chain hybridizes with the polynucleotide sequence of a), or c) nucleic acid molecules whose sequence differs of a) and / or b) due to the degeneracy of the genetic code.
  • the amino acid sequence to which the polynucleotide of the invention is translated has an identity of at least 60% with SEQ ID NO: 1.
  • the amino acid sequence to which the polynucleotide of the invention is translated it has an identity of at least 80%, more preferably, of 90% and more preferably of 95%, with SEQ ID NO: 1.
  • the polynucleotide of the invention is used to produce, in amounts not yet available, the peptide of Ia invention.
  • Another aspect of the invention relates to a genetic construction, henceforth, the first genetic construction of the invention or aid construction, which allows the expression of the peptide of the invention, as described above, in amounts so far not available Production in quantities greater than those currently available would allow the use of the peptide of the invention as a genetic tool.
  • Another aspect of the invention refers to a genetic construction, hereinafter, a second genetic construction of the invention or donor construction, which comprises the ITR regions recognized by the peptide of the invention, between which a polynucleotide of interest is inserted. Additionally, it may contain a marker, such as, but not limited to, an antibiotic resistance gene.
  • a method for obtaining a recombinant peptide of the invention which consists in placing the genetic construction described above in a suitable reaction medium.
  • This method includes the cloning and expression vectors that comprise the nucleic acid molecules of the invention.
  • expression vectors include suitable control sequences, such as, for example, control elements for translation (such as start and stop codes) and for transcription (for example, promoter-operator regions, binding sites.
  • the invention may include plasmids and viruses (comprising bacteriophages and eukaryotic viruses), in accordance with procedures well known and documented in the state of the art, and can be expressed in a variety of different expression systems, also well known and documented in the state of the art.
  • Suitable viral vectors include baculovirus and also adenovirus and vaccine viruses. Many other viral vectors are also described in the state of the art.
  • the transformed or transfected eukaryotic or prokaryotic host cells which contain a nucleic acid molecule according to the invention, as defined above, also form part of this aspect of the invention.
  • sequences (same or homologous) encoding the peptide of the invention, using a series of known techniques and expression systems, including the expression in prokaryotic cells such as E.coli and in eukaryotic cells such as yeasts or the system of baculovirus-insect cell or transformed mammalian cells and in transgenic animals and plants.
  • the fusion plasmid pMal-c2X (New England Biolabs) was used, together with the Escher ⁇ chia coli malE gene that encodes the binding protein to maltose (MBP: maltose-binding protein).
  • MBP maltose-binding protein
  • IPTG is going to express the Mboumar + MBP transposase fusion protein that will be purified thanks to the affinity that MBP has for amylose and maltose.
  • dual transposition systems have been developed. A dual system works by contacting at least two genetic constructs (which can be, for example, two plasmids), or by means of the co-transfection or introduction of these into the same cell: the donor construct and the aid construct.
  • the donor is a gene expression construct (promoter-gene-terminator) or a polynucleotide of interest, flanked by two ITRs (5 'ITR promoter-gene-terminator-3' ITR or 5 'ITR - polynucleotide of interest-marker - 3 'ITR or 5' ITR - polynucleotide of interest - 3 'ITR) and the aid Ia contains the Mboumar transposase under the control of a promoter. Additionally, the transposition can be carried out massively in vitro, producing the recombinant transposase exogenously, and putting it in contact, subsequently, with the donor construction.
  • the contact or the co-transfection of both constructions (donor and aid) in the same cell first causes the expression of the Mboumar transposase and then the Mboumar transposase cuts the donor construction by the 2 ITRs and glue all the construction in the genome of Ia host cell, or a third recipient genetic construct, which may be, for example, a plasmid (receptor plasmid).
  • the Mboumar-mediated transposition requires not only a complete and active Mboumar transposase in the aid construction, but also 2 binding sites for the transposase in the ITRs in the donor construction.
  • Another aspect of the invention relates to a method for transposition in vitro or in vivo that consists in contacting the recombinant peptide of the invention, with the genetic construction comprising the polynucleotide of interest.
  • the method for transposition in vitro or in vivo is used for the generation of random mutations.
  • the method for transposition in vitro or in vivo is used for the generation of transgenesis and random mutagenesis in the genome of prokaryotic or eukaryotic cells.
  • Another aspect of the invention relates to the use of the peptide of the invention for the generation of random mutations, both in vitro and in vivo. More preferably, the peptide of the invention is used for the generation of transgenesis and random mutagenesis in the genome of prokaryotic or eukaryotic cells, both in vitro and in vivo.
  • the function of certain genes could be identified, oncogenes identified and / or to introduce massive mutagenesis.
  • the first ITR constructs inserted in the genome in a first generation can be massively activated by the introduction of exogenous transposases. Inactivation of genes by insertional mutagenesis with ITR would result in obtaining some mutants with the alteration of the desired phenotype. In this process, the inactivated genes are also labeled with the ITRs and their sequences can then be recovered and the genes identified.
  • recombinant peptide comprising refers to the fact that the peptide includes the amino acid sequence SEQ ID NO: 1, which can be integrated with other amino acids and peptides, including fusion peptides, provided it retains its function as a transposase enzyme, and which is the subject of the present invention. It also refers to amino acid sequences that encode a homologous peptide that encodes SEQ ID NO: 1.
  • homologous refers to the similarity between two structures due to a common evolutionary ancestry, and more specifically, to the similarity between the amino acids of two or more proteins or amino acid sequences. Since two proteins are considered homologous if they have the same evolutionary origin or if they have similar function and structure, in general, it is assumed that higher values of similarity or identity of 30% indicate homologous structures. We can consider, therefore, that identity percentages of at least 80% will maintain the transposase function of said peptide, which is the subject of the present invention.
  • identity refers to the proportion of identical amino acids between two amino acid sequences that are compared. Sequence comparison methods are known in the state of the art, and include, but are not limited to, the GAG program, including GAP (Devereux et al., Nucleic Acids Research 12: 287 (1984) Genetics Computer Group University of Wisconsin, Madison, (Wl); BLASTP or BLASTN, and FASTA (Altschul et al., J. Mol. Biol. 215: 403-410 (1999). Additionally, the Smith Waterman algorithm should be used to determine the degree of identity of two sequences.
  • fragment or derivative of a peptide refers to fragments or peptides derived from the Mboumar transposase in their natural state that lack some or some amino acids, and that still act by transposing DNA. Alternatively, this term also refers to peptides derived from The natural Mboumar transposase, where one or more amino acids have been changed, eliminated, added, or less preferably, which has undergone investments or duplications. Such modifications are preferably made by recombinant DNA technology. Other modifications can also be made by chemical alterations of the transposase. The resulting peptide (or the fragments derived therefrom) can be produced recombinantly, and retain identical characteristics, or essentially identical to the natural Mboumar transposase.
  • a “recombinant peptide” as understood herein, is one that is obtained from the expression of a “recombinant polynucleotide”.
  • polynucleotide as used herein assumes a polynucleotide of genomic, cDNA, semi-synthetic, or synthetic origin which, by virtue of its origin or manipulation: (1) is not associated with the total or a portion of a polynucleotide with which is associated in nature, (2) is linked to a polynucleotide different from that to which it is linked in nature, or (3) does not occur in nature.
  • polynucleotide refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term only refers to the primary structure of the molecule. Thus, this term includes double or single stranded DNA, as well as double or single stranded RNA.
  • genetic tool refers to the tools used in genetic engineering for various applications, and encompasses tools of molecular genetics, quantitative genetics, population genetics, etc.
  • genetic tool refers to a tool useful for transposition in vitro, or for transgenesis and mutagenesis randomly, both in vitro and in vivo.
  • a "vector” is a replicon to which another polynucleotide segment has been attached, to perform the replication and / or expression of the bound segment.
  • a “replicon” is any genetic element that behaves as an autonomous unit of polynucleotide replication within a cell; that is, able to replicate under its own control.
  • Control sequence refers to polynucleotide sequences that are necessary to effect the expression of the coding sequences to which they are linked. The nature of such control sequences differs depending on the host organism; in prokaryotes, said control sequences generally include a promoter, a ribosomal binding site, and termination signals; in eukaryotes, generally, said control sequences include promoters, termination signals, enhancers and, sometimes, silencers. It is intended that the term “control sequences” includes, at a minimum, all components whose presence is necessary for expression, and may also include additional components whose presence is advantageous.
  • Operaationally linked refers to a juxtaposition in which the components thus described have a relationship that allows them to function in the intended way.
  • a control sequence "operatively linked" to a coding sequence is linked in such a way that the expression of the coding sequence is achieved under conditions compatible with the control sequences.
  • a “free reading frame” is a region of a polynucleotide sequence that encodes a polypeptide; This region may represent a portion of a coding sequence or a complete coding sequence.
  • a "coding sequence” is a polynucleotide sequence that is transcribed into mRNA and / or translated into a polypeptide when under control of appropriate regulatory sequences. The limits of the coding sequence are determined by a translation start codon at the 5 'end and a translation end codon at the 3' end.
  • a coding sequence may include, but is not limited to mRNA, cDNA, and recombinant polynucleotide sequences.
  • naturally Mboumar refers to the peptide or protein encoded by the nucleotide sequence called Mboumar-9, corresponding to SEQ ID NO: 1, cloned from the genome of the Messor ant genome bouvier ⁇ .
  • Mboumar-9 corresponding to SEQ ID NO: 1, cloned from the genome of the Messor ant genome bouvier ⁇ .
  • Mboumar means in this description, the protein resulting from the modification of the "natural Mboumar” protein, preferably to enhance the biological activity of said protein.
  • a “host” or “host cell” as used herein refers to an organism, cell or tissue that serves as a target or recipient of the transposable element to insert into themselves.
  • a host or host cell can also indicate a cell or host that expresses a recombinant protein of interest where the host cell is transformed with an expression vector containing the gene of interest.
  • transgenic is used in the context of the present invention to describe animals or plants in which a non-proprietary DNA sequence introduced by a mariner element (mariner-like element MLE) has been stably incorporated into its chromosomes of such that it can pass stably to successive generations of transgenic descendants.
  • the initial transgenic organism is known as the "founder.”
  • the "founder” animal must have the non-own DNA or transgene incorporated in all its cells or in a proportion sufficient for its progeny to establish by inheritance the transgenic.
  • the organism is referred to as a chimera.
  • the present invention also extends to animals that incorporate the transgene stable or directly into their chromosomes and which express the transgene in their somatic cells without passing the transgene to their offspring.
  • these animals would serve as genetic models, for, for example, but not limited to these uses, to identify and study the function of genes, detect oncogenes, obtain biopharmaceuticals, ... Similar models described by genetic constructs derived from transposons in mice and fish zebra.
  • Transgenesis is understood herein as the process of transferring non-own DNA to an organism, which in this way becomes known as "transgenic.”
  • FIG. 1 Plasmid pITR and plasmid pITR-Kn.
  • the pITR vector is obtained from the plasmid cloned with the Mboumar-9 pGEMT easy vector mariner (Promega) by the elimination of the ampicillin resistance gene (Ampr) and the origin of replication (ori).
  • the EcoRI targets of the pGEMT easy vector have also been removed.
  • Figure 3 Procedure for removing EcoRI targets from pGEMT easy vector. A- Digestion with E. coli and generation of protruding ends. B- Filling the protruding ends with Klenow and generating blunt ends. C- Ligating of the two ends with T4 ligase. Figure 4. Scheme of the in vitro transposition assay.
  • the in vitro production of the protein is carried out by introducing its coding region (ORF) into an expression vector.
  • ORF coding region
  • the fusion plasmid pMal-c2X New England Biolabs
  • the Mboumar ORF is introduced into the pMal-c2X vector downstream of the Escherichia coli malE gene encoding the maltose binding protein (MBP: maltose-binding protein).
  • MBP maltose binding protein
  • the Mboumar transposon ORF is amplified from the Mboumar-9 clone (Palomeque et al. 2006. Detection of a mariner-Wke element and a miniature inverted-repeat transposable element (MITE) associated with the heterochromatin from ants of the genus Messor and their possible involvement for satellite DNA evolution (Gene 371: 194-205) with two primers that incorporate two cutting targets for the restriction enzymes EcoRI and BamHI, which will allow it to be introduced in phase in the polylinker of pMal-c2X.
  • MITE inverted-repeat transposable element
  • the 100 ml of culture is introduced in a French press and the bacteria are subjected to a pressure of 1,000 Psi.
  • the lysate obtained is centrifuged at 15,000 rpm for 30 minutes.
  • the supernatant is passed through a column with 1 ml of amylase (previously washed with Buffer HSG) where the fusion protein will be retained. Once all the supernatant has passed through the amylose matrix, it is washed again with Buffer HSG.
  • the sequence of the Mboumar-9 transposase obtained from the DNA sequence of the Mboumar-9 mariner, is collected in SEQ ID NO: 1.
  • the Donor Plasmid will contain the sequence to be mobilized, which must be flanked by the ITRs of the Mboumar transposon.
  • the Receptor Plasmid can be any other where the insertion of the sequence located between the two ITRs provided by the donor plasmid can be detected.
  • the Mboumar transposase will recognize the ITRs that are located in the donor plasmid.
  • the transposase will make a cut at the ends of these ITRs and integrate this DNA fragment into a TA dinucleotide (which will undergo duplication during the process) of the recipient plasmid.
  • integration takes place at a site other than the donor plasmid itself. To avoid this inconvenience, the donor plasmid must be unable to replicate in the bacteria that will be transformed with the reaction of the transposition in vitro.
  • pITR which presents the ends of the mariner element and therefore its ITRs
  • pITR-Kn derived from the previous one, but to which the kanamycin resistance gene (Knr gene) has been incorporated ( Fig. 1).
  • the Mboumar-9 plasmid containing the Mboumar-9 mariner element cloned in the plasmid pGEMT easy vector (Promega) is used.
  • the first modification that is made on the plasmid is the elimination of the ampicillin resistance gene (Ampr) and the origin of replication (ori).
  • the plasmid has been digested with the Seal and Accl enzymes, thus releasing most of the Ampr gene and the ori. This fragment is replaced by the origin of R6K replication, which will allow this plasmid to replicate only in pir + bacteria, but not in pir- strains, which will be those transformed with the transposition reaction.
  • the plasmid is digested with the restriction enzymes Mscl and Ndel (partial digestion), and the ends generated in the vector are joined by ligation.
  • These enzymes will release the central region of the mariner leaving only 173 bp of the ends, in which the two ITRs are found.
  • a target sequence for the Apol enzyme is located, which can be used to insert a DNA sequence (Fig. 2).
  • this enzyme is also able to cut on the EcoRI targets found in the pGEM-T vector, on both sides of the insert, so it is necessary to eliminate them.
  • the plasmid is digested with this enzyme, generating two fragments, one with the insert corresponding to the mariner and the other with the vector.
  • This enzyme generates protruding ends that are filled with the help of the Klenow enzyme, thus generating fragments with blunt ends that are re-bound, thus degenerating the target for EcoRI (Fig. 3), leaving the Apol target between the two ITRs as a single target for this enzyme in the plasmid (Fig. 2)
  • Plasmid pITR-Kn is derived from the pITR to which the kanamycin resistance gene (Knr gene) has been inserted into the Apol target (Fig. 1)
  • any other that confers resistance to an antibiotic other than kanamycin and is capable of replicating in pyr- bacteria can be used.
  • the pGEMT Easy Vector (Promega) vector itself has been used.
  • Plasmid receptor (pGEM-T). - Purified Mboumar transposase.
  • Reaction buffer It is the buffer in which the necessary conditions are met for the transposition by the Mboumar transposase to be carried out.
  • Donor plasmid 9 ⁇ M.
  • This reaction is incubated at 28 0 C place overnight. During this period the transposase cleaves the Knr gene along with the ITRs that flank it and integrate it into TA dinucleotide of the recipient plasmid (or donor plasmid). The mobilization of this sequence to the recipient plasmid will generate a plasmid that will confer resistance to kanamycin and ampicillin, and that will be able to replicate in pyr- bacteria.
  • competent bacteria pir- (DH5 ⁇ ) are transformed with 8 ⁇ l of the transposition reaction and the negative control (without transposase).
  • the half of the volume of the transformation with the transposition reaction is seeded in LB plates with kanamycin and ampicillin, and the other half in plates with only ampicillin.
  • the same volume is the one that is going to be sown of the transformation with the negative control in an LB plate + ampicillin + kanamycin.
  • the efficiency with which the Mboumar transposase has carried out the transposition under the conditions described is 10 ⁇ 3 . This is calculated by dividing the number of Transformer Receptor Plasmids with the Kn R gene integrated by the total number of Transformer Receptor Plasmids.

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Abstract

L'invention concerne la production d'une transposase naturellement active de Messor bouvieri de forme recombinante, des constructions génétiques dérivées du transposon Mboumar nécessaires pour la transgénèse et la mutagénèse, ainsi que des utilisations de ces dernières.
PCT/ES2009/070044 2008-02-29 2009-02-25 Vecteurs et utilisations du transposon mboumar WO2009106668A1 (fr)

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Citations (2)

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WO2004078981A1 (fr) * 2003-01-28 2004-09-16 Centre National De La Recherche Scientifique (C.N.R.S.) TRANSPOSASES D'ELEMENTS GENETIQUES MOBILES mariner MUTANTES, NON PHOSPHORYLABLES ET HYPERACTIVES
WO2007063033A1 (fr) * 2005-11-30 2007-06-07 Centre National De La Recherche Scientifique (Cnrs) Procede de production chez les procaryotes de transposases actives et stables d'elements genetiques mobiles mariner

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