MXPA06009808A - Generation of recombinant genes in prokaryotic cells by using two extrachromosomal elements - Google Patents

Abstract

The present invention relates in general to methods for generating and detecting recombinant DNA sequences in prokaryotic cells, in particular bacteria, by using two different extrachromosomal elements and extrachromosomal elements, in particular plasmids that can be used for conducting the inventive methods. DNA sequences for which these methods are relevant include protein-encoding and non-coding sequences.

Description

GENERATION OF RECOMBINANT GENES IN PROCEDURAL CELLS USING TWO ELEMENTS EXTRACROMOSOMALES DESCRIPTIVE MEMORY The present invention relates in general to in vivo methods for generating and detecting recombinant DNA sequences in prokaryotic cells, in particular bacteria, using two different extrachromosomal elements and extrachromosomal elements, in particular, plasmids that can be used to perform the inventive methods. The DNA sequences for which these methods are relevant include sequences that encode and that do not encode proteins. Evolution is a continuous process of genetic variation and phenotypic selection. The genetic diversity of a population can be amplified by creating new mutant combinations that improve the performance of individuals within the population. The directed evolution of microorganisms has traditionally been achieved through the process of classical strain improvement, through mutagenesis and sequential random selection. The evolution directed so far has been used almost exclusively as a tool to design proteins. By mutation techniques such as site-directed mutagenesis, cassette mutagenesis, random mutagenesis and error-prone PCR, variants of the functions of the protein have been generated and libraries thus produced have been selected for their ability to perform a specific function. The recursive application of this procedure has been used successfully for the modification of physical and catalytic properties of enzymes such as optimal pH, thermotolerance, stability with the solvent, stereoselectivity, catalytic activity and substrate specificity, as well as mechanisms of resistance to toxicity in bacteria and the range of hosts and stability of the viruses. Traditional mutagenesis procedures to develop new properties in enzymes have several limitations. These methods are only applicable to genes or sequences that have been cloned and functionally characterized that have a discrete function. Also, traditional mutagenesis procedures can explore only a very limited number of the total number of permutations, even for a single gene. However, under certain circumstances, it may be necessary to modify not only one gene, but additional genes, in order to express a protein with new properties. Such additional genes may be, for example, that cooperatively confer a single phenotype or genes that have a role in one or more cellular mechanisms, such as transcription, translation, post-translational modifications, secretion or proteolytic degradation of a gene product. Trying to individually optimize all the genes that have such a function through traditional mutagenesis procedures would be a virtually impossible task. Most of the problems associated with conventional mutagenesis procedures can be overcome by recombination procedures that involve randomly recombining sequences of functional genes, allowing molecular mixing of naturally similar or randomly mutated genes. Compared with conventional mutagenesis with recombination, the probability of obtaining mutants with an improved phenotype is significantly higher. The main advantages of recombination procedures with respect to conventional DNA manipulation technologies are, in particular, the experimental simplicity and freedom from the limitations imposed by the DNA sequence. Much of what is known about recombination procedures has come from studies of simple, unicellular organisms, such as bacteria. The study of recombination in such organisms has the advantage of the ease of manipulation of the DNA sequences and the possibility of studying the specific recombination events induced synchronously in a large proportion of cells. Equally important is the growing conviction that the procedures studied in microorganisms are identical or similar in most aspects in the ways in which mammalian cells, for example, human cells, generate genetic diversity. In addition, the definition of these mechanisms has taken an added importance in the struggle to develop more efficient mechanisms of gene selection and gene replacement in mammalian cells. Although there are numerous different systems for effecting recombination in prokaryotic cells, most of these do not allow easy and reliable detection of newly recombined DNA sequences. Therefore, there is a demand in the art for efficient prokaryotic test systems, which in particular allow a rapid and simple detection of the recombinants and / or a selection of recombinants under selective pressure. Therefore, the technical problem underlying the present invention is to provide improved methods and means for a simple and efficient generation of recombinant mosaic genes in prokaryotic cells, in particular, to select and detect such recombinant sequences. The present invention solves this underlying technical problem by providing a method for generating and detecting recombinant DNA sequences in prokaryotes, comprising the steps of: a) generating a first prokaryotic cell containing a receptor DNA molecule, comprising a first sequence of DNA to be recombined, and which can be replicated autonomously in the prokaryotic cell, and a donor DNA molecule, which comprises a second DNA sequence to be recombined and at least one first marker sequence that encodes a gene product and that can not replicate autonomously in the prokaryotic cell, b) cultivate the first prokaryotic cell under selective conditions that only allow cell growth and / or propagation if the gene product of the first marker sequence is expressed, and c) isolate a second cell prokaryotic cultured and / or propagated under selective conditions and containing a hybrid DNA molecule with at least the first marker sequence and a first and second recombined DNA sequences due to recombination between the first and second DNA sequences. The present invention provides a prokaryotic system for selecting recombination events in at least two divergent or heterologous DNA sequences, or recombination substrates in vivo. The inventive system allows the generation of new advantageous DNA sequences, with improved properties in a fast and efficient way from at least two divergent DNA sequences, by a process that involves an in vivo exchange of DNA of two extrachromosomal elements that contain the two DNA sequences to be recombined. The two extrachromosomal elements that do not show homology in their nucleotide sequences are introduced into a prokaryotic host cell in the form of a receptor DNA molecule and a donor DNA molecule. The receptor molecule can replicate autonomously in the host, while the donor molecule does not have the capacity to replicate. However, the donor molecule contains at least one marker sequence that encodes a unique protein, such as a marker of resistance to an antibiotic or a nutritional marker, that is not present in the genome of the host cell or in the receptor molecule. After the introduction of both extrachromosomal elements into the prokaryotic host cell, the cell is cultured under conditions that require recombination between the two heterologous genes. These conditions may include, for example, a culture of the cell in the presence of an antibiotic to which the cell is normally sensitive or a culture of the cell in a medium lacking an essential nutrient, which the cell can not synthesize on its own. same, and therefore, has to be provided externally. Under the culture conditions applied, the cell can only grow and spread, that is, dividing, if the gene product of the respective marker sequence is expressed. A requirement for the expression of the marker sequence is, however, that the non-replicating donor molecule and the replicating receptor molecule form a cointegrate that can replicate from the origin of the receptor molecule, and therefore ensure sequence maintenance. marker The formation of this cointegrate results from the recombination between the two recombination substrates that lead to the generation of new DNA molecules, whose sequences differ from those of the original DNA sequences. The host cells cultured and propagated under the applied selective conditions therefore contain new recombined DNA sequences. The inventive method therefore provides an easy and quick screening system for identifying recombinant DNA sequences. With the inventive method, a large library of recombinant, mutated DNA sequences and variants that have acquired a desired function can be easily generated, which can be identified using an appropriate selection or screening system. The study of recombination procedures in a simple, unicellular organism, such as a prokaryote, has the obvious advantage of the ease of manipulation of the DNA sequences and the possibility of studying the specific recombination events, induced in a synchronous manner in a large proportion of cells. In addition, during the last decades a great experience has accumulated both in fermentation technology and in the basic genetics of prokaryotic organisms. Another major advantage of prokaryotic host cells is related to their very short duplication times. Thus, by using prokaryotic host cells, it is possible to make many rounds of cell division and therefore, many rounds of recombination and create a plurality of new recombinant DNA sequences over a short period of time. The inventive method can be performed either in wild-type or defective prokaryotic cells to repair mis-matching. The procedures by which the damaged DNA is repaired and the mechanisms of genetic recombination are intimately related; and it is known that the machinery for repairing the bad correspondence has inhibitory effects on the frequency of recombination between divergent sequences, that is, homologous recombination. Mutations of the mismatch repair system therefore improve, to a large extent, the total frequency of recombination events in prokaryotic cells. On the other hand, it is known that prokaryotic cells of the wild type have a recombination mechanism dependent on the repair of the bad correspondences, which is based on bad correspondences separated distally in two recombination substrates. Depending on the DNA sequences to be recombined, wild-type or defective prokaryotic cells can be used to repair the mismatches to obtain the recombined sequences. The inventive method for generating and detecting recombinant DNA sequences has the advantage that DNA sequences that diverge to a large extent can be combined. It has been unexpectedly found by the inventors, that sequences with a high degree of total divergence and that only share very few stretches of homology or identity, can recombine. An analysis of the recombined sequences revealed that the longest stretches of identity in which recombination occurred comprise only 18-22 nucleotides. Most of the recombination events occurred in homologous stretches with a length of 10-15 nucleotides. In some cases, recombination occurred in stretches with a length of only 4-5 nucleotides.

Therefore, the inventive method allows the recombination of DNA sequences derived from different prokaryotic species or different prokaryotic genera, in order to create recombinant DNA sequences with advantageous characteristics. The inventive method for generating and detecting sequences of Recombinant DNA, has the advantage that more than two divergent sequences can be recombined, so that two divergent sequences to be recombined can be preselected or unselected sequences. For example, a variety of divergent DNA sequences to a complete gene library can be inserted into the recipient DNA molecules as well as donors. Subsequently, the individual divergent DNA sequences can be casually recombined with themselves. It is also possible to insert a first set of divergent DNA sequences into a complete gene library only on the recipient DNA molecules and insert a second set of divergent DNA sequences into a complete gene library on the donor DNA molecules or vice versa, and then recombine the two sets casually one with another. In both cases, there is no selection of which pair of divergent DNA sequences will recombine. However, it is also possible to recombine several divergent, preferably pre-selected, sequences in a step-wise manner, whereby in each step a selection is made of which pair of the divergent DNA sequences will be recombined. If, for example, three divergent DNA sequences must be recombined, then in the first step, first prokaryotic cells are generated, where, for example, a DNA receptor molecule with a first DNA sequence to be recombined and a DNA molecule donor with a second DNA sequence to be recombined, they are introduced into bacterial cells of given species. These prokaryotic cells are cultured under selective conditions, so that only the cells are cultured and propagated, in which the donor and recipient DNA molecules have formed a hybrid molecule that allows the expression of the marker sequence of the donor molecule and the recombination between the two DNA sequences to be recombined. Thus, the second prokaryotic cells are obtained, which contain a hybrid molecule with a first and second recombined DNA sequences due to recombination. The first and second recombined sequences are isolated, and one of them is inserted, for example, into a receptor molecule, while the third DNA sequence to be recombined is inserted into the donor molecule. Next, in the next step, the receptor molecule comprising one of the recombined sequences and the donor molecule comprising the third DNA sequence to be recombined, is again introduced into a prokaryotic host cell and subjected to another round of recombination. If, for example, four divergent DNA sequences must be recombined, then in the first step, two different sets of first prokaryotic cells are generated. For example, a first set of first prokaryotic cells can be generated by introducing a receptor DNA molecule with a first DNA sequence to be recombined and a donor DNA molecule with a second DNA sequence to be recombined into prokaryotic cells. Likewise, a second set of first prokaryotic cells can be generated by introducing a receptor DNA molecule with a third DNA sequence to be recombined and a donor DNA molecule with a fourth DNA sequence to be recombined, in the cells thereof. species. Each set of prokaryotic cells is cultured under selective conditions to effect recombination. Thus, a first set of prokaryotic cells containing a hybrid molecule with a first and second recombined DNA sequences is obtained due to recombination, between the first and second DNA sequences to be recombined. Also, another set of prokaryotic cells containing a hybrid molecule with a third and fourth recombined DNA sequences due to recombination, between the third and fourth DNA sequences to be recombined. Next, the first, second, third and fourth recombined DNA sequences thus obtained are isolated from their respective host cells. In the next step, the first or second recombined sequence can be inserted into a donor DNA molecule, and the third or fourth sequence recombined into a recipient DNA molecule. Both the donor and recipient DNA molecules thus obtained are then introduced into a prokaryotic host cell and subjected to another round of recombination. In this way, five, six or more divergent DNA sequences can also be recombined. In a preferred embodiment of the invention, the donor DNA molecule and the recipient DNA molecules are different linear or circular DNA structures. The term "DNA structure" means a DNA molecule, for example, a vector such as a plasmid or a bacteriophage, which is characterized in that it is present after production in the prokaryotic host cell, in the form of an extrachromosomal element. In the context of the present invention, an "extrachromosomal element" is therefore a DNA molecule that does not integrate into the chromosomes of the prokaryotic host cell. The donor DNA molecule must be capable of hybridizing to the receptor DNA molecule, so that a hybrid molecule can be formed. Preferably, the donor DNA molecule and the recipient DNA molecule do not share homology in general, with the exception of the DNA sequences to be recombined. According to the invention, the receptor DNA molecule must be able to replicate autonomously in a prokaryotic host cell, after introduction into that cell. Therefore, the receptor DNA molecule must have at least one origin of replication that allows the receptor DNA molecule to replicate independently of the genetic material of the host. In the context of the present invention, an "origin of replication" or "ori" is the region of a DNA molecule that is used by cellular enzymes to initiate the replication of the DNA molecule. At the origin, the two strands of DNA pull together to form a bubble of replication, which creates a region of single-stranded DNA on each side of the bubble. The DNA polymerase machinery can move inward and begin to synthesize the new strands of DNA, using the anterior strands as a template. Small DNAs that include bacterial or bacteriophage plasmids, usually have a single origin. In one embodiment of the invention, the receptor DNA molecule is a plasmid, in particular a double-stranded circular DNA molecule. A "plasmid" is an extrachromosomal element that can be replicated independently of the genetic material of the host. In another embodiment of the invention, the receptor DNA molecule is a bacteriophage, in particular a phage, the DNA of which is present in the prokaryotic cell in the form of an extrachromosomal element. In a preferred embodiment of the invention, the receptor DNA molecule is a plasmid, which can be replicated in a host cell of E. coli. Preferably, the receptor DNA molecule is the pACYC184 plasmid of E. coli or a derivative thereof. Plasmid pACYC184 is TetR and CamR In accordance with this invention, the donor DNA molecule is a DNA molecule that can not replicate in the prokaryotic host cell, but is capable of forming a hybrid molecule with the receptor DNA. Therefore, any DNA molecule can be used as a donor molecule, provided that it contains at least one marker sequence and can not be replicated independently within a given prokaryotic host cell. Examples of a suitable donor molecule comprise, in a non-exclusive manner, linear double-stranded DNA, which can circulate, generated, for example, by PCR and containing an appropriate marker sequence, plasmids and bacteriophages. In the case that a plasmid or a bacteriophage is used as the donor DNA molecule, this molecule does not have a functional origin of replication that is in the non-functional, non-active prokaryotic host cells used. In one embodiment of the invention, therefore, the donor DNA molecule does not contain an origin of replication. This means, that the donor DNA molecule does not contain any sequence that can function in a prokaryotic host cell as an origin of replication, i.e., sequences to which the factors of the proteins involved in the initiation of replication can bind. The absence of an origin of replication may be due to the suppression of those nucleic acid sequences that function as the origin. In another embodiment of the invention, the function of the origin of replication of the donor DNA molecule is diminished by one or more mutations that abolish the function of the replication origin itself or the function of the proteins involved in replication, in particular those proteins that bind to the nucleic acid sequence of the origin, so replication starts. For example, it is known that in E. coli, the key protein for the initiation of replication, ADNA, binds to specific sequences, the so-called ADNA sequences, in the chromosomal origin of replication and fuses three direct repeats of 13 mer-rich AT. It is believed that the additional binding of the ADNA protein to the 6-mer single-stranded sequences in the open region stabilizes the open complex (for a review, see Jiang et al., PNAS, 100 (2003), 8692-8697 ). ADNA sequences and AT-rich regions are commonly found at the origin of a variety of prokaryotic replicons, and the ADNA protein has been shown to play a key role in the initiation of replication at these origins. Therefore, certain mutations of the ADNA sequences and / or AT-rich regions or corresponding sites in the sequences of the origin of replication, such as substitutions, deletions, etc., of the appropriate base, can prevent binding of the proteins required for the initiation of replication, and thus inhibit the function of the origin of an extrachromosomal element. In a preferred embodiment of the invention, therefore, the function of the origin of replication of the donor DNA molecule can be decreased by one or more mutations in the nucleic acid sequence of the origin of replication of the donor DNA molecule, in particular , in the ADNA sequences and / or the AT rich regions of the origin. Furthermore, it has been shown that the ADNA protein alone is not sufficient for the formation of an open complex at the origin of the plasmids such as RK2, P1, F, pSC101 or R6K. In these cases, efficient formation of an open complex requires the binding of the plasmid Rep protein, albeit in concert with the host ADNA protein and the HU or IHF protein (integrated host factor). The requirement of a plasmid-specific initiation protein for the formation of the open complex is key to the ability of the plasmid to exert control over the frequency of the start events at its origin of replication (for a review, see Jiang et al., PNAS, 100 (2003), 8692-8697). This means that mutations of the plasmid nucleic acid sequences encoding the protein factors with essential functions in the replication of the plasmid can also decrease the function of the origin of replication of an extrachromosomal element. In another preferred embodiment of the invention, therefore, the function of the origin of replication of the donor DNA molecule can be decreased by one or more mutations in the nucleic acid sequences encoding the protein factors with essential functions in the replication of the donor DNA molecule. In yet another preferred embodiment of the invention, the donor DNA molecule contains an origin of replication that is active only in certain prokaryotic host cells, but not in that prokaryotic cell in which the donor DNA molecule is introduced, in order to effect the recombination between the two DNA sequences to be recombined. There are numerous reports that an origin of replication that is active in particular bacterial species does not work in other bacterial species. For example, it is known that the origin of replication of Klebsiella pneumoniae (oriC) is not active in Caulobacter crescentus, Pseudomonas putida or Rhodobacter sphaeroides (O'Neill and Bender, J. Bacteriol., 170 (1988), 3774-3777). It is also known that Bacillus subtilis plasmids can not replicate in E. coli cells. In a preferred embodiment thereof, the donor DNA molecule and / or its origin of replication are derived from prokaryotic species other than the prokaryotic species in the cells in which the donor DNA molecule is introduced. In a particularly preferred embodiment, the donor DNA molecule is the plasmid pMIX91 of B. subtilis, which is a derivative of plasmid plL253, and which can be replicated in B. subtilis, but not in E. coli, and which comprises the specR marker. , the phleoR marker and the restriction sites Seal, Ppt / MI and EcoO109l to insert a foreign DNA sequence. In another particularly preferred embodiment, the donor DNA molecule is the plasmid pMIX101 of B. subtilis, which is a derivative of plasmid plL253, and which can be replicated in β. subtilis, but not in E. coli, and comprising the tcR marker sequence and the Xhol and PstI restriction sites to insert a foreign DNA sequence. In another embodiment of the invention, the origin of replication of the donor DNA molecule is functional only at a certain temperature range, for example, at a temperature of less than 45 ° C. If the donor DNA molecule contains such a temperature-sensitive origin, it will not be able to replicate under impermissible conditions, that is, at a temperature above 45 ° C, which still allows the growth of the prokaryotic host cell. In the context of the present invention, the term "marker sequences" refers to DNA sequences that are unique in a given prokaryotic cell, and that are placed in the donor DNA molecule or the receptor molecule, preferably, current up or downstream of a recombination substrate or an already recombined DNA sequence. The presence of a marker sequence of the same DNA molecule as the recombination substrate or the already recombined DNA sequence, preferably in combination with another marker sequence, which can be placed on another side of the recombination substrate, allows the substrate of recombination or the DNA sequence already recombined are recognized and selected by molecular or genetic methods. According to the invention, the donor DNA molecule comprises a first marker sequence that allows the selection of crosses involving the recombination substrates. The first marker sequence, preferably in combination with the additional marker sequences present in the donor DNA molecule or the recipient DNA molecule, they also allow for additional rounds of recombination to be carried out in an iterative manner. According to the invention, the "first marker sequence" is a DNA sequence encoding a protein, the gene product of which is essential for the prokaryotic host cell to grow and propagate under the selective conditions applied. The first marker sequence is selected from the group consisting of a nutritional marker, a marker of resistance to the antibiotic and a sequence encoding a subunit of a functioning enzyme only, if both or more subunits are expressed in the same cell. A "nutritional marker" is a marker sequence that encodes a gene product that can compensate for an autoxotrophy of an organism or cell and, therefore, can confer prototrophy on that organism or autotrophic cell. In the context of the present invention, the term "auxotrophy" means that an organism or cell must grow in a medium that contains an essential nutrient that can not be synthesized by the autotrophic organism itself. The gene product of the nutritive marker gene promotes the synthesis of this missing essential nutrient in the autotrophic cell. Therefore, after expression of the nutritive marker gene, it is not necessary to add this essential nutrient to the medium in which the organism or cell is cultured, since the organism or cell has the acquired prototrophy. A "marker for resistance to an antibiotic" is a marker gene wherein the gene product confers, upon expression to a cell in which the expression of the antibiotic marker gene takes place, the ability to grow in the presence of an antibiotic. given at a given concentration, while a cell without the marker for resistance to an antibiotic can not.

A "sequence encoding a subunit of an enzyme" can be used as a marker sequence, if the cell can not synthesize all the subunits of an enzyme that are required for assembly of the complete structure of the enzyme and thus obtain the complete activity of the enzyme, and whether the presence or absence of enzymatic activity can be verified by genetic and / or molecular means. If, for example, the activity of an enzyme is needed for an essential biochemical trajectory of the cell, which allows the growth and / or propagation of the cell in a particular medium, and the cell can not synthesize all the components of the complete structure of the enzyme, then the cell can not survive in that medium. The "sequence encoding a subunit of an enzyme" used as the marker sequence therefore allows, after expression, the assembly of the complete enzyme and the survival of the cell. Preferably, the gene product of the first marker sequence confers resistance to an antibiotic to a cell, which is sensitive to that antibiotic. In particular, the first marker sequence is specR, the gene product of which gives a cell resistance to spectinomycin, phleoR, the gene product of which gives the cell resistance to phleomycin or tcR, the gene product of which it confers the cell resistance to tetracycline. In another embodiment of the invention, the donor DNA molecule contains a second marker sequence. In yet another embodiment of the invention, the receptor DNA molecule contains a third marker sequence and optionally a fourth marker sequence. This means, in one embodiment of the invention, that the donor DNA molecule can comprise at least one first and second marker sequences, optionally, even more marker sequences, which can be arranged, for example, either upstream or downstream of the substrate of recombination. In another embodiment, the receptor DNA sequence can comprise a third and fourth marker sequences, optionally, even more marker sequences, which can be arranged, for example, upstream or downstream of the recombination substrate. These additional markers allow to increase the rigor of the selection of the recombined DNA sequences, since the hybrid molecule formed in the second prokaryotic cell and comprising the two different recombined DNA sequences, can contain at least four different different marker sequences. According to the invention, the "second, third and fourth marker sequences" are sequences that encode or do not encode a protein selected from the group consisting of nutritive markers, pigment markers, markers for antibiotic resistance, markers for sensitivity to an antibiotic, primer recognition sites, intron / exon boundaries, sequences encoding a particular subunit of an enzyme, promoter sequences, downstream gene sequences regulated and restriction enzyme sites.

A "pigment marker" is a marker gene, wherein the gene product is involved in the synthesis of a pigment, which, after expression, will stain the cell, in which the pigment marker is expressed. A cell without the pigment marker does not synthesize the pigment and therefore is not stained. The pigment marker therefore allows rapid phenotypic detection of that cell containing the pigment marker. A "marker for antibiotic sensitivity" is a marker gene, wherein the gene product destroys upon expression, the ability of a cell to grow in the presence of a cell of a given antibiotic at a given concentration. The "primer recognition sites" are annealing sites for site-specific PCR primers, which allow rapid identification of the respective marker sequences by PCR. In a preferred embodiment of the invention, the second marker sequence of the donor DNA molecule and the third and fourth marker sequences of the receptor DNA molecule, are sequences that encode the protein, the gene products of which confer resistance to an antibiotic to a cell that is sensitive to that antibiotic Preferably, the gene product of the third marker sequence confers to a cell resistance to tetracycline and the gene product of the fourth marker sequence confers to a cell resistance to chloramphenicol. In the context of the present invention, the terms "DNA sequences to be recombined" and "recombination substrate" mean any two DNA sequences that can be combined as a result of homologous or non-homologous recombination procedures. Homologous recombination events of various types are characterized by base pairing of a DNA strand damaged with a homologous partner, where the degree of interaction may involve hundreds of base pairs paired almost perfectly. In contrast, illegitimate or non-homologous recombination is characterized by the junction of DNA ends that share none or only a few complementary base pairs. In prokaryotic cells, non-homologous repair and recombination events occur at significantly lower frequencies than homologous recombination events. In a preferred embodiment of the invention, the two recombination substrates, i.e., the first and second DNA sequences to be recombined, are divergent sequences, i.e., sequences that are not identical, but show a certain degree of homology. In the context of the invention, the term "homology" denotes the degree of identity that exists between the sequences of two nucleic acid molecules, while "divergence" denotes the degree of non-identity between the sequences of two nucleic acid molecules . According to the invention, the DNA sequences to be recombined differ from each other in two or more positions with respect to their general alignment. In one embodiment of the invention, the total divergence between two recombination substrates, relative to their total length, is more than 0.1%, in particular more than 5%, and preferably more than 25%. This means that the DNA sequences to be recombined can differ by more than 30%, by more than 40% and even by more than 50%. In a particularly preferred embodiment of the invention, the two DNA sequences to be recombined share at least one or more homologous or identical regions, which, however, can be very short. According to the invention, the homologous or identical regions may comprise less than 25 nucleotides, in particular, less than 20 or less than 15 nucleotides, and even less than 10 nucleotides, for example 4, 5, 6, 7, 8 or 9 nucleotides. The recombination substrates or the DNA sequences to be recombined may have a natural or synthetic origin. Therefore, in one embodiment of the invention, the first and second DNA sequences to be recombined, are natural sequences. The natural sequences can be isolated from any natural source, including viruses, living or dead prokaryotic organisms, such as bacteria, living or dead eukaryotic organisms, such as fungi, animals, plants and humans, or parts thereof by any appropriate isolation methods. or they can be synthesized by chemical means. The natural sequences can also comprise such sequences that after isolation from a natural source were subjected to mutagenesis. In another embodiment of the invention, the first and second DNA sequences to be recombined are artificial sequences that are not found in a natural source. The artificial sequences can be synthesized by any known chemical methods. In a preferred embodiment of the invention, the DNA sequences to be recombined are sequences that encode a protein, for example, sequences that encode enzymes that can be used for the industrial production of natural or non-natural compounds. Enzymes or those compounds produced by the help of enzymes can be used for the production of drugs, cosmetics, food, etc. The sequences that encode a protein may also be sequences that encode proteins that have therapeutic applications in the fields of human and animal health. Important classes of medically important proteins include cytokines and growth factors. The recombination of the sequences encoding the protein allows the generation of new mutated sequences that encode proteins with altered functions, preferably improved and / or recently acquired functions. In this way it is possible, for example, to achieve improvements in the thermostability of a protein, change the substrate specificity of a protein, improve its activity, develop new catalytic sites and / or merge domains of two different enzymes. The DNA sequences that encode a protein to be recombined may include sequences from different species that encode the same proteins or the like, which have similar or identical functions in their natural context. The DNA sequences that encode a protein to be recombined can include sequences from the same family of proteins or enzymes. The sequences that encode the protein to be recombined can be sequences that encode proteins with different functions, for example, sequences that encode enzymes that catalyze different steps of a given metabolic path. In a preferred embodiment of the invention, the first and second DNA sequences to be recombined are selected from the group of gene sequences of the Oxa superfamily of Beta lactamases. In another preferred embodiment of the invention, the DNA sequences to be recombined are non-coding sequences such as sequences that, for example, are involved within the natural cellular context in the regulation of the expression of a sequence encoding a protein. Examples of non-coding sequences include, but are not limited to, promoter sequences, sequences that contain ribosome binding sites, intron sequences, polyadenylation sequences, etc. By recombining such non-coding sequences, it is possible to develop mutated sequences, which in a cell medium result in an altered regulation of a cellular process, for example, an altered expression of a gene. According to the invention, a recombination substrate or DNA sequence to be recombined can, of course, comprise more than one sequence encoding a protein and / or more than one non-coding sequence. For example, a recombination substrate may comprise a sequence encoding a protein plus a non-coding sequence or a combination of different sequences encoding a protein and different non-coding sequences. In another embodiment of the invention, the DNA sequences to be recombined can therefore consist of one or more sections of coding sequences with intervening and / or flanking non-coding sequences. This means, that the DNA sequence to be recombined can be, for example, a gene sequence with regulatory sequences at its 5 'terminus and / or the 3' untranslated region or a mammalian gene sequence with an exon / intron structure . In yet another embodiment of the invention, the DNA sequences to be recombined can consist of larger stretches of DNA that contain more than one unique coding sequence, optionally with intervening non-coding sequences, such as an operon. The DNA sequences to be recombined can be sequences that have already undergone one or more recombination events, for example homologous and / or non-homologous recombination events. Recombination substrates can comprise unmutated wild-type DNA sequences and / or mutated DNA sequences. In a preferred embodiment thereof, it is possible to recombine wild type sequences with already existing mutated sequences, in order to develop new mutated sequences. According to the invention, a prokaryotic cell is used as a host cell to introduce the donor and donor DNA molecules. The terms "prokaryotic cell" and "prokaryotic host cell" can include any cell, in which the genome is freely present within the cytoplasm as a circular structure, i.e., a cell, in which the genome is not surrounded by a membrane nuclear. A prokaryotic cell is also characterized in that it does not necessarily depend on oxygen and its ribosomes are smaller than those of eukaryotic cells. Prokaryotic cells include archabacteria and eubacteria. Depending on the composition of the cell wall, eubacteria can be divided into gram-positive bacteria, gram-negative bacteria and cyanobacteria. Therefore, according to the present invention, the prokaryotic cell is a cell of an archabacterium or a eubacterium, wherein in a preferred embodiment of the invention, the prokaryotic cell is a gram-negative bacterium, a gram-positive bacterium or a cyanobacterium. . Preferably, the gram negative bacterium is Escherichia coli, for example E. coli strain AB1157 or its MutS mutant, the E. coli strain MXP1, In accordance with the invention, it may be preferred to use prokaryotic host cells for the inventive procedure, which have a functional repair system.The bad correspondence repair (MMR) systems, is one of the largest contributors to avoid mutations due to DNA polymerase errors of replication. the repair of mismatches also fosters genetic stability by ensuring the fidelity of genetic recombination, whereas in bacteria and also in yeast and mammalian cells, recombination between homologous DNA substrates that contain few bad correspondences (<; 1%), occurs much less efficiently than between identical sequences, the frequency of recombination (conversion and / or gene crossings), rises dramatically in the defective lines of the MMR. This means that the high fidelity of the recombination is not only caused by the intrinsic properties of the recombination enzymes, but also by the editing of the recombination by the repair system of the bad correspondences. Thus, the machinery of repair of the bad correspondences has an inhibiting effect on the recombination between the divergent sequence. In E. coli, two proteins of the MMR system directed to methyl, namely MutS and MutL, are required for this strong antirecombination activity, while the effect of the other proteins of the MMR system, MutH and UvrD, is less pronounced. In addition to those roles in MMR and homologous recombination, MMR proteins also play an important role in eliminating non-homologous DNA during gene conversion. In another preferred embodiment of the invention, prokaryotic cells are used that are deficient in the repair system of the bad correspondences. In the context of the present invention, the term "poor in the repair system of bad correspondences" means that the MMR system of a prokaryotic cell is damaged in a transient or permanent manner. MMR deficiency of a cell or organism can be achieved by any strategy that transiently or permanently damages the MMR system, including, but not limited to, a mutation of one or more genes involved in MMR, treatment with an agent such as UV light, which results in an overall decrease of MMR, treatment with an agent such as 2-aminopurine or a heteroduplex, which contains an excessive amount of poor correspondences to transiently saturate and inactivate the MMR system, and the expression or inducible repression of one or more genes involved in MMR, for example, via adjustable promoters, which would allow transient inactivation. In a preferred embodiment of the invention, the deficiency of the ratio of the bad correspondences of the prokaryotic host cell is due to a mutation of at least one of the genes involved in the MMR. In a preferred embodiment, the prokaryotic cells have a mutated mutS gene, a mutated mutL gene, a mutated mutant gene and / or a mutated UvnD gene. In another embodiment, the prokaryotic host cell is damaged or impeded in one or more of the major recombination proteins. It has been shown that a damaged cell, for example, in the AddAB genes shows a higher frequency of homologous and non-homologous recombination. In another embodiment, the prokaryotic host cell overexpresses one of the major recombination proteins, such as recA. This protein is involved in homologous recombination promoting the renaturation of single-stranded DNA to form the heteroduplex molecule required for the recombination event and initiates the exchange of the DNA strands.

According to the invention, the first prokaryotic cell is generated simultaneously or sequentially, by introducing the receptor DNA molecule and the recipient donor molecule into the prokaryotic host cell. In one embodiment of the invention, therefore, the first prokaryotic cell can be generated by introducing in a first step the receptor DNA molecule into a particular prokaryotic host cell. After recovery of the prokaryotic host cell harboring the receptor molecule, the donor DNA molecule is introduced into a second step in the cell harboring the receptor DNA molecule. In another embodiment of the invention, both receptor and donor DNA molecules can be introduced simultaneously into the prokaryotic host cell. According to the invention, the introduction of both receptor and donor DNA molecules into the prokaryotic host cell can be effected by any suitable known method, including, but not limited to, transformation, conjugation, transduction, sexduction, infection and / or electroporation. . In the context of the present invention, the term "transformation" means the uptake of an isolated nucleic acid molecule, preferably purified, from the medium by a cell, for example, by a microbial cell. The cells of some prokaryotic species such as Bacillus or Diplococcus are naturally competent, whereas the cells of other prokaryotic species such as E. coli, have to undergo special treatments in order to make them competent, ie to induce the transfer of the nucleic acid molecule through the cell membrane. Several bacteria are known for their ability to exchange DNA through transformation. "Conjugation" means the transfer mediated by the plasmid of a bacterial plasmid from a bacterial cell in another bacterial cell through cell-to-cell contact. The transfer machinery involved is usually encoded by plasmids or conjugative transposons. Examples of such plasmids are conjugative plasmids or cooperating plasmids. Conjugative plasmids are self-transmissible plasmids carrying genes that promote cell-to-cell contact (mobilization genes). They contain the genes to generate conjugation bridges. Once a bridge is made, other plasmids and even chromosomal DNA (conjugative transposons) can also be transferred. The mobilizable plasmids contain mobilization genes, but they need the "help" of the conjugative plasmids to move between the cells. The conjugation is one of the main routes of genetic exchange between different phylogenetic groups of prokaryotic cells and between prokaryotes and eukaryotes. "Sexduction" is a procedure whereby the genetic material is transferred from a prokaryotic cell with an F factor or a plasmid F to a cell that does not contain that F factor (F cell). Factor F is usually present in the cytoplasm of a bacterial cell, but occasionally it can be incorporated into several sites of the bacterial chromosome, which lead to the formation of Hfr cells. The integration of factor F is reversible, so that after an incorrect disintegration of the plasmid, so-called F-substituted factors (F ') arise, which may contain genetic material that flanks the original F-factor integration site on the bacterial chromosome. F 'plasmid can be transferred with high frequency in F cells. "" Transduction "means the transfer of a nucleic acid molecule from a bacterial cell in another bacterial cell by a bacteriophage, comprising the release of a bacteriophage from a cell, and the subsequent infection of another cell There are two types of transduction: specialized transduction may occur during the lysogenic life cycle of a temperate bacteriophage, where the genetic material of the bacterium can replace a part of the bacteriophage genome. This piece of bacterial DNA replicates as a part of the phage genome, and can be transferred by the phage to another recipient cell. In the case of a generalized transduction, the whole genome of a lytic phage can be replaced by bacterial DNA. When this phage infects other bacteria, it injects the DNA into the receptor, where it can be exchanged for a piece of DNA from the recipient cell. "Electroporation" is a procedure in which cells are mixed with nucleic acid molecules and then briefly exposed to high voltage electrical impulses. The cell membrane of the host cell becomes penetrable, thereby allowing foreign nucleic acids to enter the host cell. In a particular preferred embodiment of the invention, the donor DNA molecule is irradiated with UV prior to introduction into the prokaryotic host cell, since it is known that irradiation leads to an increase in recombination frequencies. According to the invention, the first prokaryotic cell containing the donor and recipient DNA molecules is cultured under such conditions that they require the formation of a cointegrate or hybrid molecule between the donor and recipient DNA molecules and recombination, preferably , non-homologous recombination, between the two recombination substrates, that is, conditions that allow the selection of the recombined DNA sequences. If the first marker sequence of the donor DNA molecule is a marker for resistance to an antibiotic, the first prokaryotic cell containing the recipient and donor DNA molecules is cultured in the presence of an antibiotic, to which the prokaryotic host cell it is sensitive, and to which the gene product of the first marker sequence confers resistance. In a particularly preferred embodiment, the first prokaryotic cell is cultured in the presence of spectinomycin if the first marker sequence present in the donor DNA molecule is specR, the gene product of which gives a cell resistance to spectinomycin. In another particularly preferred embodiment, the first prokaryotic cell is cultured in the presence of phleomycin if the first marker sequence present in the donor DNA molecule is phleoR, the gene product of which gives a cell resistance to phleomycin. In another particularly preferred embodiment, the first prokaryotic cell is cultured in the presence of tetracycline if the first marker sequence present in the donor DNA molecule is tcR, the gene product of which gives a cell resistance to tetracycline. If the first marker sequence of the donor DNA molecule is a nutritive marker, the first prokaryotic cell containing the donor and recipient DNA molecules is cultured in a medium that lacks the particular essential nutrient that can not be synthesized by the host cell itself, and that is supplied to the host cell after the introduction of the donor molecule and the expression of the first marker gene contained in the donor molecule. After the expression of the first nutritional marker gene, it is not necessary to add this essential nutrient to the medium in which the prokaryotic cell is cultured. If the second marker sequence, the third marker sequence and / or the fourth marker sequence are markers for resistance to an antibiotic, then in a preferred embodiment, the first prokaryotic cell can be further cultured in the presence of a second, a third and / or a fourth antibiotic to which the gene products of the second marker sequence, the third marker sequence and the fourth marker sequence, respectively, confer resistance. Preferably, the first prokaryotic cell is cultured not only in the presence of phleomycin or spectinomycin or tetracycline, but also also in the presence of chloramphenicol. More preferably, the first prokaryotic cell is cultured and propagated in the presence of phleomycin, spectinomycin, chloramphenicol and tetracycline. After cultivation of the prokaryotic cells under selective conditions, the second prokaryotic cells containing a cointegrate or a hybrid molecule formed by the donor and recipient DNA molecules are isolated. This cointegrate contains the recombinant DNA sequences. The presence of cointegrated and / or recombinant DNA sequences can be verified and detected by various means, such as restriction profile analysis, PCR amplification and / or sequencing. If, for example, the second marker sequence of the donor molecule and the third or fourth marker sequences of the receptor molecule are unique primer recognition sequences, then the specific cointegrated fragments can be amplified by PCR using appropriate primers that recognize these marker sequences. . In order to detect the presence of the respective marker combination. If a cointegrate has not been formed, that is, recombination has not occurred, these fragments can not be detected. If, for example, the second marker sequence of the donor molecule and the third or fourth marker sequences of the receptor molecule are cleavage sites of the single restriction enzyme, then the cointegrate can be subjected to an analysis of the restriction enzyme with the order to detect the specific DNA fragments. If a cointegrate has not been formed, that is, recombination has not occurred, these fragments can not be detected. According to the invention, the first and second recombined DNA sequences contained in the hybrid DNA molecule of the second prokaryotic cell can be isolated and / or isolated and / or selected. The first and second recombined DNA sequences obtained can be isolated, for example, by PCR amplification or cleavage with appropriate restriction enzymes. An analysis of the first and second recombined DNA sequences contained in the hybridized DNA molecule can be performed, for example, by sequencing methods. According to the invention, the first and second isolated recombinant DNA sequences can be inserted back into a donor DNA molecule and a recipient DNA molecule, respectively, and subjected to another round of recombination using the inventive method. Another aspect of the present invention relates to a method for generating novel proteins, enzymes and non-coding sequences with novel or improved functions and properties, whereby the known protein coding sequences or the known non-coding sequences are subjected to a or more rounds of recombination, using the inventive method to generate and detect recombinant DNA sequences in a prokaryotic host cell. The present invention also relates to the proteins, enzymes and non-coding sequences, generated by any of the inventive methods. The present invention also relates to plasmid pMIX91 of B. subtilis, which is a derivative of plasmid plL253 and which can be replicated in B. subtilis, but not in E. coli, and which comprises the specR marker and the phleoR marker and the restriction sites Seal, PpuMI and EcoO109l to insert a foreign DNA sequence. The present invention also relates to the plasmid P-MIX101 from B. subtilis, which is a derivative of plasmid plL253 and which can be replicated in B. subtilis, but not in E. coli, and which comprises the tcR marker sequence and the Xhol and PstI restriction sites to insert a sequence of strange DNA. The present invention also relates to the DSM4393 strain of B. subtilis, which contains the plasmid pMIX91 of B. subtilis (deposited at the DSMZ, Deutsche Sammiung für Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany, on February 21, 2005, SB202pMIX91 ), and strain 1A423 from B. subtilis, which contains plasmid pMIX101 from B. subtilis (deposited at DSMZ, Deutsche Sammiung für Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany, February 21, 2005, 1A423pMIX101). Another aspect of the present invention relates to the use of β pMIX91 plasmid. subtilis and pMIX101, respectively, as donor DNA molecules in the inventive method, ie, the use of plasmid pMIX91 or pMIX101, to generate and / or detect the recombinant DNA sequences in a prokaryotic host cell, preferably in a E. coli cell. Another aspect of the present invention relates to the use of the plasmid pACYC184 of E. co / or pMIX100 or derivatives thereof as the receptor DNA molecule in the inventive method, ie, the use of the plasmid pACYC184 or pMIX100 to generate and / or detecting recombinant DNA sequences in a prokaryotic host cell, preferably, in an E. coli cell. Another aspect of the present invention relates to equipment that can be used to perform the inventive method, to generate and detect recombinant DNA sequences in a prokaryotic host cell. In a first embodiment, the kit comprises at least one first container comprising cells from E. coli strain AB1157 as the prokaryotic host cell, a second container containing cells from E. coli strain AB1157, containing the plasmid pACYC184 of E. co // or plasmid pMIX100 of E. coli, which can be used as the receptor DNA molecule, and a third container comprising the cells of strain DSM4393 of B. subtilis, which contains plasmid pMIX91 of β. subtilis, or the cells of strain 1A423 of B. subtilis, which contain the plasmid pMIX101 of β. subtilis, which can be used as the donor DNA molecule. In a second embodiment of the invention, the kit comprises at least one first container comprising cells of the E. Coli MXP1 strain, which is a MutS mutant of strain AB1157, as the prokaryotic host cell, a second container comprising cells of E. coli strain AB1157, which contains the plasmid pACYC184 or pMIX100, and a third vessel comprising the cells of the DSM4393 strain of. subtilis, which contains the plasmid pMIX91, or cells of the strain 1A423 of. containing the plasmid pMIX101. In yet another embodiment, the kit comprises at least one first container comprising other cells of the E. coli strain AB1157 or the MXP1 strain of E. coli, a second container comprising the DNA of plasmid pACYC184 from E. co // or pMIX100, and a third container comprising the plasmid DNA pMIX91 from B. subtilis or the plasmid pMIX101 from B. subtilis Another aspect of the present invention relates to a method for producing c) a hybrid gene and / or a protein encoded by a hybrid gene in a prokaryotic cell. The method for producing a hybrid gene and / or the encoded protein, comprises the step of performing the inventive method for generating and detecting recombinant DNA sequences, wherein the hybrid gene and / or the protein encoded by the hybrid gene, is produced in the prokaryotic cell. After expression, the hybrid gene and / or the encoded protein are selected in the prokaryotic cell and / or isolated therefrom. The present invention also relates to a hybrid gene that is obtained by the inventive method, producing a hybrid gene or the inventive method for generating and detecting recombinant DNA sequences. The present invention also relates to a protein, which is encoded by a hybrid gene, which is obtained by the inventive method for producing a hybrid gene or the inventive method for generating and detecting recombinant DNA sequences, and / or which is obtained by the inventive method to produce a protein encoded by a hybrid gene. The present invention is illustrated by the following list of sequences, appended figures and examples. Figure 1 schematically illustrates the inventive method for generating and / or detecting recombination between two DNA sequences. The first sequence, the oxa7 gene, is present in the pTG2-phleo plasmid of B. subtilis as the donor DNA molecule. PTG2-ph! Eo carries the specR and phleoR markers that confer resistance to spectinomycin and phleomycin. By electroporation, pTG2-phleo is introduced into the E. coli host cell which contains the plasmid pTG3 as the receptor DNA molecule. pTG3 contains the second DNA sequence, the oxa11 gene, and the cmR marker that confers resistance to chloramphenicol. After the introduction of pTG2-phleo, the cell is cultured in the presence of spectinomycin and phleomycin, which requires the formation of a cointegrate between the two plasmids and the concomitant recombination between the genes. Therefore, after culturing, an E. coli cell containing a dimeric plasmid containing the newly recombined DNA sequences R1 and R2 is obtained. Recombinant DNA sequences can be analyzed by restriction profile analysis, PCR amplification of the R1 and R2 recombinant genes and / or sequencing of R1 and R2. Figures 2A-2D show the physical maps of the plasmids pUC19-phleo, pic156, pACYC184 and plL253, used to construct plasmids suitable for performing the inventive method. Figures 3A-3F show the physical maps of the plasmids that are constructed to carry out the inventive process. Plasmids pMIX96 and pMIX97 are not described, since they are like pMIX95, but with oxa5 and oxal, respectively, instead of or a11. pMIX99 is like pMIX98, but with oxa instead of oxal 1. Figure 4 shows the structure of the genes obtained by the inventive method of in vivo recombination between divergent oxa genes by 22%. The identity sections of the sequence in which the crossing takes place are detailed. Figure 5 shows the structure of plasmid pMIX100 of E. coli, which can be used as the receptor DNA molecule in an E. coli host cell. PMIX100 carries the origin of replication of plasmid pACYC184, as well as the chloramphenicol resistance gene thereof.

PMIX100 also carries the lacZ gene of pBluescript SK +.

Figure 6 shows the structure of β pMIX101 plasmid. subtilis, which is a derivative of plasmid plL253 of B. subtilis, and which can be used as the donor DNA molecule in an E. coli host cell. pMIX101 carries the ErmR marker that confers resistance to erythromycin and TcR that confers resistance to tetracycline. The tetracycline resistance gene was amplified from plasmid pACYC184. Figure 7 shows the structure of the plasmid control of transformation efficiency pMIX102. pMIX102, a derivative of pBluescript SK +, contains the amplified TcR gene of plasmid pACYC184. In pMIX102, the TcR gene is driven by the plac promoter. Figure 8 shows the plasmid structure of the pMIX103 transformation efficiency control. pMIX103, a derivative of pBluescript SK +, contains the amplified TcR gene of plasmid pACYC184. In pMIX103, the TcR gene is cloned in the opposite direction to plac. Therefore, the gene is expressed from its own promoter. Figure 9 schematically illustrates the strategy for cloning the oxa7, oxa11 and oxa5 genes in plasmid pMIX100 of E. co // and in plasmid pMIX101 of B. subtilis. To clone oxa7, oxa11 and, in pMIX100, the genes were amplified using primers containing Psti or Xho \ at their 5 'ends. After digestion with these enzymes, the amplified DNA fragments were ligated independently with pMIX100, which was previously cut with Psfí + Xho. The DHB10 competent cells of E. coli were electroporated with the ligand mixtures, and the colonies harboring the positive clones were phenotypically selected by chloramphenicol resistance / white color, on LB plates containing Cm (30 μg / ml) + X-Gal (80 μg / ml) + IPTG (0.5 mM). Plasmid DNAs were obtained and analyzed by restriction map formation. The results confirmed that pMIX104 carries oxa7, pMIX106 carries oxa11 and pMIX107 carries oxa5. For cloning in plasmid pMIX101 of ß. subtilis, oxa7 was obtained as a 0.9 kb fragment from pMIX104 by restriction with Pst \ and Xho \, and ligated with pMIX101, which was previously cut with the same enzymes. After transformation of the competent 1A423 cells of B. subtilis, with the ligated product, the cells were selected in LB containing 0.5 μg / ml erythromycin (Erm). SEQ ID No. 1 and 2 show the sequences of the primers OLG1 and OLG2, respectively, for the amplification of oxa7 and the introduction of the restriction sites Seal and Ppu S at the 5 'and 3' ends, respectively. SEQ ID No. 3 and 4 show the sequences of primers OLG3 and OLG4, respectively, for the amplification of oxa11 and the introduction of the restriction sites ßamHI and EcoO109l at the 5 'and 3' ends, respectively. SEQ ID No. 5 and 6 show the sequences of the primers OLG5 and OLG6, respectively, for the amplification of oxa5 and the introduction of the ßamHI and EcoO109I restriction sites at the 5 'and 3' ends, respectively.

SEQ ID No. 7 and 8 show the sequences of the primers OLG7 and OLG8, respectively, for the oxal amplification and the introduction of the ßamHI and EcoO109I restriction sites at the 5 'and 3' ends, respectively. SEQ ID No. 9 and 10 show the sequences of the primers OLG9 and OLG10, respectively, for the amplification of oxa11 and the introduction of the restriction sites Seal and EcoO109l at the 5 'and 3' ends, respectively. SEQ ID No. 11 shows the sequence of the OLG11 primer, which was used together with the OLG8 primer (SEQ ID No. 8), for the oxal amplification and the introduction of the Seal and EcoO109l restriction sites at the 5 'ends and 3 ', respectively. SEQ ID Nos. 12 and 13 show the sequences of the primers OLG12 and OLG13, respectively, for the amplification of the recombinant R1 gene, contained in the hybrid pMIX93 plasmid. SEQ ID No. 14 shows the sequence of primer OLG14, which was used in conjunction with primer OLG12 (SEQ ID No. 12), for the amplification of the recombinant gene R1 contained in one of the plasmids pMIX95, pMIX96 and pMIX97 hybrids. SEQ ID No. 15 shows the sequence of the OLG15 primer, which was used together with the OLG17 primer (SEQ ID No. 17), for the amplification of the recombinant R2 gene, contained in the hybrid pMIX93 plasmid.

• SEQ ID No. 16 shows the sequence of primer OLG16, which is used together with primer OLG17 (SEQ ID No. 17), for the amplification of the recombinant R2 gene, contained in one of the pMIX95 plasmids, pMIX96 and pMIX97 hybrids. SEQ ID No. 17 shows the sequence of the OLG17 primer.

EXAMPLE 1 In vivo recombination of heterologous genes using a double plasmid system (E. coli / B. subtilis), whereby the recombinant DNA sequences are selected for resistance to spectinomycin and / or phleomycin 1. Materials and methods 1. 1 Bacterial strains and plasmids The bacterial strains and plasmids used in this example are shown in Table 1 and Table 2, respectively.

TABLE 1 Bacterial strains TABLE 2 Plasmids 1. 2 Growing conditions and culture medium Both strains of E. coli and B. subtilis were grown at 37 ° C in LB medium (Difco Laboratories, Detroit, USA). When the medium was used on the plates (LBA), 15 g of agar (Difco) per liter was added. When required, the medium was supplemented with antibiotics, the final concentrations were as follows: tetracycline (Sigma-Aldrich Chimie, St. Quentin Fallavier, France), 12.5 μg / ml; chloramphenicol (Sigma-Aldrich Chimie), 30 μg / ml; ampicillin (Sigma-Aldrich Chimie), 100 μg / ml; erythromycin (Sigma-Aldrich Chimie), 0.5 μg / ml; Spectinomycin (Sigma-Aldrich Chimie), 75 μg / ml; phleomycin (Euromedex, Strasbourg, France), 2 μg / ml. When LBA was supplemented with spectinomycin plus phleomycin, the final concentrations were 60 and 1 μg / ml, respectively. 1. 3 Manipulation of DNA and microorganisms Transduction The MIXP1 strain of E. coli was constructed using P1-mediated transduction (3).

Transformation Plasmids were introduced into E. coli strains by electrotransformation, using an Eppendorf 2510 Electroporator (Eppendorf AG, Hamburg, Germany), and according to the supplier's instructions. The competent cells of ß. subtilis were prepared and transformed as described by Yasbin et al. (9) DNA manipulations The established protocols for molecular biology techniques were followed (4). Enzymes for DNA manipulations were purchased from New England Biolabs (Beverly, MA, USA), MBI Fermentas (Vilnius, Lithuania), Promega (Madison, Wis.) Or Stratagene (La Jolla, CA, USA), and were used as recommended by the manufacturers. When necessary, the DNA digested with restriction endonucleases was purified with agarose gels using the NuceloSpin Extract Kit (Machery-Nagel). The plasmid DNA was isolated from E. coli using the NucleoSpin equipment (Machery-Nagel GmbH &Co., Duren, Germany), according to the manufacturer's instructions. To isolate the DNA from the β plasmid. subtilis, the same equipment was used, but a first lysis step was performed incubating the cells with 2 mg ml of lysozyme for 30 minutes at 37 ° C. The primers used were synthesized by Proligo France SAS (Paris, France). The nucleotide sequences were determined in both directions by Genome Express (Meylan, France) The sequences were analyzed with the Infobiogen package (Genopole d'Evry, Evry, France) The ClustalW program was used for the comparisons of the sequences.

Amplifications with PCR PCR reactions were performed using a Mastercycler Gradient (Eppendorf AG, Hamburg, Germany). The reactions were carried out in a volume of 50 μl, using the DNA polymerase improved with high fidelity Herculase (Strategene), under the following conditions: 96 ° C for 3 minutes, 35 cycles of 96 ° C for 30 seconds, temperature annealing for 30 seconds, 72 ° C for 1 minute, and final elongation step at 72 ° C for 10 minutes. The annealing temperature was determined by subtracting 5 ° C at the lowest Tm of the primers used. When necessary, the PCR products were purified using the NucleonSpin Extract (Machery-Nagel) kit. The products of the amplification were analyzed by electrophoresis in 0.7% agarose gels (Sigma). 2. General strategy This experiment was carried out in order to generate new molecules that exhibit advantageous properties by in vivo recombination of two original genes, which share different degrees of sequence identity. The strategy employed is as follows: The genes that will recombine are carried by two different plasmids that do not show homology in their nucleotide sequences. The first is a replicating plasmid of E. coli that confers resistance to chloramphenicol (Cm) or to tetracycline (Te). It is based on the standard cloning vector pACYC184, a plasmid with a low copy number, obtained from New England Biolabs.

The second is a plasmid of Bacillus subtilis derived from plL253 (Simon and Chopin, 1988). It is not capable of replicating in E. coli and carries two markers of resistance to an antibiotic for spectinomycin (Spc) and phleomycin (Phleo), respectively. To recombine the heterologous gene pairs carried by these two vectors, the β plasmid. subtilis is introduced by electroporation into the E. coli strains harboring the replicating plasmid. This is shown schematically in Figure 1. Such strains are either proficient (+) or deficient (-) for the mismatch repair system (MMR), which controls both mutagenesis and recombination. After electroporation, the transformants are selected with antibiotics for which the plasmid of B. subtilis confers resistance (Spc and Phleo). Such selective pressure forces recombination between the heterologous genes, since only the cells harboring a cointegrate formed between the plasmids of B. subtilis and E. coli can grow under these conditions. The hybrid plasmid confers resistance to Spc and Phleo, and replicates the origin of E. coli, since that of B. subtilis is not functional. In addition, it carries the two recombinant genes R1 and R2. The first step was the cloning in the vectors of E. coli and B. subtilis of the genes initially chosen as objective, to evaluate the efficiency of the recombination.

Recombination experiments were performed between the oxa genes in both strains of the wild type and deficient in the repair of the bad correspondences. Experiments were performed between pairs of identical or divergent genes. The plasmid DNAs were irradiated with UV before electroporation, since it was shown that the irradiation leads to a 10-fold increase in the recombination frequencies. Recombination was also performed on strains that were temporarily mutated by treatment with 2-aminopurine. 2-aminopurine is an adenine analogue that is incorporated into the DNA during the growth of bacteria and saturates the MMR system. Thus, a transient mutator phenotype is generated, since after eliminating 2-aminopurine, a state of the wild type is recovered. This temporal control of the repair activity of the bad correspondences, provides a stable base for the strains used in the recombination, avoiding the accumulation of mutations in their genomes. 3. Results Construction of the B. subtilis vector In order to construct a vector of ß. subtilis to carry the target genes for recombination, two gene markers that confer resistance to the antibiotics spectinomycin (specR) and phleomycin (phleoR), respectively, were cloned into the plasmid plL253 after two steps of the cloning strategy. First, the specR gene was obtained as a Sacl fragment of 1294 pbs in length from plasmid pic156. The fragment was purified and then ligated to Sacl digested with plL253. The competent DS4393 cells of ß. Subtilis were transformed with the ligand mixture and the transformants were selected in LBA plates containing 75 μg mi "1 of spectinomycin Restriction analyzes of the plasmid DNA obtained from the transformants, confirmed that they harbor the p1LF253 derivatives carrying the gen specR In a second step, the phleoR gene was cloned into plL253-spec. The plasmid pUC19-phleo was digested with the restriction enzymes EcoRI and Sa / I and the 574 bps fragment corresponding to phleoR was gel purified and ligated to plL253-spec, previously digested with the same two enzymes. The DSM4393 competent cells of B. subtilis were transformed with the ligand mixture, and the transformants were then selected on LBA plates containing 60 μg ml "1 of phleomycin Restriction analyzes of the plasmid DNA obtained from the transformants showed that host the expected 6.69 kb plasmid, which carries both the specR and phleoR genes.The plasmid was named pMIX91 (see Figures 3A-3F).

Construction of the vector controlling the efficiency of the transformation A vector was constructed to be used as a control of the transformation efficiency of the E. coli strains under the selection of spectinomycin and phleomycin in recombination experiments. The vector was constructed as follows: the spectinomycin resistance gene (specR) was obtained as a 1.25 kb fragment after digestion of pid 56 with BamHI and EcoR1. It was cloned into the corresponding sites of pUC-phleo, then, with the phleomycin resistance gene (phleoR). After electroporation of the cells of the DH10B component of E. coli with the binding mixture, the colonies resistant to spectinomycin and phleomycin were selected in LBA plates containing both antibiotics at final concentrations of 60 μg / ml and 1 μg / ml. ml, respectively. Restriction analyzes of the plasmid DNA isolated from the transformants showed that they correspond to the expected 4.46 kb long construct, which was named pM! X92 (see Figures 3A-3F).

Cloning of genes encoding ß-lactamases in E. coli vectors. and B. subtilis Four genes encoding ß-lactamases were chosen as targets for evaluating recombination efficiencies in wild type E. coli strains and MutS mutants. "Such genes, oxa7 (accession number of GenBank X75562), oxa11 (access number of GenBank Z22590), oxad (access number of GenBank X58272) and oxal (access number of GenBank J02967), show different degrees of divergence in their nucleotide sequences, the oxal sequence and the oxa5 sequences, oxa7 and oxal 1, respectively, differ by 40% The sequence of oxa5 and the sequences of oxa7 and oxa11, respectively, differ by 22% The sequence of oxa7 and the sequence of oxal 1 differ by 5% The four oxa genes they were cloned into plasmid pACYCI 84 from E. coli, while oxa7, oxa11 and oxal were cloned into the plasmid pMIX91 from ß. subtilis as well.The cloning of the genes was done as follows: oxa7 was amplified by PCR with primers designed for enter l Seal and Ppi / MI sites at the 5 'and 3' ends, respectively, of the amplified DNA (primers OLG1 with SEQ ID No. 1 and OLG2 with SEQ ID No. 2). The PCR product was digested with these restriction enzymes, and the resulting fragment of 991 bps in length was ligated to pMIX91, previously cut with the same enzymes. The DSM4393 competent cells of B. subtilis were transformed with the ligand mixture and the selection of the transformants was made in LBA plates containing 75 μg mi "1 of spectinomycin Restriction analysis of the plasmid DNA obtained from the transformants, demonstrated housing the 6.89 kb pMIX94 plasmid carrying oxa7 (see Figures 3A-3F). To clone oxa7 in the pACYC184 vector of E. coli, the PCR product described above, as well as pACYC184, were digested with PpuM and Seal The blunt ends were generated from the digested DNAs using the Klenow fragment of DNA polymerase I, and ligated together The competent DH1 B cells from E. coli were electroporated with the ligand mixture, and the transformants LBA plates containing 12.5 μg mi "1 of tetracycline were selected. Restriction analyzes of the plasmid DNA isolated from the transformants showed that they correspond to the expected 4.33 kb long construct, which was named pMIX93 (see Figures 3A-3F). To clone oxa11, oxad and oxal in pACYC184, genes were amplified by PCR using primers designed to introduce the ßamHI and EcoO109l sites at the 5 'and 3' ends, respectively, of the amplified DNA. The pairs of primers OLG3 (SEQ ID No. 3) / OLG4 (SEQ ID No. 4), OLG5 (SEQ ID No. 5) / OLG6 (SEQ ID No. 6) and OLG7 (SEQ ID No. 7) / OLG8 (SEQ ID No. 8), were used to amplify oxal 1, oxa 5 and oxal, respectively. The PCR products were digested with SamHI and EcoOl 09I and the resulting fragment of 997 pbs (oxal 1) and 830 pbs (oxad) and 936 pbs (oxal) were ligated independently to pACYC184, previously cut with the same enzymes. The DH10B competent cells of E. coli were electroporated with the ligand mixtures, and the transformants were selected on LBA plates containing 30 μg ml of chloramphenicol.1 Restriction analyzes of the plasmid DNA isolated from the transformants showed that they correspond to the expected 6.89 kb plasmids pMIX95 (3.72 kb), pMIX96 (3.55 kb) and pMIX97 (3.66 kb) (see Figures 3A-3F).

To clone oxa11 and oxal in pMIX91, the genes were amplified by PCR using primers designed to introduce the Seal and EcoO109I sites at the 5 'and 3' ends, respectively, of the amplified DNA. The pairs of primers OLG9 (SEQ ID No. 9) / OLG10 (SEQ ID No. 10), and OLG11 (SEQ ID No. 11) / OLG8 were used to amplify oxa11 and oxal, respectively. The PCR products were digested with Seal and EcoO109l, and the resulting fragment of 995 pbs (oxa11) and 934 pbs (oxal), were ligated independently to pMIX91, previously cut with the same enzymes. The DSM4393 competent cells of ß. subtilis were transformed with the ligand mixture, and the selection of the transformants was made in LBA plates containing 75 μg mi "1 of spectinomycin Restriction analyzes of the plasmid DNA obtained from the transformants, showed that they correspond to the expected plasmids pMIX98 (6.89 kb) and pMIX99 (6.83 kb) See Figures 3A-3F.

In vivo recombination of the oxa genes in wild-type E. coli compared to the MutS mutant "In a first step, the competent AB1157 cells of E. co // and its mutant MutS", MXP1 of E. coli, were transformed independently by electroporation with plasmids derived from pACYC184, which carry the oxa genes, pMIX93, pMIX95, pMIX96 or MIX97. Transformants were selected based on their resistance to tetracycline or chloramphenicol. The presence of the appropriate plasmids was subsequently confirmed by restriction analysis and / or PCR. In a second step, the competent cells of the wild-type and MutS "strains harboring the replicating plasmids were prepared from selective medium containing tetracycline or chloramphenicol, Subsequently, such competent cells were transformed independently by electroporation with the B. subtilis plasmids carrying the oxa genes, pMIX94, pMIX98 or pMIX99 After the electroporation, the transformants were selected in LBA plates containing antibiotics for which the plasmid of. subtilis confers resistance: spectinomycin and phleomycin, at concentrations At the end of 60 μg mi "1 and 1 μg mi" 1, respectively, such selective pressure forces recombination between the oxa genes, since only the cells that host a hybrid plasmid formed between the plasmids of B. subitilis and E. coli can grow under these conditions.The plates were incubated overnight at 37 ° C, and subsequently the plasmid DNA from The transformants were analyzed by digestion with restriction enzymes, in order to confirm that they host the hybrid plasmids (approximately 10.5 kb in length), which carry two recombinant genes, R1 and R2. In some cases, the recombinant genes were amplified by PCR and sequenced. If the resident plasmid was pMIX93, R1 was amplified with OLG12 (SEQ ID No. 12) / OLG13 (SEQ ID No. 13) and R2 with OLG15 (SEQ ID No. 15) / OLG17 (SEQ ID No. 17); if the resident plasmid was pMIX95, pMIX96 or pMIX97, R1 was amplified with OLG12 / OLG14 (SEQ ID No. 14) and R2 with OLG16 SEQ ID No. 16) / OLG17. In the recombination experiments, the plasmid pMIX91 of B. subtilis is used as a negative control, while the plasmid pMIX92 of E. coli is used as a control of transformation efficiency under selection with spectinomycin and phleomycin. pMIX92 can be replicated in strains harboring plasmids derived from pACYC184, since their origins of replication (ColE1 and p15, respectively), are compatible. The plasmid DNAs were irradiated with UV before electroporation (200 J / m2), since it was shown that the irradiation leads to a 10-fold increase in the recombination frequencies. The recombination frequencies were calculated by dividing the efficiency of the transformation obtained with the B. subtilis plasmids by the efficiency of the transformation obtained with the control vector pMIX92. The efficiencies of the transformation were calculated in turn, as the number of units that form colonies (cfu), obtained per μg of DNA under the conditions previously described. The results obtained are summarized in Table 3. In the wild type strain, the recombinants were obtained in experiments using identical or diverging oxa genes by 5%, whereas in the mutant MutS ", recombination also passed between divergent genes by 22%.

TABLE 3 In vivo recombination frequencies obtained between oxa genes in strains of wild type E. coli and MutS ' In vivo recombination of the oxa genes in wild-type E. coli compared to E. coli treated with 2-aminopurine (2-AP) In a first step, the competent E. coli AB1157 cells were transformed by electroporation with derived plasmids of pACYC184, which carry the oxa1 1 gene, pMIX95. The transformants were selected based on their strength. The presence of suitable plasmids was confirmed by restriction and / or PCR analysis. In a second step, the competent cells of this strain were prepared in the presence of 200 μg / ml of 2-AP and subjected to electroporation independently with the plasmid pMIX94 of B. subtilis, which carries oxa7 or pMIX98 which carries oxal 1 . The pMIX91 plasmid from B. subtilis was used as a negative control, while the plasmid pMIX92 from E. coli, which carries the SpcR and PhleoR markers, was used as a control of the efficiency of the transformation. The plasmid DNAs were irradiated with UV prior to electroporation in order to increase the frequency of recombination. The results are summarized in Table 4. In the wild-type strain, the recombinants were obtained in experiments using identical oxa genes, whereas in the strain treated with 2-AP, recombination also passed between divergent genes by 5%.

TABLE 4 In vivo recombination frequencies obtained between oxa genes in wild type E. coli strains and treated with 2-AP It should be noted that recombination occurred between genes with a divergence of 22%, in which the longest stretch of sequence identity was 22 nucleotides (oxa5 / oxa11) and 18 nucleotides (oxad / oxa7), respectively (see Figures 5 and 6). It was reported that recombination becomes inefficient below a minimum length of homology, known as MEPS (minimum efficient processing segment). The length of MEPS varies depending on the recombination path, but has been described as varying from 23 to 90 base pairs (5).

Sequence analyzes of the R1 and R2 genes carried by 54 hybrid plasmids obtained in the experiments involving genes with a divergence of 5% or 22%, showed that 46 of these hybrid plasmids were different from one another. This result indicates that they correspond in different recombination events and, consequently, that a high degree of genetic diversity was generated by in vivo recombination. Most of the recombinant genes were generated by unique non-reciprocal crosses in different stretches of sequence identity, ranging from 4 to 101 nucleotides. In some cases, multiple crosses were observed, producing genes R1 or R2 mosaic. The recombinant genes obtained between oxa7 / oxa5 and oxa11 / oxa5 are described in Figure 4. Comparison of the DNA and deduced amino acid sequences of the recombinant genes also revealed that 53% of them correspond to new oxa genes (see Table 4). Since no frame shifts or stop co were generated during recombination, they can putatively encode 38 new functional β-lactamases.

TABLE 4 Comparison of the nucleotide and deduced amino acid sequences of recombinant oxa genes obtained by in vivo recombination EXAMPLE 2 In vivo recombination of heterologous genes to a double plasmid system (Escherichia colíl Bacillus subtilis) so that the sequences of Recombinant DNA are selected by resistance to tetracycline Another double plasmid system was designed to verify the results obtained in Example 1 and facilitate the cloning and recombination of the genes. This system is based on the selection of cells resistant to tetracycline. 1. Bacterial strains and plasmids The bacterial strains and plasmids used in this example are shown in Table 5 and Table 6, respectively.

TABLE 5 Bacterial strains TABLE 6 Plasmids 2. Results 2. 1 Construction of pMIXlOO plasmid from E. coli Plasmid pMIXlOO carries the origin of replication of pACYC184, as well as its chloramphenicol resistance gene. pMIXl OO also carries the lac gene? of pBluescript SK +, which facilitates selection in cloning experiments. facZ encodes a fragment of the β-galactosidase that provides complementation for the blue / white selection of the recombinants in a medium containing X-Gal and IPTG. Thus, the colonies that harbor pMIXlOO must be blue in this medium, those that carry the plasmid with a gene inserted in the polylinker (MCS) must be white. The physical map of the pMIXlOO plasmid is shown in Figure 5. 2. 2 Construction of plasmid pMIX101 of B. subtilis Plasmid pMIX101 is a derivative of plasmid plL253 of B. subtilis. Since the ErmR marker of plL253 conferring resistance to erythromycin is not useful in E. coli, the tetracycline resistance marker that allows the selection of hybrid molecules in the recombination experiments was introduced. The tetracycline resistance gene was amplified from plasmid pACYC184. Thus, pMIX101 carries two markers: ErmR, as a selection marker in β. subtilis to clone the target genes, and 7cR, to select the recombinant hybrid molecules in E. coli. The physical map of plasmid pMIX101 is shown in Figure 6. 2. 3 Construction of plasmids of the transformation efficiency control pMIX102 and PMIX103 Since the vector of B. subtilis is not capable of replicating in E. coli, it is necessary to control the efficiency of the transformation to estimate the frequency of recombination . Since the recombinants will be selected by resistance to tetracycline, the same marker must be present in the control vector. Plasmid pMIX102, a derivative of pBluescript SK + contains the amplified TcR gene of pACYC184. In pMIX102, the TcR gene is driven by the plac promoter. The physical map of plasmid pMIX102 is shown in Figure 7. In the second control vector pMIX103, the 7cR gene is cloned in the opposite direction to plac. Therefore, this gene is expressed from its own promoter. The physical map of plasmid pMIX103 is shown in Figure 8. 2. 4 Cloning of oxa7, oxa11 and oxad in plasmid pMIXlOO of E. coli To verify the results of the recombination experiments between divergent oxa genes 0%, 5% and 22%, obtained in example 1, oxa7, oxa11 and oxad cloned in the E. coli pMIXlOO plasmid. These genes were amplified using primers that contain either Psfl or Xho \ at their 5 'ends. After digestion with these enzymes, the amplified DNA fragments were ligated independently with pMIXlOO, which was previously cut with Psíl + Xho. The DHB10 competent cells of E. coli were electroporated with the ligand mixtures, and the colonies harboring the positive clones were phenotypically selected by chloramphenicol / white color resistance on LB plates containing Cm (30 μg / ml ) + X-Gal (80 μg / ml) + IPTG (0.5 mM). For each oxa cloning, five such transformants were analyzed. The plasmid DNAs were obtained and analyzed by restriction map formation. The results confirmed that pMIX104 carries oxa7, pMIX106 carries oxa11 and pMIX107 carries oxad. For cloning in plasmid pMIX101 of B. subtilis, oxa7 was obtained as a 0.9 kb fragment of pMIX104 by restriction with Pst \ and Xho \, and ligated with pMIX101, which was previously cut with the same enzymes. After transformation of the competent 1A423 cells of B. subtilis with the binding product, the cells were selected in LB containing 0.5 μg / ml erythromycin (Erm). The plasmid DNA was obtained from 24 transformants and analyzed by restriction. The results confirmed that all clones contained plasmid pMIX105 of 7 kb. The strategy to clone the genes oxa7, oxa? and oxa5 in plasmid pMIX100 of E. coli and plasmid pMIX101 of B. subtilis, is shown in Figure 9. 2. 5 Recombination in vivo recombination of the oxa genes in E. coli wild-type compared to the mutant MutS "Competent cells AB1157 hsdR" from E. coli and its mutant MMR ", AB1157 hsdR" CAmutS from E. coli, were transformed independently by electroporation with the plasmids pMIX104 (oxa7), pMIX106 (oxal 1) and pMIX107 (oxad). To recombine the oxa genes, the strains of E. coli harboring these plasmids were electroporated with pMIX105 (oxa7). Plasmid pMIX101 of ß. subtilis was used as a negative control, while the vector pMIX102 of E. coli, which carries the TcR marker, was used as the control of transformation efficiency under selection with tetracycline. All the DNAs were irradiated with UV before electroporation in order to increase the frequency of recombination. The recombination frequencies were calculated by dividing the efficiency of the transformation obtained with the β plasmids. subtilis by the efficiency of the transformation obtained with the control vector pMIX92. The efficiencies of the transformation were calculated in turn as the number of units forming colonies (cfu), obtained per μg of DNA under the conditions previously described. The results obtained are summarized in Table 7. In the wild-type strain, the recombinants were obtained in experiments using oxa genes identical or divergent at 5%, whereas in the MutS mutant, recombination also passed between divergent genes at the 22% TABLE 7 In vivo recombination frequencies obtained between the oxa genes in strains of wild type E. coli and MutS " The recombination frequencies are consistent with those obtained with the previous double plasmid system. To confirm recombination, the transformants of pMIX105 were cultured in a liquid medium containing tetracycline (12.5 μg / ml). Restriction analyzes of the plasmid DNA obtained from these cultures showed that they harbor dimers carrying the oxa recombinant genes, together with the resident plasmids (either pMIX104 or pMIX106). In addition, the R1 and R2 recombinant genes were successfully amplified by PCR of both colonies and the plasmid DNA using specific primers. The results demonstrate that the recombinants were generated with a second double plasmid system, using the resistance to tetracycline as the selective pressure.

Claims (48)

NOVELTY OF THE INVENTION CLAIMS

1. - A method for generating and detecting recombinant DNA sequences in prokaryotes comprising the steps of: a) generating a first prokaryotic cell containing an extrachromosomal receptor DNA molecule, comprising a first DNA sequence to be recombined, and which can be replicated autonomously in the prokaryotic cell, and an extrachromosomal donor DNA molecule, comprising a second DNA sequence to be recombined and at least a first marker sequence that encodes a gene product and that can not be replicated autonomously in the prokaryotic cell b) cultivate the first prokaryotic cell under selective conditions, which require the formation of a cointegrated or hybrid molecule between the recipient and donor DNA molecules and the recombination of two DNA sequences to be recombined, and which only allow growth and / or the propagation of the cell if the gene product of the first marker sequence is expressed, and c) isolating a second prokaryotic cell cultured and / or propagated under selective conditions and containing a hybrid DNA molecule with at least a first marker sequence and a first and second recombined DNA sequence, due to recombination between the first and the second DNA sequences, in which the prokaryotic cell is transiently or permanently deficient in the repair system of the bad correspondences.

2. The method according to claim 1, further characterized in that the donor DNA molecule and the DNA receptor molecules are different linear or circular DNA structures, in particular, different plasmids or bacteriophages.

3. The method according to any of claims 1 or 2, further characterized in that the donor DNA molecule does not have an origin of replication.

4. The method according to any of claims 1 or 2, further characterized in that the donor DNA molecule has a non-functional origin of replication.

5. The method according to any of claims 1 to 4, further characterized in that the donor DNA molecule is a Bacillus subtilis plasmid, which can not be replicated in E. coli.

6. The method according to claim 5, further characterized in that the donor DNA molecule is the β pMIX91 plasmid. subtilis comprising the specR marker and the phleoR marker or the pMIX101 plasmid of B. subtilis comprising the tcR marker.

7. The method according to any of claims 1 to 6, further characterized in that the first marker sequence of the donor DNA structure is selected from the group consisting of a nutritional marker, a marker for resistance to an antibiotic and a sequence that encodes a subunit of an enzyme.

8. The method according to claim 7, further characterized in that the gene product of the first marker sequence confers resistance to an antibiotic to a cell that is sensitive to that antibiotic.

9. The method according to claim 7 or 8, further characterized in that the first marker sequence is specR, the gene product of which gives a cell resistance to spectinomycin, or phleoR, the gene product of which it confers a cell resistance to phleomycin, or tcR, the gene product of which gives a cell resistance to tetracycline.

10. A method for generating and detecting recombinant DNA sequences in prokaryotes, comprising the steps of: d) generating a first prokaryotic cell containing an extrachromosomal receptor DNA molecule, comprising a first DNA sequence to be recombined, and which can be replicated autonomously in the prokaryotic cell, and an extrachromosomal donor DNA molecule, comprising a second DNA sequence to be recombined and at least one first marker sequence encoding a gene product and which can not be replicated autonomously in the prokaryotic cell, e) culturing the first prokaryotic cell under selective conditions, which require the formation of a cointegrated or hybrid molecule between the recipient and donor DNA molecules and the recombination of two DNA sequences to be recombined, and which only allow growth and / or cell propagation if the gene product of the first marker sequence is expressed, and f) isolating a second prokaryotic cell cultured and / or propagated under selective conditions and containing a hybrid DNA molecule with at least the first marker sequence and the first and second recombined DNA sequences, due to recombination between the first and second DNA sequences, wherein the donor DNA molecule is the pMIX91 plasmid of B. subtilis comprising the specR marker and the phleoR marker or the plasmid pMIX101 of B. subtilis comprising the tcR marker.

11. The method according to claim 10, further characterized in that the receptor DNA molecule is a linear or circular DNA structure, in particular a plasmid or a bacteriophage.

12. The method according to claim 10 or 11, further characterized in that the prokaryotic cell has a system of repair of the functional mismatches.

13. The method according to claim 10 or 11, further characterized in that the prokaryotic cell is transiently or permanently deficient in the repair system of the bad correspondences.

14. The method according to any of claims 1 to 13, further characterized in that the receptor DNA molecule is a plasmid, which can be replicated in Escherichia coli.

15. The method according to claim 14, further characterized in that the receptor DNA molecule is the plasmid pACYC184 of E coli or the pMIX100 plasmid of E. coli or a derivative thereof.

16. The method according to any of claims 1 to 15, further characterized in that the donor DNA molecule and / or its origin of replication are derived from a prokaryotic species different from the prokaryotic species in cells of which the molecule of Donor DNA is introduced.

17. The method according to any of claims 1 to 16, further characterized in that the function of the origin of replication of the donor DNA is damaged by a mutation.

18. The method according to any of claims 1 to 17, further characterized in that the donor DNA molecule contains a second marker sequence.

19. The method according to any of claims 1 to 18, further characterized in that the receptor DNA molecule contains a third marker sequence and optionally a fourth marker sequence.

20. The method according to claim 18 or 19, further characterized in that the second, third and fourth marker sequences are sequences that encode or do not encode a protein, selected from the group consisting of nutritive markers, pigment markers, markers for resistance to an antibiotic, markers for sensitivity to a antibiotic, restriction enzyme sites, primer recognition sites and sequences that encode a subunit of an enzyme.

21. The method according to claim 20, further characterized in that the gene products of the third and fourth marker sequences of the receptor DNA molecule confer resistance to an antibiotic to a cell that is sensitive to that antibiotic.

22. The method according to claim 21, further characterized in that the gene product of the third marker sequence gives a cell resistance to tetracycline.

23. The method according to claim 21, further characterized in that the gene product of the fourth marker sequence gives a cell resistance to chloramphenicol.

24. The method according to any of claims 1 to 23, further characterized in that the first and second DNA sequences to be recombined diverge by at least two nucleotides.

25. The method according to any of claims 1 to 24, further characterized in that the first and second DNA sequences to be recombined are natural sequences.

26. The method according to claim 25, further characterized in that the first and / or the second DNA sequences to be recombined are derived from viruses, bacteria, plants, animals and / or humans.

27. The method according to any of claims 1 to 24, further characterized in that the first and / or the second DNA sequences to be recombined are artificial sequences.

28.- The method according to any of claims 1 to 27, further characterized in that each of the first and second DNA sequences to be recombined comprises one or more sequences encoding a protein and / or one or more non-coding sequences .

29. The method according to any of claims 1 to 28, further characterized in that the first prokaryotic cell is generated by simultaneous or sequential introduction of the receptor DNA molecule and the donor DNA molecule into a prokaryotic cell.

30. The method according to claim 29, further characterized in that the receptor and donor DNA molecules are introduced into the prokaryotic cell via transformation, conjugation, transduction, sexduction and / or electroporation.

31.- The method according to any of claims 1 to 30, further characterized in that the first prokaryotic cell is cultured in the presence of at least one antibiotic to which the gene product of the first marker sequence confers resistance.

32. - The method according to claim 31, further characterized in that the first prokaryotic cell is further cultured in the presence of a second, a third and / or a fourth antibiotic to which the gene products of the second marker sequence, the third Marker sequence and fourth marker sequence, respectively, confer resistance.

33. The method according to any of claims 1 to 32, further characterized in that the prokaryotic cell is a cell of an archabacterium or a eubacterium.

34.- The method according to claim 33, further characterized in that the eubacterium is a gram negative bacterium, a gram positive bacterium or a cyanobacterium.

35.- The method according to claim 34, further characterized in that the gram negative bacterium is Escherichia coli.

36.- The method according to claims 1 to 9 and 13, further characterized in that the transient or permanent deficiency of the system of repair of the bad correspondences, is due to a mutation, a suppression and / or an expression or inducible repression of one or more genes involved in the repair system of the bad correspondences, a treatment with an agent that saturates the repair system of the bad correspondences and / or a treatment with an agent that globally cancels the repair of the bad correspondences.

37. - The method according to claims 1 to 9, 13 and 36, further characterized in that the prokaryotic cell has a mutated mutS gene and / or a mutated mutL gene.

38.- The method according to any of claims 1 to 37, further characterized in that the first and second recombined DNA sequences contained in the hybrid DNA molecule of the second prokaryotic cell are selected and / or isolated and / or analyzed. .

39.- The method according to claim 38, further characterized in that the first and second recombined DNA sequences are isolated by cleavage with restriction enzymes.

40.- The method according to claim 38, further characterized in that the first and second recombined DNA sequences are amplified by PCR.

41.- The method according to any of claims 38 to 40, further characterized in that the first and second isolated recombinant DNA sequences are inserted into a molecule of Donor DNA and a receptor DNA molecule, respectively, and undergo another round of recombination.

42.- The pMIX91 plasmid of Bacillus subtilis comprising the specR marker and the phleoR marker and the restriction sites Seal, PpuM \ and EcoO109l to insert a foreign DNA sequence.

43. The pMIX101 plasmid of Bacillus subtilis comprising a tcR marker sequence and the Xho and Pst restriction sites to insert a foreign DNA sequence. 44.- The use of plasmids pMIX99 or pMIX101 of B. subtilis as donor DNA molecules in a method as claimed in any of claims 1 to 41, for generating and / or detecting recombinant DNA sequences in a prokaryotic host cell , preferably in an E. coli cell. 45.- The use of plasmids pACYC184 or pMIXlOO of E. coli or a derivative thereof, as a receptor DNA molecule in a method as claimed in any of claims 1 to 41, for generating and / or detecting sequences of recombinant DNA in a prokaryotic host cell, preferably, in an E. coli cell. 46.- A kit comprising at least one first container comprising cells of the E. coli strain AB1157 or the E. coli strain MXP1 or the E. coli strain DHB10, a second container comprising cells of the E. coli strain AB1157, which contains the plasmid pACYC184 or cells of the E. coli strain DHB10 which contain the plasmid pMIXlOO and a third container comprising cells of the DSM4393 strain of β. subtilis, which contains plasmid pMIX91 or cells of strain 1A423 of B. subtilis which contains plasmid pMIX101. 47.- A kit comprising at least one first container comprising cells of the E. coli strain AB1157 or the MXP1 strain of E. co // or the E. coli strain DHB10, a second vessel comprising the plasmid DNA pACYC184 or the plasmid pMIXlOO and a third vessel comprising the plasmid DNA pMIX91 or the plasmid pMIX101. 48.- A process for producing a hybrid gene and / or a protein encoded by the hybrid gene in a prokaryotic cell, further characterized in that a method according to any of claims 1 to 41 and the hybrid gene and / is carried out. or the protein encoded by the hybrid gene is produced in the prokaryotic cell and the hybrid gene and / or the protein encoded is selected in the prokaryotic cell and / or is isolated therefrom after expression.