FR2831185A1 - Conversion of hybrid cloning vectors, useful for cloning nucleic acids, where conversion of competent cells is done in bulk, on a solid support - Google Patents

Abstract

Converting (M1) hybrid cloning vectors using at least one conversion-competent strain of cells, in which conversion is done in bulk, at least partly on a solid support, is new. Independent claims are also included for: (1) cloning (M2) selected nucleic acid sequences, using the new method; and (2) kits for performing M1 and M2.

Description

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Method and kit for converting hybrid solid-phase cloning vectors and method for cloning nucleotide sequences of interest by applying
The field of the invention is that of cloning of nucleotide sequences of interest, in particular for the purposes of purification, amplification and / or production of nucleotide libraries, these libraries being able to be composed eg of genomic sequences, of sequences of CDNA or synthetic sequences.

More specifically, the present invention relates to a process for mass conversion (in solid or pasty phase) of hybrid cloning vectors belonging to a binary vector / competent cell strain (preferably bacterial) conversion.

 The present invention also relates to the methods of cloning and "automatic" subcloning of nucleotide sequences integrated into hybrid cloning vectors belonging to a binary hybrid vector / conversion bacteria system.

Finally, the present invention relates to kits containing in particular the hybrid cloning vectors and the solid supports, pre-seeded or not with the conversion bacteria, allowing mass conversion.

 Cloning consists in inserting a DNA fragment of interest into a vector, and in multiplying this vector in a competent cell strain such as a bacterial, yeast or any other organism allowing the multiplication of this vector. The cultivation of these cells, then the extraction and purification of the vectors, makes it possible to produce almost unlimited quantities of the cloned DNA fragment.

* pour purifier une ou plusieurs séquences nucléotidiques d'intérêt * ou pour construire des banques nucléotidiques, composées par exemple, de séquences génomiques, de séquences d'ADNc ou de séquences synthétiques. These cloning processes are notably applied:

* to purify one or more nucleotide sequences of interest * or to construct nucleotide libraries, composed for example of genomic sequences, cDNA sequences or synthetic sequences.

 A cloning vector must be able to integrate nucleotide sequences of interest, to replicate in a competent bacterial strain, and to be easily detected and isolated in a host cell. The vectors commonly used by those skilled in the art for the production of nucleotide libraries are mainly phage and cosmid type vectors (Sambrook et al., 1989: all the bibliographic references in italics cited in the present description are defined in more detail in a summary below). Although these vectors have interesting characteristics for cloning, their use nevertheless has certain limits (Sambrook et al., 1989; Meese et al., 1990). Indeed, the nucleotide libraries produced in phage-type vectors can be screened quickly, but are a source of drawbacks as regards the analysis and subcloning of the nucleotide insert. Regarding the banks built in type vectors

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 cosmid, the screening and purification methods for specific clones are not very efficient and require a significant investment in time.

 To facilitate the isolation and characterization of a sequence of interest integrated into the genome of these vectors, a subcloning procedure is often implemented.

(en général un vecteur phage k) à l'aide des enzymes de restriction adaptées, puis par l'insertion de cette séquence nucléotidique dans une autre famille de vecteur plus facilement analysable, préférentiellement de type plasmidique. This subcloning technique consists in transferring, by molecular biology techniques, the sequence of interest into a second more functional vector, this second vector generally being of plasmid type. Traditionally, subcloning is obtained by the method comprising in particular the following steps: excision of the nucleotide region cloned in the initial cloning vector

(generally a phage k vector) using suitable restriction enzymes, then by inserting this nucleotide sequence into another vector family which is more easily analyzed, preferably of the plasmid type.

This subcloning process is a technically difficult and long process, and the yield of which is very variable. Obtaining vectors allowing simpler subcloning with high yield is therefore a constant concern.

système moléculaire de recombinaison site spécifique, l'ADN cloné étant intégré au niveau de ladite région plasmidique. Ce type de vecteur hybride permet alors un sousclonage de la séquence d'intérêt de manière "automatique". L'insert initialement intégré dans une structure de caractéristique phage est transformé, après excision et circularisation de la région plasmidique par un processus de biologie moléculaire, en une structure plasmide fonctionnelle (figure 1 annexée). Cette figure illustre les étapes a. et b. de conversion d'un vecteur de type phage : a. Insertion de l'ADNc cloné dans le site de multiclonage b. Conversion du vecteur hybride de clonage par excision de la région plasmidique contenant l'ADNc cloné. To go in this direction, a class of vectors highly appreciated for the production of nucleotide banks has thus been proposed, namely the "phage / plasmid" hybrids also called hybrid cloning vectors. These hybrids are phage constructs comprising a plasmid region which can be excised by a

site specific recombination molecular system, the cloned DNA being integrated at the level of said plasmid region. This type of hybrid vector then allows subcloning of the sequence of interest in an "automatic" manner. The insert initially integrated into a phage characteristic structure is transformed, after excision and circularization of the plasmid region by a process of molecular biology, into a functional plasmid structure (FIG. 1 attached). This figure illustrates steps a. and B. conversion of a phage type vector: a. Insertion of the cloned cDNA into the multicloning site b. Conversion of the hybrid cloning vector by excision of the plasmid region containing the cloned cDNA.

The materials c. to 1. implemented in these steps are: c. Hybrid phagemid cloning vector d. Specific LoxP e recombination site. Plasmid region f. Multicloning site g. Left arm of the phagemid vector

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h. Right arm of the phagemid vector i. CDNA insert j. Plasmid obtained by conversion k. DNA sequence of interest
1. LoxP homologous recombination site This "conversion" of a phage characteristic vector into a plasmid characteristic vector requires the excision of the plasmid region initially integrated into the vector. In the state of the art, the molecular mechanism used to obtain excision of the plasmid sequence is in general a site-specific recombination system, and still preferentially the molecular mechanism of Cre / Lox type as described in US patent B-4.959, 317.

conversion (Palazzolo et al., 1990 ; Elledge et al, 1991). La région plasmidique du vecteur phagemide est comprise entre deux sites Lox. La conversion de vecteur ou l'excision et la circularisation de la région plasmidique, est obtenue par infection par ledit vecteur d'une souche bactérienne dite de"conversion"comportant une activité enzymatique Cre, telle la lignée BM25.8 (Palazzolo et al., 1990). Conversion processes, applying this Cre / Lox molecular mechanism, are encountered in particular in the phagemid / bacterial strain binary systems.

conversion (Palazzolo et al., 1990; Elledge et al, 1991). The plasmid region of the phagemid vector is between two Lox sites. Vector conversion or excision and circularization of the plasmid region is obtained by infection with said vector of a so-called "conversion" bacterial strain comprising a Cre enzymatic activity, such as the line BM25.8 (Palazzolo et al. , 1990).

 There are commercial kits comprising a binary hybrid vector cloning / bacterial conversion strain system and greatly facilitating this subcloning step. Mention may in particular be made of the "SMART cDNA Library Construction Kit" from ClontechTM.

un vecteur phagemide pouvant être converti (ì"TriplEx2TM, Clontech ; SCREENM ou BlueSTAR, Novagen), et une bactérie de conversion comportant une activité Cre et capable d'exciser la région plasmidique du phagemide, telle la souche BM25.8 (Palazzolo et al.,
1990). These bank building kits typically contain:

a phagemid vector which can be converted (ì "TriplEx2TM, Clontech; SCREENM or BlueSTAR, Novagen), and a conversion bacterium comprising a Cre activity and capable of excising the plasmid region of the phagemid, such as the strain BM25.8 (Palazzolo et al .,
1990).

In this type of kit, the hybrid cloning vectors comprising the cloned sequence or sequences are initially multiplied in a bacterial strain adapted on an agar culture medium. The multiplication of hybrid cloning vectors then makes it possible to obtain lysis ranges, each lysis range corresponding to a vector containing a different cloned sequence. Each lysis range is then recovered individually and then brought into contact in a liquid medium with the conversion bacteria. In the end, each medium comprises conversion bacteria transformed by the construction originating from the nucleotide bank in a vector of plasmid type.

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BlueSTARM Vector System et XSCREENTM Vector Manual, Novagen), les séquences plus courtes sont sur-représentées en raison d'un biais d'amplification, les inserts dont l'expression est toxique pour dans la bactérie de conversion, ne peuvent pas être convertis en plasmides et sont perdus. The kits using this liquid conversion procedure, although saving a certain amount of time, still have certain drawbacks: - the number of manipulations (one per clone) to achieve subcloning remains significant, - the handling time increases with the number of hybrid vectors to be converted, a number which can become significant when the person skilled in the art works on a cDNA or genomic bank, - the conversion yield is relatively low (10 to 20% according to the manual

BlueSTARM Vector System and XSCREENTM Vector Manual, Novagen), the shorter sequences are over-represented due to an amplification bias, the inserts whose expression is toxic for in the conversion bacterium, cannot be converted into plasmids and are lost.

 Despite the numerous problems posed by the conversion processes in liquid medium, the person skilled in the art always uses, for lack of anything better, this process for the automatic subcloning of nucleotide banks.

 Under these circumstances, one of the essential objectives of the present invention is to provide a method making it possible to improve the conversion of the hybrid cloning vectors, in particular in terms of ease of implementation and speed of execution, as well as of conversion efficiency.

 Another essential objective of the present invention is to propose a process for subcloning hybrid vectors of simple cloning which is rapid and efficient and which also makes it possible to solve the particular problem of the inserts of nucleotide sequences to be cloned, expressing toxicity in bacteria of conversion, which cuts short the replication process of the considered sequences.

 Another essential objective of the present invention is to propose a method for cloning nucleotide sequences of interest, involving hybrid cloning vectors which are multiplied in cells cultured on gelled growth medium, which are then converted that we correctly subclone at almost industrial rates.

 Another essential objective of the present invention is to provide a kit for cloning nucleotide sequences of interest and / or for constructing genomic libraries, which comprises a binary system: hybrid cloning vector / bacterial conversion strain and which is simple, economical, fast and efficient.

 These objectives, among others, are achieved by the invention, which relates first of all, to a process for converting hybrid cloning vectors by means of at least one competent cell strain of conversion, characterized in that this conversion is produced en masse, at least in part on at least one solid support.

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 It is to the credit of the inventors to have found, quite surprisingly and unexpectedly, that the simplification of the process requires work in the solid phase so as to guarantee segregation between the cloning lines.

 Within the meaning of the invention, the qualitative "solid" includes several solid states of variable hardness: solid stricto sensu, semi-solid, gelled or pasty. In any event, this corresponds to non-liquid states of matter unfit for excessive circulation and diffusion of liquids such as aqueous suspension solutions, so as to avoid mixtures of populations of biological entities.

 It follows that according to a preferred characteristic of the invention, it is ensured that the internal flows of liquids are non-existent or limited, so as to avoid the exchange of material between the differentiated zones of the medium comprising distinct nucleotide sequences to be cloned cl to cn, This method of converting hybrid cloning vectors makes it possible to significantly improve, both qualitatively and quantitatively, the individual conversion process (clone by clone) in liquid medium, traditionally applied in commercial kits.

 By "conversion process" is meant an automatic process, preferably a molecular biology process, allowing the excision, in an initial vector, of a sub-vector and the transformation of this sub-vector, into a converted vector more easily exploitable (preferably a plasmid).

 By "vector" is meant a nucleotide construct, conventionally encountered in genetic engineering, and preferably chosen from the family of plasmids, cosmids, bacteriophages or viruses.

 A hybrid cloning vector is obtained by genetic engineering techniques known to those skilled in the art (Sambrook et al., 1989). For the synthesis of hybrid cloning vectors, it is also possible to take into account patent applications WO 88/05085, US 5286636, US 5851808 and WO 01/04288.

 In the present description, a "hybrid cloning vector" is an initial vector, into the genome of which is integrated another vector sequence which can be excised and transformed into another functional vector, for example by circularization. We will speak of a subcloning vector for the vector excised from the hybrid cloning vector and transformed during the conversion.

 In a preferred embodiment of the invention, the hybrid cloning vector is a phagemid vector. This vector then corresponds to a hybrid vector for cloning of phage characteristic comprising a vector of subcloning of the plasmid type, integrated into its genome.

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 According to alternatives, the hybrid cloning vector could no longer be a virus but, for example, a plasmid propagated by a bacterium or a yeast, or a simple DNA molecule.

 The conversion of the hybrid cloning vector is obtained automatically by the integration of said vector into a suitable bacterial strain of conversion. It is said that the hybrid cloning vector and the bacterial conversion strain constitute a suitable binary vector / bacterial conversion strain system because the conversion of the hybrid vector requires the use of an adequate bacterial strain. A binary system is therefore understood to mean a system comprising a hybrid cloning vector as described above, and a suitable bacterial strain comprising the enzymatic activity or activities allowing excision and circularization of the subcloning vector.

 In order for conversion to take place, the cloning vector must be able to penetrate the conversion strain. Viral cloning hybrid vectors can invest in the bacterial strain of infection.

Modes of biological transfer by sexual reproduction, such as conjugation, are suitable as soon as the cloning vector is propagated by a yeast or a bacterium. The transfection will be used in the case where a simple DNA molecule is used.

 In the case where we are dealing with phagemids, their automatic conversion is based on molecular biology mechanisms, and preferably recombination processes. The subcloning vector (phagemid) should preferably be surrounded by two recombination sites allowing its excision and its circularization when the hybrid cloning vector is integrated into a suitable bacterial conversion strain. These recombination sites are in particular of the Cre / Lox type as described in patent US4959317 (Du Pont, 1987), or of the RecE / T and Rec alpha / beta type as described in patent WO 01/04288 (Europe Molecular Biology Laboratory, 1999).

In one embodiment of the invention, these recombination sites are, for example, of the specific recombination site type, and more particularly still of the Cre / Lox type. The hybrid cloning vector then comprises two specific Lox recombination sites framing the subcloning vector, which allows the excision of this vector and the obtaining of a functional subcloning vector.

The bacterial conversion strain used in this binary system then expresses a Cre enzymatic activity and, more preferably, the strain is chosen from the line BM25.8 (Palazzolo et al., 1990). Among the hybrid cloning vectors used in commercial kits, there may be mentioned the phagemid ATriplEx2TM from Clontech, or the hybrid cloning vectors ASCREENTM and ABlueSTARTM from Novagen (Doherty et al., 1993).

Mention may also be made of the hybrid cloning vectors which can be obtained by

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 WO 88/05085. A person skilled in the art is able to choose the phagemid vector which is known to him, and suitable for the application sought by it.

 These vectors have in particular the advantage of having a strong power of replication. Under these conditions, the multiplication of hybrid cloning vectors takes place by bringing them into contact with a suitable bacterial strain, that is to say sensitive to said viral vectors. Preferably, the bacterial strain is the XLI-Blue strain (Bullock et al., 1987). In a semi-solid medium, the transfected XLI-Blue bacteria lead to the formation of lysis plaques, each lysis plaque corresponding to a phagemid clone.

 Preferably, these cloning and subcloning vectors contain multi-cloning sites. By multi-cloning sites is meant a region comprising a succession of unique restriction sites in the nucleotide sequence of these vectors.

citer pour exemple les sites de restriction Sau3A I, BamH I, Xho I. These multi-cloning sites allow the insertion of nucleotide sequences of interest into a determined site while using the most suitable restriction enzyme. We can

cite as an example the restriction sites Sau3A I, BamH I, Xho I.

 However, before proceeding with the conversion, it is important that the hybrid cloning vectors and their contents, namely the various nucleotide sequences of interest CI to cn, have proliferated in a compartmentalized manner.

 Thus, in accordance with the invention and prior to the conversion: - hybrid cloning vectors V ci to Vcn are multiplied which correspond to nucleotide sequences of interest to clone cl to Cn, by means of at least one strain competent cell, preferably at least one multiplication bacterium, cultivated in and / or on at least one solid multiplication medium, - and simultaneously collected, in and / or on this solid multiplication medium, by means of at least one solid support, multiplied Vci to Vcn cloning vectors.

au moins un support solide, avantageusement sous forme de feuille, au moins un milieu solide, pour la culture de cellules (e. g bactéries) de multiplication porteuses (de préférence infectées par) des vecteurs hybrides de clonage comprenant des séquences nucléotidiques distinctes à cloner ci à cn, * au moins un milieu solide pour la culture de cellules (e. g bactéries) de conversion porteuses (de préférence infectées par) des vecteurs hybrides de clonage issus du milieu de multiplication. In the preferred embodiment of the invention, the following in particular are used as essential material means:

at least one solid support, advantageously in the form of a sheet, at least one solid medium, for the culture of multiplying cells (eg bacteria) carrying (preferably infected with) hybrid cloning vectors comprising distinct nucleotide sequences to be cloned ci à cn, * at least one solid medium for the culture of conversion cells (e. g bacteria) carrying (preferably infected with) hybrid cloning vectors derived from the multiplication medium.

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 Preferably, the same solid support is used for the collection of the hybrid Vel to Vcn cloning vectors multiplied and for the conversion. This solid support (eg a leaf) is in this case seeded with conversion cells (preferably bacteria). This corresponds to the interesting operating protocol given below: multiplication on at least one solid culture medium, of bacteria comprising (preferably infected with) hybrid cloning vectors carrying the nucleotide sequences of interest Ci to Cn to be cloned, so to obtain a plurality of lysis plaques each corresponding to a sequence of interest to be cloned Cx, use of at least one solid support seeded with conversion bacteria, simultaneous collection of the hybrid cloning vectors contained in the various plaques lysis of the solid (eg gelled) multiplication medium, by means of a solid support seeded with conversion bacteria, cultivation of this (or these) solid support (s), comprising the hybrid cloning vectors and conversion bacteria, on at least one selective solid medium (eg gelled or pasty) allowing the selection and isolation of colonies of bacteria of conversion containing clones of hybrid vectors converted into plasmids carrying the sequences of interest cl to cn.

 The bacterial colonies comprising the converted hybrid vectors advantageously form on the solid conversion support. Each colony formed on the solid support comes from a bacterial clone having integrated a hybrid cloning vector. These colonies formed by the conversion bacteria, which have proliferated and which have thus multiplied the hybrid cloning vectors in a converted form.

 The solid multiplication culture medium containing the hybrid cloning vectors is a gelled culture medium, for example agar, allowing the maintenance or the multiplication of said vector. In the preferred embodiment, namely that in which the vector is of the phagemid type, the following process can be implemented: the hybrid cloning vector is brought into contact in a liquid medium with the bacterial strain which can be infected with this vector and allowing its multiplication; after a determined contact time, the hybrid cloning vector / bacterial strain mixture is diluted in a hot liquid medium (42 C) transforming into a cold semi-solid medium such as LB / 0.7% agar medium; - The agar culture medium has a certain density of lysis range, each lysis range corresponding to a clone of the hybrid cloning vector of interest.

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 The bacterial strain allowing the multiplication of the vector can in particular be chosen from XLl-Blue strains (Bullock et al., 1987), ER1647 (Wyman et al. 1986; Wertman et al. 1986; Raleigh et al. 1989; Doherty et al . 1993).

 The invention, in its preferred implementation protocol, proceeds from the surprising idea of integrating the host cell allowing the conversion of the hybrid cloning vector onto the solid conversion support.

 This invention allows in particular the simultaneous conversion of all of the hybrid cloning vectors present on the same Petri dish.

 The preferred route of mass conversion on solid support of the invention overcomes the drawbacks encountered by conventional methods of conversion in liquid medium and in particular: - to obtain a simultaneous conversion of several clones of hybrid cloning vectors while reducing the number of steps - to improve the conversion yield - to eliminate the amplification bias - to allow the safeguarding of toxic clones.

 This process of conversion onto a solid support of the invention allows direct obtaining of bacterial colonies on a solid support, each colony containing a hybrid vector for cloning the library in a converted form.

 In terms of conversion efficiency, the comparison between the known liquid phase conversion methods and the bulk or solid phase method according to the invention can be made on the basis of the following data: - the individual phase conversion method liquid makes it possible to obtain a hundred conversion of hybrid vectors per day and per manipulator, knowing that each clone converted then corresponds to coring, dilution in an Eppendorfg tube, a liquid culture and then spreading on a dish; - the globalized, mass conversion process on solid support, according to the invention, gives nearly 12,000 bacterial clones per manipulator per day, which corresponds to the conversion of as many hybrid cloning vectors, which requires no handle only 40 boxes and 40 membranes.

 These results clearly demonstrate the advantages of the solid-phase mass conversion technique of the invention compared to the technique conventionally used in liquid phase conversion kits.

 Preferably, the preliminary step of seeding the solid support with the conversion bacteria can be carried out as follows:

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 the solid support is sown, preferably in sheet form, by impregnation, preferably over the entire surface of the solid support sheet, with a liquid suspension containing the conversion bacteria, the impregnated solid support is dried before serve for the collection of hybrid cloning vectors.

 More concretely still, the impregnation of the solid support (e. G. Of the sheet) is carried out using the suspension of conversion bacteria on one of the faces of this sheet of solid support. In fact, the required volume (for example a drop) of conversion bacteria suspension is deposited on a surface, preferably hydrophobic, and one of the faces of the solid support sheet is applied to this volume of suspension. . The surface in question can be a plastic or glass plate or the lid of a petri dish.

 The seeding of a specific face of the solid support makes it possible in particular: a) to favor the contact of conversion cells / hybrid cloning vectors, by placing the seeded face downwards, during the collection of said vectors on the multiplication medium; b) limit the contamination of the gelled selection medium (eg agar) and allow replication in the form of bacterial colonies during the conversion phase, by placing the seeded side up.

 After the impregnation, the solid support seeded with the bacterial conversion strain is recovered on a single face, and the pre-seeded solid support is dried.

 Advantageously, the drying of the solid support sheet is carried out using absorbent paper, of the "Whatman" type paper or any other suitable blotting paper.

As another conceivable drying means, mention may be made of aspiration, air or gas flow, lyophilization.

 As regards the quantity of conversion seeds deposited on the solid support, it is determined by a volume rate of between 5 and 20 III of suspension per cm 2 of solid support, the suspension having an OD of between 0.5 and 1, 5, for example 1; which corresponds to a determined and sufficient number of germs.

 Insofar as the solid support (eg the sheet) advantageously has a size and a shape corresponding to those of the surface on which the collection is

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 performed, it is important that the impregnation of the solid support is as complete as possible.

 Furthermore, in order to respect one of the foundations of the invention, which is the absence of streams of liquids capable of jeopardizing the segregation between the different clones, care must be taken to ensure that the solid support dries correctly, after impregnation.

 In a variant of the method according to the invention, the seeding of the solid support (for example by impregnation and drying) can be done, not immediately before collecting the hybrid cloning vectors, but in advance. The seeded support is then stored under conditions such that it remains in a good state of conservation.

To this end, the solid support seeded can optionally be subjected to a treatment which makes it possible to keep the support in a pre-seeded form for later use. The treatments allowing the conservation of the support are known to the person skilled in the art, and can in particular be chosen from lyophilization or freezing.

The solid support thus pre-seeded is thus ready for use.

 For the collection of hybrid cloning vectors, the solid support inoculated is advantageously deposited on the surface of the multiplication culture medium, then the whole is left to incubate, advantageously from 1 to 180 min, more especially from 15 to 120 min.

 After incubation and transfer of the hybrid cloning vectors onto the solid support, the latter is deposited on the surface of the selective conversion culture medium, then the whole is cultured under appropriate conditions.

 According to an advantageous characteristic of the invention, the impregnation face of the solid support sheet is that which is brought into contact with the surface of the multiplication culture medium to collect the hybrid cloning vectors to be converted, while the face opposite of the solid support sheet is that which is brought into contact with the surface of the selective conversion culture medium.

 As a result, the colonies of conversion bacteria form on the upper face of the solid support placed on the selective conversion culture medium. This corresponds to a preferred method, but this does not exclude variants in which the face of the solid support brought into contact with the multiplication medium would also be that applied to the surface of the conversion culture medium.

 Similarly, according to another variant, it could be envisaged to use as solid support only the conversion culture medium, which would be pre-seeded on its surface with the conversion bacteria, as described above, then the latter. would then be brought into contact with the multiplication medium to collect the multiplied hybrid cloning vectors.

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 It appeared quite appropriate that the solid support used, when it consists of a means distinct from the conversion culture medium, be porous (preferably with a porosity of between 0.1 and 1 μm), so as to be permeable with nutrients and absorbent. Biocompatibility is also a required specification for the solid support. Indeed, it must allow the anchoring of the conversion bacteria and hybrid cloning vectors, the growth of the said conversion bacteria (in particular on a suitable selective culture medium) and, moreover, the passage of the nutrients intended for the conversion bacteria. growing.

 The solid support, which is at least partly the site of the conversion of the cloning vector, must also have sufficient mechanical strength, that is to say be capable of withstanding a series of manipulations carried out by a person skilled in the art. In particular, it must not tear when handled in a humidified state.

 In addition, this support should not limit the integration of the cloning vectors into the conversion bacteria, and therefore should not limit the process of conversion of the hybrid cloning vectors.

 Thus, the material constituting the solid support is preferably selected from the group comprising: - cellulose and its derivatives, preferably nitrocellulose, - nylon and its derivatives - supports based on silica or glass fibers.

 In practice, the solid support is preferably a filter or a nitrocellulose membrane. This type of membrane has the advantage of being widespread, of allowing good adsorption of microorganisms, of having good resistance to handling, of allowing the passage of nutrients, of being biocompatible and of not interfering with integration of vectors into conversion bacteria.

For example, the nitrocellulose membrane used is a membrane from the brand Shleicher & Schnell &commat; (Optitran &commat; called 100% nitrocellulose) or Amersham (S (nylon membrane) with average porosity 0.45 µm.

 Another important characteristic of the invention is that at least one selection marker is used in the selective conversion culture medium, so as to identify the conversion bacteria that have integrated and converted a hybrid cloning vector. .

peut citer notamment les gènes conférant une résistance aux antibiotiques tels que : - le gène npt II pour la résistance à la kamamycine (Bevan et al., 1983), Thus, the vectors for cloning and subcloning can in particular contain selection genes or reporter genes. Those skilled in the art will use the selection marker that they think is most suitable. Among the marker genes commonly used,

may include in particular the genes conferring resistance to antibiotics such as: the npt II gene for resistance to kamamycin (Bevan et al., 1983),

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- ou le gène hph pour la résistance à l'hygromycine (Gritz et al., 1983).

- or the hph gene for resistance to hygromycin (Gritz et al., 1983).

(Gerdes et al., 1996), la ss-glucuronidase (Jefferson, 1987), ou le gène ccdB (Bernard P. et al, 1994). It is also possible to use in the construction of the vector, a reporter gene which will allow a simple and rapid "screening" of the cells having integrated into their genome a heterologous sequence. This gene can in particular be luciferase, GFP

(Gerdes et al., 1996), ss-glucuronidase (Jefferson, 1987), or the ccdB gene (Bernard P. et al, 1994).

un gène conférant une résistance aux antibiotiques, -un gène rapporteur choisi dans le groupe comprenant les gènes codant pour : la luciférase, la GFP, ou la ss-glucuronidase, le gène ccdB. It follows that according to a preferred characteristic of the invention, this marker is chosen from the group comprising:

a gene conferring resistance to antibiotics, a reporter gene chosen from the group comprising the genes coding for: luciferase, GFP, or ss-glucuronidase, the ccdB gene.

The method of conversion to solid support of the invention therefore allows the conversion of all of the hybrid cloning vectors present in a solid culture medium, advantageously agar, and this in a single manipulation. This method also has the advantage, when using the phagemid vector, of making it possible to obtain an exact replica of each lysis range in the form of a bacterial colony.

 According to one example, the microorganisms of the binary conversion system are as described above, namely a binary system comprising the bacterial strain BM 25.8 and a phagemid. The solid support, for its part, is preferably a nitrocellulose filter.

(bactéries de conversion et vecteurs hybrides de clonage) sur les différents supports..., l'homme du métier peut être amené, en pratique, à procéder à certaines adaptations en s'appuyant sur les conditions optimales préférées selon l'invention et définies ci-dessus. Ces conditions optimales éventuellement adaptées, doivent permettre d'obtenir le meilleur rendement de conversion des vecteurs hybrides de clonage ainsi que le développement le plus important des micro-organismes de conversion sur le support solide. Concerning the variable parameters of the protocol, and in particular the contact times of the solid support with the culture media, the density of microorganisms

(conversion bacteria and hybrid cloning vectors) on the various supports, etc., those skilled in the art may, in practice, have to make certain adaptations based on the preferred optimal conditions according to the invention and defined above. These optimal conditions, possibly adapted, must make it possible to obtain the best conversion efficiency of the hybrid cloning vectors as well as the greatest development of the conversion microorganisms on the solid support.

 According to another of its aspects, the invention also relates to a method for cloning of nucleotide sequence (s) of interest, using hybrid cloning vectors preferably of the phagemid type, in which: the sequences ( s) nucleotide (s) of interest CI to Cn are inserted into hybrid vectors of cloning Vcl to Vcn,

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Vc, à Vcn et sont mises en culture à des fins de multiplication, les vecteurs hybrides de clonage Vcl à Vcn multipliés sont mis en contact avec des souches cellulaires compétentes (de préférence des bactéries) de conversion des inserts CI à Cn en plasmides, les souches cellulaires compétentes (de préférence des bactéries) de conversion sont mises en culture, pour que la conversion s'opère et pour produire in fine des colonies de bactéries de conversion correspondant aux clones plasmidiques ci à cn, caractérisé en ce qu'il intègre le procédé de conversion tel que défini ci-dessus. competent cell strains (preferably bacteria) comprising (preferably infected with) these hybrid cloning vectors

Vc, to Vcn and are cultured for multiplication, the hybridized cloning vectors Vcl to Vcn are brought into contact with competent cell strains (preferably bacteria) for converting the CI to Cn inserts into plasmids, competent cell strains (preferably bacteria) of conversion are cultured, so that the conversion takes place and to ultimately produce colonies of conversion bacteria corresponding to the plasmid clones ci to cn, characterized in that it incorporates the conversion process as defined above.

 By definition, such a cloning method includes an automatic sub-cloning module for nucleotide sequences. Also, the invention relates, in addition, to a method of automatic subcloning of nucleotide sequences, incorporating the conversion as defined above.

 By "automatic subcloning method" is meant a method consisting in transferring a polynucleotide of interest initially integrated in one type of vector to another type of vector. This process is said to be automatic since this hybrid vector can directly give the subcloning vector by excision and circularization via a process of molecular recombination.

 In the context of a hybrid cloning vector, the nucleotide sequence of interest must be introduced into the subcloning vector so that automatic subcloning can take place. The sequence may be unique or be part of a nucleotide library. The polynucleotide (s) may then be of genomic, complementary or synthetic DNA type.

 The polynucleotide (s) of interest will be introduced into the hybrid cloning vector at the multicloning site. Those skilled in the art are able to select the appropriate restriction enzymes allowing them to integrate the heterologous sequence of interest.

 In the context of the invention, automatic subcloning is obtained by the conversion of the hybrid cloning vector, comprising at least one nucleotide sequence of heterologous interest, by the appropriate bacterial conversion strain.

 In a preferred embodiment of the invention, the conversion to solid support uses the biological material and the solid support as described above. The cloning vector of the binary system must be as described previously in the invention, and preferably be of the phagemid type. This vector must preferably include at least one multicloning site in the region of the subcloning vector.

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 to allow insertion of the nucleotide sequence of interest. The conversion then allows automatic sub-cloning of the sequences making up the nucleotide library in plasmid vectors. The automatic conversion system preferably uses the Cre / Lox site-specific recombination system. As described above, the binary system is composed of a hybrid phagemid-type cloning vector containing Lox sites surrounding the plasmid region, and of a bacterial strain of BM 25.8 conversion. The solid support, for its part, will preferably be a nitrocellulose membrane.

* isolement des vecteurs hybrides de clonage toxiques, * amplification des vecteurs hybrides de clonage toxiques, * extraction de l'ADN ou PCR, * puis séquençage direct de l'insert toxique Ci ou sous-clonage dans un vecteur vis à vis duquel l'insert n'est pas toxique, * récupération des clones de séquences toxiques Ct.. According to a singular modality of the invention, in the context of the aforementioned cloning, subcloning of any heterologous toxic sequences Ci is carried out with respect to the competent cell strains (preferably bacteria) for converting the inserts cl to Cn in plasmids, using the following steps: * comparison of the lysis ranges of the gelled multiplication culture medium and of the colonies of conversion bacteria corresponding to the plasmid clones cl to Cn and present on the solid support (sheet) and / or on the conversion culture medium, in order to identify any lysis ranges which do not have their corresponding cloning colonies,

* isolation of hybrid toxic cloning vectors, * amplification of hybrid toxic cloning vectors, * DNA extraction or PCR, * then direct sequencing of the toxic insert Ci or subcloning in a vector with which the insert is not toxic, * recovery of clones of toxic sequences Ct.

 The method of the invention therefore allows the conversion of toxic clones, that is to say clones comprising a polynucleotide which. if it is expressed in the conservation bacteria causes toxicity. Indeed, by mass conversion on solid medium as provided for in accordance with the invention, the toxic hybrid vectors do not give bacterial colonies. It is then possible to recover the hybrid vectors not adsorbed on the filter to use an alternative subcloning process known to those skilled in the art. The alternative subcloning method can be chosen from one of the following subcloning methods: - amplification of the phage and extraction of the DNA sequence of interest from the hybrid cloning vector, or - PCR reaction and direct sequencing if alone a probe is sought.

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* au moins un vecteur hybride de clonage, * au moins une souche bactérienne de conversion adaptée au vecteur hybride de clonage, sous forme de suspension prête à l'emploi et/ou sous forme d'inoculum permettant la préparation d'une suspension, * au moins un support solide pour les bactéries de conversion et permettant la collecte des vecteurs hybrides de clonage sur les plages de lyse du milieu de culture de multiplication, * et éventuellement des moyens de séchage du support solide après imprégnation. The subject of the invention is also a kit, in particular for implementing the method as defined above, characterized in that it comprises in particular the following elements:

* at least one hybrid cloning vector, * at least one bacterial conversion strain adapted to the hybrid cloning vector, in the form of a ready-to-use suspension and / or in the form of an inoculum allowing the preparation of a suspension, * at least one solid support for the conversion bacteria and allowing the collection of hybrid cloning vectors on the lysis ranges of the multiplication culture medium, * and optionally means for drying the solid support after impregnation.

According to a variant, the kit is characterized in that it comprises in particular the following elements: * at least one hybrid cloning vector, * at least one solid support stable during storage-preferably in lyophilized form-pre-seeded with at least a bacterial conversion strain adapted to the hybrid cloning vector and allowing the collection of hybrid cloning vectors on the lysis ranges of the multiplication culture medium,
Advantageously, the solid support is a nitrocellulose membrane, preferably of substantially the same shape and the same dimensions as the surfaces of the multiplication culture medium and of the conversion culture medium, on which it is intended to be deposited.

 This concerns nucleotide sequence cloning kits or nucleotide library construction kits using hybrid cloning vectors allowing automatic subcloning of the cloned nucleotide sequences. This kit will also allow the subcloning of toxic sequences which cannot be obtained by the process of conversion into a liquid medium usually employed by a person skilled in the art.

 By solid support is meant any solid support as described above and which can be used in the process of the invention such as a nitrocellulose membrane.

We speak of a pre-seeded storable support to designate a support comprising bacteria belonging to the bacterial strain of conversion, said support being able to be kept for a determined period. These conversion bacteria must be able to

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 multiply after a period of latency on the solid support. The methods making it possible to conserve bacteria on a solid support, preferably of the nitrocellulose membrane type, are known to those skilled in the art. Those skilled in the art will preferably use a lyophilization process, and potentially a freezing process.

 According to a preferred embodiment of the invention, the kit comprises a binary system using the Cre / Lox site-specific recombination process. More preferably, the phagemid hybrid vector is a ÂTriplEx2TM, ÂSCREENTM or BlueSTAR vector and the nitrocellulose membrane is pre-seeded with the bacterial conversion strain BM25.8 (Palazzolo et al., 1990).

 Optionally, the conversion kit may in particular contain media allowing the multiplication of bacteria (LB medium, for example), an in-vitro packaging system in the case of the use of phage vectors, a competent bacterial strain allowing the multiplication of vectors (E. coli XLI-Blue: Bullock et al., 1987; ER1647: Wyman et al. 1986, Wertman et al. 1986, Raleigh et al.

1989, Doherty et al. 1993), enzymes allowing the integration of the inserts into the vectors (restriction enzymes: Sau3A, BamH 1, Xho 1, DNA ligase), a medium for selection of the bacterial conversion strains containing the converted vector (LB / carbenicillin or LB / ampicillin).

 The invention also relates to the use of the kit, as described above, for improving the efficiency of subcloning of one or more different sequences present in hybrid cloning vectors. By improving the subcloning yield is meant more particularly the possibility of performing a subcloning simultaneously of all of the hybrid cloning vectors present in a culture dish. This type of kit also makes it possible to increase the percentage of conversion of the hybrid cloning vectors on solid support, in comparison with the conversion process in liquid medium.

 The invention also relates to the use of a kit, as described above, to allow the subcloning of one or more sequences toxic for conversion bacteria as described above.

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Description des Figures

Fi, 1 : Cette figure illustre : o les étapes a. et b. de conversion d'un vecteur de type phage o les matériels c. à 1. mis en oeuvre dans ces étapes Fleure 2 :
Cette figure illustre les étapes composant le procédé de conversion d'un vecteur hybride de clonage de type plasmide sur un filtre.

Description of Figures

Fi, 1: This figure illustrates: o the steps a. and B. conversion of a phage type vector o the materials c. to 1. implemented in these steps Flower 2:
This figure illustrates the steps making up the process for converting a hybrid plasmid-type cloning vector onto a filter.

Caption:
Steps: a. Seeding of one face of the solid conversion support b. Moderate drying c. Contact with the lysis ranges d. Turn the membrane over and put on the box with antibiotic e. Cultivation f. Obtaining colonies corresponding to the converted phages
Materials: g. nitrocellulose filter h. drop of a culture of conversion bacteria i. plastic support j. Whatman k type paper. box with lysis ranges
1. storage

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Exemples

Exemple 1 : Conversion d'un vecteur phac A fplEx2en plasmid 3TriplEx2'ur SMPPO Sû/ < /t ? M ) (matériel biologique provient du kit Clontech, SMARTTM Library Construction Kit : Triplex2, BM25.8)
Une banque primaire d'ADNc réalisée dans un phage TriplEx2, préalablement titrée, est étalée selon les protocoles recommandés par le fabricant du vecteur à une densité de 2-2, 2 pfu/cm2 et mise en culture une nuit (Clontech, SMARTTM cDNA Library Construction Kit, User Manual, 2000). Parallèlement, une préculture de la souche d'E. coli BM25.8 est réalisée à 31 C dans du LB + MgS04 10 mM également pendant une nuit. Il est important de s'assurer que l'humidité des boîtes de phage n'est pas trop importante sinon un séchage adéquat sera réalisé sous une hotte stérile.

Examples

Example 1: Conversion of a phac A vector fplEx2 into plasmid 3 TriplEx 2 'on SMPPO Sû / </ t? M) (biological material comes from the Clontech kit, SMARTTM Library Construction Kit: Triplex2, BM25.8)
A primary cDNA library produced in a TriplEx2 phage, previously titrated, is spread according to the protocols recommended by the vector manufacturer at a density of 2-2, 2 pfu / cm2 and cultured overnight (Clontech, SMARTTM cDNA Library Construction Kit, User Manual, 2000). In parallel, a preculture of the strain of E. coli BM25.8 is carried out at 31 ° C. in LB + MgS04 10 mM also overnight. It is important to ensure that the humidity of the phage boxes is not too high otherwise an adequate drying will be carried out under a sterile hood.

10mM en comptant 15ul/cm2 de boîte à convertir et ceci jusqu'à une DO"" de 1 en partant d'une dilution au 1/40ème de la culture overnight. La culture est alors supplémentée en MgCl2 à 1M à raison de lOul/ml soit une concentration finale de 10mM. The next day, a culture of the strain BM25.8 is carried out in LB + MgS04

10mM by counting 15ul / cm2 of box to convert and this up to an OD "" of 1 starting from a dilution to 1 / 40th of the overnight culture. The culture is then supplemented with MgCl2 at 1M at a rate of 10 μl / ml, ie a final concentration of 10 mM.

haut) et placé sur une boîte de milieu contenant l'ampicilline à lOOg/mI minimum et mis en culture une nuit à 31 C. Le lendemain, chaque plage de lyse aura donné naissance à une colonie portant le plasmide correspondant. Chaque colonie peut alors être repiquée dans du milieu sélectif pour être stockée ou utilisée selon le besoin du manipulateur. Cette manipulation doit être réalisée dans les 3 à 4 semaines suivant la conversion, si la membrane est bien conservée à 4 C, pour éviter que les bactéries de conversion ne sèchent sur le support. A nitrocellulose filter of suitable size is placed on a drop (15 μl / cm 2) of bacteria BM25.8 deposited on a sterile hydrophobic surface (flexible plastic or lid of petri dish for example). The entire filter must be impregnated when this step is carried out, the filter is turned over and allowed to dry on Whatman type paper (filter paper). It takes a few seconds and at the end no trace of liquid should be perceptible. The inverted filter (bacteria down) is placed on the box carrying the lysis ranges and incubated for 15 minutes at 31 ° C. (may be extended up to 1 hour). The filter is then removed by one end, turned over (phages towards the

above) and placed on a box of medium containing ampicillin at lOOg / mI minimum and cultured overnight at 31 C. The next day, each lysis range will have given rise to a colony carrying the corresponding plasmid. Each colony can then be subcultured in selective medium to be stored or used according to the needs of the manipulator. This manipulation must be carried out within 3 to 4 weeks following the conversion, if the membrane is well stored at 4 C, to prevent the conversion bacteria from drying out on the support.

 Each colony can be subcultured using a yellow Gilson pipetman cone in 100 ml of medium in a microtiter plate for culture and stored in the form of stock glycerol.

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Example 2: Test of different solid supports
To determine the solid support allowing an optimal conversion yield, various solid supports were tested with the protocol as described above in example 1. These supports are: - bench-top paper; - Whatman paper; filter paper; - Amershamg Hybond-N nylon membrane (porosity 0.45 u. M); - Millipore nitrocellulose membrane (D (0.45 M); - Shleicher & Schuell # and Amersham nitrocellulose membrane (R) (0.45 M).

The conversion presents an optimal yield on the membranes of nitrocellulose Schleicher & Schuell # (Optitran # called 100% nitrocellulose) or Amersham (R) of porosity 0.45pu.

Example 3: Extemporaneous preparation and conservation of the pre-seeded support
2 ml of BM 25.8 bacteria are cultured at 31 ° C. overnight until an optical density close to 1. This suspension is then used for inoculating Schleicher & Schuell &commat; nitrocellulose membranes. These membranes are finally prepared for long-term storage under different conditions: dried and stored at room temperature; - lyophilized 5 minutes and stored at 4 C; - lyophilized 20 minutes and stored at 4 C.

 After 2 days of storage under each of the conditions, the filters were returned to culture on dishes: - LB agar for monitoring the survival of the conversion bacteria, - LB agar + ampicillin in the mass conversion process.

 The membranes pre-seeded and preserved by lyophilization allow conversion of the hybrid vectors, while the membrane dried and preserved at room temperature is unable to convert the hybrid cloning vectors.

Lyophilization is suitable for the conservation, on membrane, of BM 25.8 conversion bacteria.

Example 4: Mass method / liquid method comparison Effectiveness of the different types of liquid / solid phase conversion

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In liquid phase
1. Individual conversion is 100% effective but very tedious
2. The conversion on the phage sweater can save time but the efficiency is very low: around 15 to 20%.

 In the solid phase on a nitrocellulose filter: the efficiency is around 100%, each clone being treated individually.

Efficiency of simultaneous conversion of several clones / individual conversion
Individual conversion to liquid phase: 100 clones can be made per manipulator and / day knowing that 100 Eppendorf tubes (g) and 100 boxes must be handled.

 Mass conversion in solid phase: we can reasonably make 40 boxes per manipulator and / day knowing that a box has 300 clones: that makes 12,000 clones / day.

 It is therefore necessary to handle 40 boxes and 40 membranes.

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 References - Bernard P. et al. : "Positive selection vectors using the F plasmid ccdB killer gene".

 (1994) Gene 148,71-74.

- Bevan et al. :
Nature, 1983, Vol 304, p 1184-187.

- Bullock et al. :
XL-1-Blue: a high efficiency plasmid transforming recA Escherichia coli strain with beta-galactosidase selection.

 BioTechniques (1987) 5: 376-379.

- Doherty et al: (1993) Gene 124,29-35.

- Elledge et al. :
Lambda YES: "a multifunctional cDNA expression vector for the isolation of gene for the complementation of yeast and Escherichia coli mutations", Proc. Natl. Acad. Sci.

 USA 88 (1991) 1731-1735).

- Gerdes et al. :
Green fluroescent protein: applications in cell biology.

- Gritz et al. :
Gene, 1983, Vol 25, p 179-188.1.

- Jefferson RA et al. :
GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.

 EMBO J. 1987 Dec. 20; 6 (13): 3901-7.

- Meese et al. : (1990) Quick method for high yields of lambda bacteriophage DNA, Nucleic Acids
Res, 18.19-23.

- Palazzolo et al. : (1989) Phage lambda cDND cloning vectors for subtractive hydridization, fusion-protein synthesis and Cre-loxP automatic plasmid subcloning, Gene, 88 (1990) 25-36.

- Raleigh et al. :
Genetic and physical mapping ofthe mcrA (rglA) and mcrB (rglB) loci of Escherichia coli K-12.

 Genetics. 1989 Jun; 122 (2): 279-96.

- Sambrook et al. : (1989) A laboratory manual 2nd ed, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY.

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- Whertman KF et al. :
Host / vector interactions which affect the viability of recombinant phage lambda clones.

 Uncomfortable. 1986; 49 (2): 253-62.

- Wyman AR et al. :
Factors which equalize the representation of genome segments in recombinant libraries.

 Uncomfortable. 1986; 49 (2): 263-71.

Claims (1)

 -l- Method for converting hybrid cloning vectors using at least one competent cell strain for conversion, characterized in that this conversion is carried out in bulk, at least in part on at least one solid support. - 2-A method according to claim 1, characterized in that one makes sure that the internal flows of liquids are non-existent or limited, so as to avoid exchanges of material between the differentiated zones of the medium comprising distinct nucleotide sequences to clone cl to Cn. - 3-A method according to claim 1 or 2 characterized in that, prior to conversion: we multiply hybrid vectors cloning Vcl to Vcn which correspond to nucleotide sequences of interest to clone cl to cn, using d '' at least one competent cell strain, preferably at least one mutiplication bacterium, cultivated in and / or on at least one solid multiplication medium, and is collected simultaneously, in and / or on this solid multiplication medium, by means of 'at least one solid support, the hybrid vectors of cloning Vcl to Vcn multiplied. - 4-A method according to any one of claims 1 to 3, characterized in that one uses the same solid support for the collection of hybrid Vc to Vcn cloning vectors multiplied and for the conversion. - 5-A method according to any one of claims 1 to 4, characterized in that it comprises in particular the following steps: multiplication on at least one solid culture medium, of bacteria comprising (preferably infected with) hybrid vectors of cloning carrying the nucleotide sequences of interest C to Cn to be cloned, so as to obtain a plurality of lysis plaques each corresponding to a sequence of interest to be cloned cx, use of at least one solid support seeded with bacteria conversion, <Desc / Clms Page number 25>

 simultaneous collection of hybrid cloning vectors contained in the different lysis ranges of the solid multiplication medium, by means of a solid support inoculated with conversion bacteria, cultivation of this (or these) solid support (s) ), comprising the hybrid cloning vectors and the conversion bacteria, on at least one solid selective medium allowing the selection and the isolation of colonies of conversion bacteria containing clones of hybrid vectors converted into plasmids carrying the sequences of interest cl to cn. - 6-A method according to claim 5, characterized in that it comprises a prior step of seeding the solid support with the conversion bacteria. - 7-A method according to claim 6, characterized in that: the seeding of the solid support-preferably in the form of sheet is carried out by impregnation-preferably of the entire surface of the solid support sheet-with a liquid suspension containing the conversion bacteria, the impregnated solid support is dried before being used for the collection of phagemids. - 8-A method according to claim 7, characterized in that the impregnation of the solid support sheet is carried out using the suspension of conversion bacteria on one of the faces of this solid support sheet, preferably at a rate of 5 to 20 μl / cm ', the suspension having an OD of between 0.5 and 1.5. - 9-A method according to claim 7 or 8, characterized in that the impregnation is carried out by depositing the required volume of suspension of conversion bacteria on a surface, preferably hydrophobic, then by applying one of the faces of the solid support sheet on the suspension. - 10-A method according to any one of claims 3 to 9, characterized in that: for the collection of phagemids, the solid support inoculated is deposited on the surface of the multiplication culture medium, then the whole is left to incubate advantageously from 1 to 180 min, more especially from 15 to 120 min, after incubation and transfer of the phagemids to the solid support, the latter is deposited on the surface of the selective conversion culture medium, then the whole is placed in culture under appropriate conditions. <Desc / Clms Page number 26>  - 11-A method according to claim 10, characterized in that the impregnation face of the solid support sheet is that which is brought into contact with the surface of the multiplication culture medium to collect the phagemids to be converted, while the opposite side of the solid support sheet is that which is brought into contact with on the surface of the selective conversion culture medium.  - 12-A method according to any one of claims 3 to 11, characterized in that the solid support used is porous (preferably of porosity between 0, 1 and 1 m), so as to be permeable to nutrients, absorbent, biocompatible, and preferably selected from the group comprising: - cellulose and its derivatives, preferably nitrocellulose, - nylon and its derivatives,

 supports based on silica or glass fibers. - 13-A method according to any one of claims 3 to 12, characterized in that the drying of the solid support sheet is carried out using absorbent paper.  - 14-Process according to any one of claims 3 to 13, characterized in that at least one selection marker is used in the selective conversion culture medium, so as to identify the conversion bacteria which have integrated and converted a phagemid; this marker being chosen from the group comprising:

 a gene conferring resistance to antibiotics, a reporter gene chosen from the group comprising the genes coding for: luciferase, GFP, or ss-glucuronidase the ccdB gene. - 15-Method for cloning a nucleotide sequence (s) of interest, using hybrid cloning vectors preferably of the phagemid type, in which: the nucleotide sequence (s) of interest cl to Cn are inserted into hybrid vectors for cloning VCI to Vcn, # competent cell strains (preferably bacteria) comprising (preferably infected with) these hybrid vectors for cloning VCI to Vcn and are cultured for multiplication purposes , # the multiplied hybrid cloning vectors Vc1 to Vcn are brought into contact with competent cell strains (preferably bacteria) for conversion of the inserts C1 to Cn into plasmids, # the competent cell strains (preferably bacteria) for conversion cultivated, for the conversion to take place and for <Desc / Clms Page number 27>

 ultimately produce colonies of conversion bacteria corresponding to the plasmid clones cl to cn, characterized in that it incorporates the conversion process according to any one of claims 1 to 14. - 16-A method according to claim 15, characterized in that one carries out a subcloning of any heterologous toxic sequences with respect to competent cell strains (preferably bacteria) for conversion of the CI to Cn inserts into plasmids, by putting implementing the following steps: * comparison of the lysis ranges of the gelled multiplication culture medium and of the colonies of conversion bacteria corresponding to the plasmid clones cl to Cn and present on the solid support (sheet) and / or on the culture medium conversion, in order to identify any lysis plaques which do not have their corresponding cloning colonies, * isolation of the hybrid toxic cloning vectors, * amplification of the hybrid toxic cloning vectors, * DNA extraction or PCR, * then direct sequencing of the toxic insert Ci or subcloning in a vector with respect to which the insert is not toxic, * recovery of the clones of se toxic consequences ct. - 17-Kit in particular for implementing the method according to any one of claims 1 to 16, characterized in that it comprises in particular the following elements: * at least one hybrid cloning vector, * at least one strain bacterial conversion adapted to the hybrid cloning vector, in the form of a ready-to-use suspension and / or in the form of an inoculum allowing the preparation of a suspension, * at least one solid support for the conversion bacteria and allowing the collection hybrid cloning vectors on the lysis ranges of the multiplication culture medium, * and optionally means for drying the solid support after impregnation. 18-Kit in particular for implementing the method according to any one of Claims 1 to 16, characterized in that it comprises in particular the following elements: * at least one hybrid cloning vector, <Desc / Clms Page number 28>  * at least one solid support stable during storage - preferably in lyophilized form - pre-seeded with at least one bacterial strain of conversion adapted to the hybrid cloning vector and allowing the collection of hybrid cloning vectors on the lysis ranges of the medium of multiplication culture.  - 19-Kit according to any one of claims 17 or 18, characterized in that the solid support is a nitrocellulose membrane, preferably substantially of the same shape and the same dimensions as the surfaces of the multiplication culture medium and the medium of conversion culture, on which it is intended to be deposited.