ES2389792A1 - System for assembly of genetic pieces. (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

System for assembling genetic pieces. The present invention describes an in vitro assembly system for genetic pieces. The assembly of dna fragments forms the basis of genetic engineering and synthetic biology. For the design of new genetic circuits, both disciplines tend towards the generation of collections of interchangeable and recyclable genetic pieces (that is, susceptible to being used in different laboratories and to generate different genetic combinations), which can be joined together through the use of a standard method of assembly. It is particularly necessary to develop methods that allow great efficiency and versatility in the assembly of pieces in the ranges that go between 5 and 50 individual pieces, since the modular nature of the genetic interactions means that a good part of the engineering is developed around to genetic designs that encompass these size ranges. (Machine-translation by Google Translate, not legally binding)

Description

System for assembling genetic parts

The present invention is comprised in the technical sector of Biotechnology, Synthetic Biology and by extension to Agricultural Biotechnology and Plant Synthetic Biology for the creation of transgenic, cisgenic and / or intragenic plants with new agronomic characters.

STATE OF THE PREVIOUS TECHNIQUE

The assembly of basic functional DNA structures (hereinafter referred to as "pieces" or "parts") to produce new combinations (referred to hereafter as "modules", or "devices" if they result from the assembly of two or more modules ) is the basic tool of Synthetic Biology.

Traditionally, multiple assembly of DNA fragments has been carried out on plasmids containing a multiple cloning site (MCS). The MCS consists of a DNA sequence with a variable number of type II restriction enzyme targets (ERTII). In this methodology, the assembly of one or more fragments to form structures of increasing complexity is carried out by successive insertions of new pieces, using the restriction targets present in the MCS. Multiple clones based on traditional MCS have important limitations, mainly the presence of internal restriction targets in the new fragments to be assembled, the finite number of targets of the MCS, the technical complexity, which includes electrophoretic separation / purification of the fragments, or the permanence of the target sequences in the junction zones between the assembled parts (hereinafter referred to as "assembly seams" or simply "seams"). All this means that the traditional MCS method is not susceptible to standardization and is therefore capable of being improved.

As an alternative to traditional MCS, new assembly methods have been developed. The general tendency is to bring the genetic assembly closer to the usual mechanisms in other engineering, and for this four basic characteristics are required:

(i)

standardization, that is to say that the rules of assembly can be applied independently of the identity of the parts;

(ii)

 recyclability, meaning that new assemblies can be reused to make more complex assemblies;

(iii) efficiency, that is to say that allows the construction of complex assemblies quickly and safely; and finally

(iv) simplicity, that is to say that the assembly works with a minimum set of simple rules to facilitate the automation and adoption of the system by the end user.

The methods currently developed are fundamentally of two types: multipartite, in which the multiple assembly is carried out in a single step by adding two or more parts in tandem; or binary, in which the multiple assembly grows step by step through two to two unions of previously assembled fragments.

One of the most commonly used methods is the so-called "Gateway Cloning", subject to US Patent Nos. 5888732, 6143557, 6171861, 6270969, 6277608, 6720140 and other pending patents belonging to Invitrogen Corporation. Gateway cloning is based on homologous in vitro recombination mediated by a mixture of recombinases called BP and LR. In a further elaboration, the Multisite Gateway (Invitrogen) system was developed, which allows multipartite assembly of up to four parts in a directional way into a target vector. Several multigenic assembly vectors have been developed from Gateway technology (Chen et al., 2006, Chung et al., 2005). Despite its high efficiency, the assembly by Gateway cloning is limited to a maximum of four parts, which prevents forming higher order modules and / or devices. In addition, recombination leads to the formation of seams (attB sites) of 25 nucleotides.

An alternative set of methodologies (SLIC, Gibson, CPEC) is based on the generation of simple chain overlapping fragments between the “parts” to be assembled. In this way the assembly is stabilized by mating between the complementary chains, which is subsequently covalently closed by ligases or by the host cell itself. In all these technologies, overlapping extensions are introduced by PCR, subsequently generating the single chain fragments by different techniques. SLIC (Sequence and Ligase Independent Cloning), produces the single chain fragments by means of the T4 ligase exonuclease activity (Aslanidis and de Jong, 1990, Li and Elledge, 2007). Gibson's method uses T5 polymerase pursued by Phusion polymerase to generate and repair overlapping sequences (Gibson et al., 2009). Finally, the method called Circular Polymerase Extension Cloning (Quan and Tian, 2009), is based on the mutual plasmid primer activity and insert to assemble parts by PCR without the addition of oligonucleotides. All these assembly systems are multipartite and generate seamless assemblies. However, they are quite susceptible to

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introduce errors, since they are based on PCR. In addition, because they are based on pairing of complementary sequences, they are poorly compatible with the existence of areas with high secondary structure, repetitive sequences, etc. Other multipartite methodologies use different tools such as the use of restriction enzymes with long recognition sequences (rare cutters) (Goderis et al., 2002) "homing endonucleases", recombination sequences (Lin et al., 2003), or a combination of two or more of them as the ACEM kit (Bieniossek et al., 2009) (Berger, 2010) (WO / 2010/100278).

Another multipartite assembly methodology recently described is the so-called Golden Gate method (Engler et al., 2009, Engler et al., 2008). It is based on the use of restriction enzymes of type IIS (ERTIIS). Unlike conventional ERTIIs, in which the recognition site is palindromic and coincides with the digestion site, ERTIIS do not have a cutoff target but digest some nucleotides beyond the recognition site, regardless of their sequence, leaving extremes usually 4 nucleotide bulges. In Golden Gate, the parts to be assembled are flanked by ERTIIS targets oriented inside the fragment. In this way the target disappears after assembly, facilitating a seamless connection. For assembly only a small overlapping region of 4 nucleotides between adjacent parts is required, which coincides with the ERTIIS cutting area. In a Golden Gate reaction the fragments to be assembled are incubated in the presence of ERTIIS and T4 ligase in digestion and ligation cycles in a single tube. This technique significantly increases the efficiency of assemblies because it avoids having to perform gel extraction of the fragments to be cloned. Golden Gate is easily standardizable, however in its initial development, the constructions were not reusable, which prevents its adoption as a standard in Synthetic Biology.

In a subsequent technical innovation, called MoClo (Weber, Engler et al. 2011), two new restriction enzymes of the IIS type were introduced in the target plasmids, which allows the use of these enzymes for a second round of multipartite assembly. In order to approach the objective of recyclability, the MoClo system introduces an assembly level called "intermediate" which consists in adding a negative selection cassette as one more piece at the end of the assembly. This allows to create versions of multiple “open” constructions, which although they cannot be directly transferred to the cell since they contain a superfluous piece (selection cassette), they can be used as a mold on which to add new parts and do so Build construction in the future. The MoClo system is therefore a standardized modular system capable of automation. Although MoClo allows the theoretically indefinite growth of Golden Gate assemblies, the resulting design presents a series of drawbacks that complicate its adoption by the user and are listed below:

(i)

 the system requires the use of three restriction enzymes to ensure potential indefinite growth of the system; for the system to work properly, the parts must be free of internal restriction targets. For this it is necessary to mutagenize (domesticate) those original pieces that present internal restriction targets. The use of three different enzymes increases the probability of finding internal targets in the original parts and therefore increases the domestication requirements.

(ii)

 the modules assembled in MoClo are not completely recyclable given the “closed” character of the constructions: if the additional selection cassette is not added, they cannot continue to increase in size;

(iii) the system as a whole is complex: it consists of 36 plasmids (42 if the reverse growth option is used) and a large number of assembly rules;

(iv) the topology of the system is basically linear with different levels of assembly and lateral branches corresponding to the intermediate levels (Figure 1A).

Finally, the most representative proposal that allows binary growth is the Biobricks assembly standard developed at MIT (Che, 2004, Knight, 2003), and recently marketed by New England Biolabs and Ginkgoo Bioworks. Biobricks are, by virtue of their simplicity, a very successful proposal of standardization of the rules assembling parts in Synthetic Biology. Biobricks are basic genetic pieces (biopieces) flanked by 4 type II restriction enzymes, A and B at their 5 'end, and C and D at their 3' end. By virtue of the compatibility between B and C, the union between two basic parts generates an indivisible higher order part (module) that contains the same flanking sites as its initial constituents. This property is called "idempotence," and is the maximum simplification in assembly rules, since a single rule governs all types of assemblies. This simplicity facilitates the automation and exchange of genetic pieces between laboratories, since any genetic pieces generated by this methodology can in turn be assembled together regardless of their origin and nucleotide sequence. However, the use of type II enzymes generates “seams”, which is problematic in the generation of “clean” assemblies such as those required in fusions of coding regions. On the other hand, this strategy makes use of 4 different restriction enzymes, which forces to synthesize or mutagenize ("domesticate") a large number of basic pieces that contain recognition sites for these enzymes in their original sequences. Finally, since it is a system that does not incorporate multipartite unions, a large number of binary unions are necessary to perform complex assemblies, which makes it a slow and expensive methodology for many applications.

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BRIEF DESCRIPTION OF THE INVENTION

Current assembly methodologies are of two types: multipartite or binary. Binary methods can be compatible with the use of interchangeable and recyclable parts. In particular, Biobricks systems are idempotent, that is, they use a single rule for any assembly, which greatly facilitates their standardization and automation. However, the methods described so far produce seams between the assembled parts. In addition, being binary assemblies in any case the construction of complex circuits from basic parts is more laborious than in the case of multipartite assemblies.

Multipartite methods are fast because they allow simultaneous assemblies of more than two elements. However, current methods suffer from certain limitations. Some are limited in the number of parts to assemble (multisite Gateway). Others require the synthesis of de novo pieces in each assembly reaction, usually by PCR, in order to add overlapping regions between the parts to be assembled that did not necessarily exist in the original parts. The generation of large overlapping regions ensures seamless assembly but makes interchangeability and recyclability of parts difficult. In addition, it is necessary to check the integrity of the part after each assembly, since de novo synthesis can generate copy errors. Therefore, these multipartite methodologies are not suitable for use as an assembly standard in collections of interchangeable and recyclable genetic pieces.

Recently multipartite methodology known as Golden Gate was described, based on the use of restriction enzymes of the IIS type. Golden Gate is highly efficient, and has as its main feature that allows a seamless assembly without using PCR in the assembly process. However, in its original description, Golden Gate has as its main drawback the lack of recyclability of its parts, which makes its use as a standardized assembly system in Synthetic Biology difficult. This lack of Golden Gate has been partially overcome more recently by MoClo technology, which extends the multi-party assembly capabilities of Golden Gate indefinitely. To this end, MoClo expands the Golden Gate technology using at least two type IIs restriction enzymes in A12B orientation, with A and B being cut sites of the first enzyme and 1 and 2 recognition sites of the second enzyme (eg BpI-BsaI-BsaI -BpI). This allows you to carry out two levels of assembly: at level 1 the basic "parts" are assembled using the first of the enzymes (BsaI), while the second enzyme is used for assembly of transcriptional units (BpI). In order to open the system indefinitely to further assemblies, there is the possibility of leaving "level 2" open by adding, as one more piece, a new selection cassette. The MoClo system therefore has a linear structure with lateral branches (Figure 1A). These branches allow leaving the structure open to new assemblies, although said open structure is not directly usable for introduction into the cell since it contains a selection cassette outside the assembly. Therefore, the MoClo strategy is a partial solution to the problem of recyclability of Golden Gate multipartite assemblies, and is weighed down by great complexity, far removed from the idempotence of BioBricks: the complete system contains a total of 42 plasmids including adapters , linkers and plasmids to perform reverse assemblies.

The present invention consists of a new modular assembly system that allows the Golden Gate multipartite constructions to be combined indefinitely using very simple rules, close to idempotence and with a very small number of elements. After a first multipartite assembly stage in which basic parts are joined in a standardized manner, the resulting multipartite assemblies (devices) are joined together indefinitely by binary unions. As a whole, the cloning system can be described as a double loop that allows a quasi-idempotent assembly of multipartite assemblies. It is therefore an alternative solution to MoClo that adds simplicity and recyclability to multipartite assemblies, facilitating their application to Synthetic Biology.

Briefly, the invention consists of an in vitro assembly system for genetic parts based on the use of IIS type restriction enzymes. In the prior art, the MoClo standardized assembly, also based on restriction enzymes of the IIS type, used two targets of two restriction enzymes in defined orientation (A12B), which resulted in a linear design of successive hierarchical levels , with optional side branches that allow to leave assemblies open for the addition of additional parts (Fig 1A). The present invention provides a simple alternative solution to the linear design: a loop cloning design that makes use of only two levels that alternate with each other (Figure 1B). This is achieved through the use of 2 restriction enzymes type IIS (E1 and E2), but unlike MoClo, in the present invention, both enzymes are used at both levels and with inverted orientations. Thus at level α the orientation of the enzymes is of the general type A12B, while at the next level (level Ω), the orientation is of the general type 1AB2, where the numbers represent cut-off sites of the first enzyme and where the letters represent cutting sites of the second enzyme. In order to achieve the recyclability of the resulting assemblies (i.e., that these can continue to be assembled together), the present invention requires only two target plasmids for each level (α and Ω). The resulting system has a double-loop structure in which the input plasmids of the first round (level α) of assembly become target plasmids of the second round (level Ω) and vice versa, which allows to assemble new units of indefinitely The only limitations will be those imposed by the size and / or composition of DNA that can be propagated by a certain vector. The present invention therefore allows

4

Golden Gate standardization for use in Synthetic Biology. In addition, potentially indefinite growth is achieved with a small set of tools and a limited number of assembly rules.

Plasmid pDGB_K_C12B (CECT 7899)

One of the target plasmids objects of the present invention for the in vitro assembly of double stranded DNA pieces, has been deposited on March 21, 2011, in the Spanish Type Culture Collection (CECT) in Building 3 CUE of the scientific park of the University of Valencia, Professor Agustín Escardino nº9, 46980 Paterna, Valencia (Spain), by D. Diego Orzáez, of the Institute of Molecular and Cellular Biology of Plants (IBMCO), Higher Council of Scientific Research (CSIC), University Polytechnic of Valencia, Fausto Elio Avenue S / N 46022, Valencia (Spain).

The target plasmid for the in vitro assembly of deposited double-stranded DNA pieces whose reference is pDGB_K_C12B, was received by the CECT with the access number CECT 7 899 once said International Deposit Authority declared that the plasmid in question was viable.

The CECT 7899 plasmid is a circular plasmid and contains a LacZ positive selection cassette flanked at its 5 'end by sites C and 1 and at its 3' end by sites 2 and B. The rest of the structural elements of the plasmid are: an origin of pSa replication, an origin of ColE1 replication and a kanamycin resistance gene. (SEQ.ID.NO:11).

Plasmid pDGB_K_A12C (CECT 7900)

One of the target plasmids objects of the present invention for the in vitro assembly of double stranded DNA pieces, has been deposited on March 25, 2011, in the Spanish Type Culture Collection (CECT) in Building 3 CUE of the scientific park of the University of Valencia, Professor Agustín Escardino nº9, 46980 Paterna, Valencia (Spain), by D. Diego Orzáez, of the Institute of Molecular and Cellular Biology of Plants (IBMCO), Higher Council of Scientific Research (CSIC), University Polytechnic of Valencia, Fausto Elio Avenue S / N 46022, Valencia (Spain).

The target plasmid for the in vitro assembly of deposited double-stranded DNA pieces whose reference is pDGB_K_A12C, was received by the CECT with the accession number CECT 7 900 once said International Authority for Deposit declared that the plasmid in question was viable.

The CECT 7900 plasmid is a circular plasmid and contains a LacZ positive selection cassette flanked at its 5 'end by sites A and 1 and at its 3' end by sites 2 and C. The remaining structural elements of the plasmid are: an origin of pSa replication, an origin of ColE1 replication and a kanamycin resistance gene. (SEQ.ID.NO:10).

Plasmid pDGB_K_3AB2 (CECT 7901)

One of the target plasmids objects of the present invention for the in vitro assembly of double stranded DNA pieces, has been deposited on April 1, 2011, in the Spanish Type Culture Collection (CECT) in Building 3 CUE of the scientific park of the University of Valencia, Professor Agustín Escardino nº9, 46980 Paterna, Valencia (Spain), by D. Diego Orzáez, of the Institute of Molecular and Cellular Biology of Plants (IBMCO), Higher Council of Scientific Research (CSIC), University Polytechnic of Valencia, Fausto Elio Avenue S / N 46022, Valencia (Spain).

The target plasmid for the in vitro assembly of deposited double-stranded DNA pieces whose reference is pDGB_K_3AB2, was received by the CECT with the access number CECT 7 901 once said International Authority for Deposit declared that the plasmid in question was viable.

The CECT 7901 plasmid is a circular plasmid and contains a LacZ positive selection cassette flanked at its 5 'end by sites 3 and A and at its 3' end by sites B and 2. The rest of the structural elements of the plasmid are: an origin of pSa replication, an origin of ColE1 replication and a spectinomycin resistance gene. (SEQ.ID.NO:13).

Plasmid pDGB_K_1AB3 (CECT 7902)

One of the target plasmids objects of the present invention for the in vitro assembly of double stranded DNA pieces, has been deposited on March 21, 2011, in the Spanish Type Culture Collection (CECT) in Building 3 CUE of the scientific park of the University of Valencia, Professor Agustín Escardino nº9, 46980 Paterna, Valencia (Spain), by D. Diego Orzáez, of the Institute of Molecular and Cellular Biology of Plants (IBMCO), Higher Council of Scientific Research (CSIC), University Polytechnic of Valencia, Fausto Elio Avenue S / N 46022, Valencia (Spain).

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The target plasmid for the in vitro assembly of deposited double-stranded DNA pieces whose reference is pDGB_K_1AB3, was received by the CECT with the accession number CECT 7 902 once said International Authority for Deposit declared that the plasmid in question was viable.

The CECT 7902 plasmid is a circular plasmid and contains a LacZ positive selection cassette flanked at its 5 'end by sites 1 and A and at its 3' end by sites B and 3. The rest of the structural elements of the plasmid are: an origin of pSa replication, an origin of ColE1 replication and a spectinomycin resistance gene. (SEQ.ID.NO:12).

DETAILED DESCRIPTION OF THE INVENTION

The invention describes an in vitro assembly system based on the use of restriction enzymes of the IIS type. The Golden Gate system described above allows seamless multipartite assembly based on restriction enzymes of the IIS type by using targets of a single restriction enzyme (E1). The present invention allows, by virtue of a special design of the target plasmids in which at least 2 type IIs restriction enzymes (E1, E2) are used, in addition to multipartite assemblies such as those previously described, the possibility of combining them indefinitely in binary form.

The system consists of four target plasmids, pDGBs. Each of the target plasmids incorporates a reporter cassette that allows negative selection (Sel), such as LacZ or ccdB, or any other marker that allows the growth and selection of those clones that do not contain said marker. Said marker is flanked by a minimum of 4 targets of at least 2 restriction enzymes of type IIS, (E1 and E2). E1 and E2 enzymes can be restriction enzymes of the IIS type such as BsaI, BsmBI, BsbI, FokI, BsmAI, AarI, etc., or any other restriction enzyme with the following characteristics:

(i)

that the enzyme recognition and cleavage sites are spatially separated, and preferably that the recognition site consists of a specific sequence of five or more nucleotides;

(ii)

that the enzyme cutting site does not have sequence requirements, so that each enzyme can be assigned any cutting sequence consisting of a series of n nucleotides (preferably four or more) where n is a characteristic of the enzyme itself;

(iii) that the digestion of DNA with the enzyme results in the formation of cohesive ends of DNA (preferably of 4 or more nucleotides), which will coincide with the cutoff sequence assigned by the invention.

Taking advantage of the ERIIs characteristic of having different recognition and cutting sites and the fact that the cutting zone is determined by the orientation of the restriction enzyme recognition sequence, an arrangement was designed as shown in the Figure 2A The recognition targets of the restriction enzymes show their recognition sequences in the 5 ’→ 3’ direction as indicated in Figure 2A with solid arrows. These are in inverted orientations so that the recognition sites can be directed as indicated in Figure 2A by the dashed arrows.

The ERTII cleavage sites are any nucleotide sequences, where the number of nucleotides that make up the target, usually four, may vary depending on the particular enzyme in question. For simplicity, the specific nucleotide sequences assigned to the E1 enzyme cleavage sites in the plasmids of the invention are denoted with numerical nomenclature (1,2,3 ...) and those of E2 own an alphabetic nomenclature (A, B, C ... ).

Since ERTIIs do not have requirements with respect to the cut sites (only those of recognition), the cut sites 1, 2, 3, A, B and C assigned to the different enzymes in the different plasmids of the invention may consist in any nucleotide sequence provided that:

(i)

The number of nucleotides in each specific sequence is equal to the number of nucleotides in the cut sequence of its corresponding enzyme (E1 or E2). Usually it will be sequences of 4 or more nucleotides;

(ii)

each specific cutting sequence is located at the correct distance from the recognition site of its enzyme and in the correct orientation with respect to it, so that it can be digested by its corresponding enzyme resulting in the generation of a single chain cohesive end;

(iii)

sequences 1 2 and 3 are different from each other, and preferably differ in 2 or more positions;

(iv)

the sequences A, B and C are different from each other, and preferably differ in 2 or more positions;

(v)

Do not deal with palindromic sequences.

In the system, at least two different positive selection markers (M1 and M2) are used in the plasmid structures, giving rise to two groups of plasmids, α level plasmids, which contain the M1 marker and the

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Ω level plasmids, containing the M2 marker. The positive selection markers used can be any type of marker that allows the growth and selection of those clones that do carry the marker, such as antibiotic resistance genes such as ampicillin, kanamycin, rifampicin, spectinomycin, streptomycin, tetracycline, gentamicin , carbenicillin, etc.

Thus, the first pair of plasmids, corresponding to the α level (pDGB_ A12C and pDGB_ C12B), contain E2 targets in position 5 ’with respect to E1 targets, with orientations being inverted.

one.

Plasmid pDGB_A12C contains a selection cassette that allows negative selection with the following structure: 5'- E2 recognition site-E2 type A cut site-E1 type cut site 1- E1-Casset recognition site Selection-Site of recognition of E1- Site of cutting of E1 type 2-Site of cutting of E2 type C- Site of recognition of E2 -3 '. (Figure 2B).

2.

Plasmid pDGB_C12B contains a selection cassette that allows negative selection with the following structure: 5 '-E2 recognition site -E2 type C cutting site-E1 type cutting site 1- E1-Casset recognition site Selection-Site of recognition of E1- Site of cutting of E1 type 2-Site of cutting of E2 type B- Site of recognition of E2 -3 '. (Figure 2C)

The Ω level plasmid pair (pDGB_ 1AB3 and pDGB_ 3AB2) contains E1 targets in position 5 ’with respect to E2 targets, also with inverted orientations.

one.

Plasmid pDGB_1AB3 contains a selection cassette that allows negative selection with the following structure: 5'- E1 recognition site-E1 type 1 cutting site-E2 type A cutting site- E2-Cassete recognition site Selection-Site of recognition of E2- Site of cutting of E2 type B-Site of cutting of E1 type 3- Site of recognition of E1-3 '. (Figure 2D)

2.

Plasmid pDGB_3AB2 contains a selection cassette that allows negative selection with the following structure: 5'-E1 recognition site-E1 type 3 cutting site-E2 type A cutting site- E2-Cassete recognition site Selection-Site of recognition of E2- Site of cutting of E2 type B-Site of cutting of E1 type 2- Site of recognition of E1 -3. (Figure 2E) ´.

The starting point of the system is a multipartite assembly of basic parts (pieces) of double-stranded DNA, flanked by single-chain ends (tails), so that the tail at 3 'of each piece appears with the tail at 5' of the subsequent piece, the 5 'tail of the first piece matches the type 1 cutting site, and the 3' tail of the last piece matches the type 2 cutting site of the α-type plasmids (Figure 3A) . In a particular embodiment, a

or several of the pieces can be presented in the form of precursors, either linear, or forming part of a circular plasmid (entry plasmid or pENTR). Each precursor contains the original piece flanked by additional sequences containing E1 targets. In this way, after digestion with the enzyme E1, the original piece is detached from the precursor, being flanked by protuberant ends that constitute in themselves the tails necessary for assembly. In the example illustrated in Figure 3A, three pieces of DNA are assembled together (PR, CDS and TM), flanked by protruding ends represented with labels 1, IV, III and 2. It will be possible to assemble the desired number of pieces provided that the first precursor contains a cut-off site for the E1 compatible with the site 1, the last precursor contains a cut-off site for the E1 compatible with the site 2 and the adjacent pieces have compatible ends. It is recommended that the pieces to be assembled do not contain targets for the E1 and E2 enzymes in their sequence to increase the efficiency of the cloning. It is also recommended that the PENTRs contain a different selection marker (M3) than the pDGB.

In each assembly reaction, the α level target plasmid (pDGB_A12C or pDGB_C12B) loses its negative selection cassette (eg LacZ), instead incorporating the assembled module through a ligation reaction between the protruding ends (1 and 2 in the extremes, and as many ligaments as parts you want to join) left after digestion with E1. After ligation, the resulting plasmid, which contains the assembled module, loses all the E1 targets, but maintains the E2 targets, inherent in the plasmid.

Next, the modules assembled on the α level plasmids can be considered as input plasmids for a new Ω level assembly (Fig 3B). Two modules assembled on pDGB_A12C and pDGB_C12B respectively can in turn be assembled together, this time using the E2 enzyme and a Ω level plasmid. In this second round of assembly, all E2 sites are lost, but the E1 sites of the resulting plasmid are maintained.

In a third round of assembly, and similar to the previous case, modules constructed with plasmids of level Ω can be assembled together again, using the enzyme E1 and a plasmid of level α, closing an iterative and indefinite loop of hierarchical assemblies (see Figure 1 B).

Similarly to the previous example, the incorporation of modules into the multipartite assembly system can be carried out directly on the plasmids of the Ω level. Once the module is assembled on a target plasmid of the Ω level, the assembly procedure is the same as described above. This possibility does not alter the

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normal operation of the system but it gives it greater versatility.

The system as a whole is composed of only 4 target plasmids and a simple set of assembly rules that can be formally defined as follows:

1. EGB RULES for BINARY assemblies

one.

pE [1 (Xi) 3] + pE [3 (Xj) 2] + pDGB (A12C) = pE [A (Xj + Xj) C]

2.

pE [1 (Xi) 3] + pE [3 (Xj) 2] + pDGB (C12B) = pE [C (Xi + Xj) B]

3.

pE [A (Xi) C] + pE [C (Xj) B] + pDGB (1AB3) = pE [1 (Xi + Xj) 3]

Four.

pE [A (Xi) C] + pE [C (Xj) B] + pDGB (3AB2) = pE [3 (Xi + Xj) 2] Where

•

(Xi) and (Xj) are pieces of DNA to assemble, usually expression cassettes.

•

(Xi + Xj) is an idempotent assembly of (Xi) and (Xj), that is, that (Xi + Xj) can in turn be assembled with other parts following these same rules.

•

1, 2, 3, are sequences of 4 nucleotides that flank the Xi pieces and become protuberant ends after a cut with BsaI.

•

A, B and C are sequences that flank the Xi pieces and become protruding ends after a cut with BsmBI.

•

pE [] is any plasmid (input plasmid) that houses a piece of Xi, flanked by the cutting and recognition sites indicated within the bracket.

•

pDGB [] is a binary plasmid with a selection cassette that allows a negative selection (target plasmid) flanked by the cutting and recognition sites indicated within the bracket. There are only 4 binary plasmids, which are those that appear in the assembly rules.

Starting from standardized genetic pieces and by applying these four assembly rules, the four plasmid system of the invention allows to generate new fully recyclable assemblies of indefinitely increasing complexity for use in Synthetic Biology applications.

The main advantages of the system compared to the current state of the art are:

(i)

 Recyclability / reusability of its parts: all assemblies can be used directly for transformation into cells or can be integrated into more complex constructions, without PCR amplification or subsequent modifications;

(ii)

 Speed: the starting point of the system is a multi-party assembly so that it is accelerated with purely binary systems such as BioBricks .;

(iii) Precision: seamless assemblies are generated thanks to the use of type IIs enzymes.

(iv) Simplicity: the system object of the invention can theoretically perform infinite assemblies with only four plasmids and four assembly rules.

The present invention refers to a target plasmid (pDGB) for in vitro assembly of double stranded DNA pieces, characterized in that it comprises any skeleton of a double stranded DNA plasmid comprising:

a) at least one origin of replication,

b) at least one selection marker, preferably positive,

c) at least one cassette that allows selection, preferably negative,

d) at least 2 recognition sites for a first restriction enzyme of type IIS (E1) each giving rise to a cut-off site of 4 or more nucleotides selected between 1, 2 and 3, being 1, 2 and 3 preferably any four nucleotide sequences any different from each other,

e) at least 2 recognition sites for a second restriction enzyme of type IIS (E1) each giving rise to a cut-off site of 4 or more nucleotides selected from A, B and C, A, B and C preferably

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sequences of any four nucleotides distinct from each other, so that the cut and recognition sites cited in (d) and the cut and recognition sites cited in (e) flank the selection cassette cited in (c) at their 5 'ends and 3´ with inverted orientations.

In a preferred embodiment the present invention refers to a target plasmid (pDGB) for in vitro assembly of double stranded DNA pieces, characterized in that the inverted orientations of the cutting and recognition sites cited in (d) and the sites of cutting and recognition cited in (e) flanking the selection cassette cited in (c) at its 5'and 3'-ends are preferably selected from all possible combinations between 1, 2, 3, A, B, and C that meet the characteristics described above, and more preferably are selected from: A12C, C12B, 1AB3, and 3AB2;

In a preferred embodiment, the present invention refers to a target plasmid (pDGB) for the in vitro assembly of double stranded DNA pieces, characterized in that the orientation of the two cutting and recognition sites mentioned in (d) and the two cutting and recognition sites cited in (e) is A12C, deposited in the Spanish Type Crops Collection as CECT 7900.

In a preferred embodiment the present invention refers to a target plasmid (pDGB) for the in vitro assembly of double stranded DNA pieces according to claim 2, characterized in that the orientation of the two cutting and recognition sites mentioned in (d ) and the two cutting and recognition sites mentioned in (e) is C12B, deposited in the Spanish Type Culture Collection as CECT 7899.

In a preferred embodiment, the present invention refers to a target plasmid (pDGB) for the in vitro assembly of double stranded DNA pieces, characterized in that the orientation of the two cutting and recognition sites mentioned in (d) and the two cutting and recognition sites cited in (e) is 1AB3, deposited in the Spanish Type Crops Collection as CECT 7902.

In a preferred embodiment, the present invention refers to a target plasmid (pDGB) for the in vitro assembly of double stranded DNA pieces, characterized in that the orientation of the two cutting and recognition sites mentioned in (d) and the two cutting and recognition sites cited in (e) is 3AB2, deposited in the Spanish Type Crops Collection as CECT 7901.

In a preferred embodiment the present invention refers to an in vitro assembly method of double-stranded DNA pieces characterized in that it comprises an indefinite assembly loop that alternates the level α and the level Ω of quasi-idempotent binary assembly, where the level α preferably comprises the following steps:

i) construction of the α-level input plasmids (pE), each α-level input plasmid (pE) comprises a piece of double stranded DNA selected from (Xi) and / or (Xj), or (Xi + Xj ), flanked at its 5´ and 3´ ends by two cutting and recognition sites selected between 1, 2 and 3, for a first restriction enzyme of type IIS (E1):

pE [1 (Xi) 3] and pE [3 (Xj) 2]

ii) construction of the target plasmids (pDGB) of level α:

pDGB (A12C) (CECT 7900) and pDGB (C12B) (CECT 7899)

iii) enzymatic digestion of the input plasmids obtained in i) and the target plasmids obtained in ii) in the presence of E1, preferably generating at its ends 3 'and 5' single stranded DNA terminal sequences of at least 3 or 4 nucleotides, also called protuberant ends,

iv) ligation with T4 ligase between the single-chain terminal sequences of the input and target plasmids digested in iii) forming the assembled plasmids of level of closed circular double-stranded DNA α:

pE [1 (Xi) 3] + pE [3 (Xj) 2] + pDGB (A12C) (CECT 7900) = pE [A (Xi + Xj) C]

pE [1 (Xi) 3] + pE [3 (Xj) 2] + pDGB (C12B) (CECT 7899) = pE [C (Xi + Xj) B];

and where the Ω level comprises the following stages:

v) construction of the Ω level input plasmids (pE) each Ω level input plasmid (pE) comprises a piece of double stranded DNA selected from (Xi) and / or (Xj), flanked at its 5 'ends and 3 'by two cut and recognition sites selected from A, B and C, for a second restriction enzyme of type IIS (E2):

pE [A (Xi) C] and pE [C (Xj) B]

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vi) construction of target plasmids (pDGB) of Ω level:

pDGB (1AB3) (CECT 7902) and pDGB (3AB2) (CECT 7901)

vii) enzymatic digestion of the input plasmids obtained in v) and the target plasmids obtained in vi) in the presence of E2, generating at its ends 3´ and 5´ single stranded DNA terminal sequences of at least 4 nucleotides, also called protruding ends

viii) T4 ligase ligation between the single-chain terminal sequences of the input and target plasmids digested in vii) forming the assembled level A plasmids of closed circular double-stranded DNA:

pE [A (Xi) C] + pE [C (Xj) B] + pDGB (1AB3) (CECT 7902) = pE [1 (Xi + Xj) 3]

pE [A (Xi) C] + pE [C (Xj) B] + pDGB (3AB2) (CECT 7901) = pE [3 (Xi + Xj) 2];

when the assembly method starts at the α level and continues with the Ω level, the level A input plasmids pE [A (Xi) C] and pE [C (Xj) B] defined in v) are replaced by Level 1 assembled plasmids pE [A (Xj + Xj) C] and pE [C (Xi + Xj) B] obtained in iv); Y

when the assembly method starts at the Ω level and continues with the α level, the α level entry plasmids pE [1 (Xi) 3] and pE [3 (Xj) 2] defined in i) are replaced by assembled plasmids of level Ω pE [1 (Xi + Xj) 3] and pE [3 (Xi + Xj) 2] obtained in viii).

In a preferred embodiment the present invention refers to an in vitro assembly method of double stranded DNA pieces characterized in that (Xi) and / or (Xj) is selected from a promoter, a subcellular localization sequence, a coding sequence, a copy of DNA is an interfering RNA sequence, a termination sequence, or an expression cassette of proteins, enzymes, transcription factors, markers, antibodies, regulatory proteins, or an expression cassette of functional RNAs or chromatin structural elements .

In a preferred embodiment the present invention refers to a method of in vitro assembly of double stranded DNA pieces characterized in that the target plasmids (pDGB) are preferably binary plasmids for the genetic transformation of plants mediated by Agrobacterium tumefaciens.

In a preferred embodiment the present invention refers to an in vitro assembly method of double stranded DNA pieces according to claim 7, characterized in that (Xi + Xj) is preferably formed by a kanamycin gene, a gene encoding the chain light of an antibody and a gene that codes for the heavy chain of an antibody.

In a preferred embodiment the present invention refers to a method of in vitro assembly of double stranded DNA pieces characterized in that (Xi + Xj) is formed by four expression cassettes preferably constituting three genes encoding three fluorescent proteins and a fourth gene that encodes the p19 protein.

In a preferred embodiment the present invention refers to a plant transformed by Agrobacterium tumefaciens by the assembly method described in the invention and characterized in that said plant expresses a recombinant antibody of the IgA type.

In a preferred embodiment the present invention refers to a plant transiently transformed by Agrobacterium tumefaciens by the assembly method described in the invention and characterized in that said plant expresses, preferably simultaneously, the said three fluorescent proteins.

DESCRIPTION OF THE FIGURES

Figure 1. Comparison of topologies between MoClo and the system object of the invention. (A) Hierarchical topology of the MoClo assembly. At level 0 the subparts are assembled flexibly to form basic parts, while allowing the domestication of these parts. At level 1, multipartite assembly of basic parts is carried out to give rise to transcriptional units. At level 2-1 the multipartite assembly of transcriptional units takes place, giving rise to a final non-recyclable structure. Alternatively, level 1 can be branched to level 2-1i (intermediate) by joining a tail adapter, resulting in an open structure (although not functional), which can house the assembly of new transcriptional units (level 2-2). The creation of successive intermediate levels will ensure the indefinite growth of the cloning system. (B) Double loop structure of the system object of the invention. Plasmids of level α receive multipartite assemblies of basic parts giving rise to transcriptional units. Two transcriptional units assembled at level α can be assembled together giving rise to two alternative multigenic complexes of level Ω. In turn, two multigenic complexes of level Ω can be assembled together at level α. The general structure of the system is an iterative double loop that ensures the indefinite growth of assemblies.

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Figure 2 (A) Scheme of arrangement of the recognition and cutting sites of the E1 and E2 enzymes in the pDGB. The recognition sites are arranged in an inverted manner, so that their restriction action can be directed as needed for the development of the invention. (B) Disposition of recognition and cutting sites of enzymes in plasmid pDGB_A12C. (C) Provision of recognition and cutting sites of enzymes in plasmid pDGB_C12B (D) Provision of recognition and cutting sites of enzymes in plasmid pDGB_1AB3 (E) Provision of recognition and cutting sites of enzymes in the plasmid pDGB_3AB2

Figure 3. Mechanism of the system object of the invention. (A) Multipartite assembly of standard parts (PR, promoters; CDS, coding regions; TM, terminators) flanked by E1 restriction sites (represented by Arabic and Latin numbers). The E1 recognition sites disappear after assembly (represented by a crossed out box). The new assembled module (DEV, represented as an arrow to simplify the scheme) is flanked by BsmBI restriction sites (represented by circles and capital letters). (B) Two modules assembled in complementary α-type plasmids can be recycled as input vectors (pEGB) for subsequent binary assemblies in Ω type vectors since they have a common cutting site (labeled with circle C). Similarly, constructions made on complementary plasmids of type Ω can be used for subsequent assemblies on vectors of type α since both contain a common cutting site (square 3). The α and Ω levels can alternate indefinitely as indicated by the arrows that cross the loop, creating increasingly complex structures. Antibiotic resistance genes are represented with vertical grated circles (kanamycin resistance) or horizontal (spectinomycin resistance).

Figure 4. Domestication of target plasmids. Assembly of the pieces that make up the skeleton of the GoldenBraid system pDGB plasmids with the negative selection (LacZ flanked from BsaI and BsmBI sites) and positive (kanamycin or spectinomycin resistance genes). An example plasmid is shown for each level of the system.

Figure 5 Structure d the cassette L acZ in the system. The system plasmid collection comprises four target plasmids (pDGBs), grouped into two levels, α and Ω. The vectors incorporate a flanked LacZ selection cassette of four type IIs restriction enzyme recognition sites (BsaI, BsmBI) in inverted positions and orientations. To facilitate the design visualization, each 4 nucleotide cutting site is labeled as follows: BsaI cutting sites with squares and Arabic numerals (1, 2, 3); BsmBI cutting sites with circles and capital letters (A, B, C).

Figure 6 (A) Restriction analysis of plasmid pDGB_K_A12C with restriction enzymes BsaI (lane 2) and BsmBI (lane 3). (B) Restriction analysis of plasmid pDGB_S_1AB3 with restriction enzymes BsmBI (lane 2) and BsaI (lane 3).

Figure 7 Co-tra nsformation of fluorescent fluorescent devices. (A) Itinerary followed for the assembly of the YFP, p19, BFP and DsRED transcription units in a single T-DNA. (B) Spatial patterns of expression of BFP, YFP and DsRED in leaves of N. bent hamiana agroinfiltrated with pEGB_A-YFP-P19-BFP-DsRed-C (upper captures, 1, 2 and 3) or with a mixture of individual devices pEGB_A-YFP-C, pEGB_C-p19-B, pEGB_A-BFPC and pEGB_C-DsRed-B (lower captures 4, 5 and 6).

Figure 8 Selection of isotypes of a human IgA constructed with the system object of the invention. (TO)

Strategy followed in the assembly of different isotypes of human IgA. First, the multi-party assembly phase consisted of the combination of different basic parts each occupying a fixed position in the assembly (P1-P5). Individual immunoglobulin chains were assembled into plasmid pDGB_C12B to generate all four isotypes of IgA. In the figure, 35S represents the CamV35S promoter; SP represents the signal peptide of a tomato pectate lyase; CHα1 and CHα2 are constant domains of the heavy chain; Cλ and Cκ are constant domains of the heavy chain; VH and VL are variable regions of the heavy and light chains of an antibody against the VP8 * peptide of rotavirus. (B) BglII digestion of two colonies of each of the four final constructs: α1λ, pEGB_C-Igα1-Igλ-B; α1κ, pEGB_C-Igα1-Igκ-B; α2λ, pEGB_C-Igα2-B-Igκ-B, α2κ, pEGB_C-Igα2-Igκ-B. (C) Western Blot analysis of the transient expression of the different constructions in Nicotiana benthamiana. The leaves of the plant infiltrated the four previous constructions. The samples were resolved under reducing conditions (left) and non-reducing conditions (right) and were revealed with antibodies against the heavy chain, light λ and light κ respectively. The HS run contains a human serum control. (D) Titrated endpoint ELISA of the four IgA combinations tested by transient expression. All samples were titrated against VP8 * or bovine serum albumin (BSA) and compared with equivalent samples derived from wild leaves.

BIBLIOGRAPHY

-

 Aslanidis C & PJ de Jong (1990) Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res 18: 6069-74.

-

 Berger I (2010) Nuclear acids for Cloning and Expressing Multiprotein Complexes. IN (EMBL), E. L. F. M. (Ed.

-

 Bieniossek C, Y Nie, D Frey, N Olieric, C Schaffitzel, I Collinson, C Romier, P Berger, TJ Richmond, MO Steinmetz & I Berger (2009) Automated unrestricted multigene recombineering for multiprotein complex production. Nat Meth 6: 447-450.

-

 Che A (2004) Synthetic Biology: BioBricks / 3A assembly. BioBricks ++ Assembly Scheme. Tech. Rep., MIT Synthetic Biology Working Group Technical Reports

-

 Chen QJ, HM Zhou, J Chen & XC Wang (2006) A Gateway-based platform for multigene plant transformation. Plant Mol Biol 62: 927-36.

-

 Chung SM, EL Frankman & T Tzfira (2005) A versatile vector system for multiple gene expression in plants. Trends Plant Sci 10: 357-61.

-

 Engler C, R Gruetzner, R Kandzia & S Marillonnet (2009) Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS One 4: e5553.

-

 Engler C, R Kandzia & S Marillonnet (2008) A one pot, one step, precision cloning method with high throughput capability. PLoS One 3: e3647.

Gibson DG, L Young, R-Y Chuang, JC Venter, CA Hutchison & HO Smith (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Meth 6: 343-345.

-

 Goderis IJWM, MFC De Bolle, IEJA François, PFJ Wouters, WF Broekaert & BPA Cammue (2002) A set of modular plant transformation vectors allowing flexible insertion of up to six expression units. Plant Molecular Biology 50: 17

eleven

27.

Hellens RP, EA Edwards, NR Leyland, S Bean & PM Mullineaux (2000) pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Mol Biol 42: 819-32.

-

 Karimi M, D Inze & A Depicker (2002) GATEWAY vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 7: 193-5.

Knight TF (2003) Idempotent Vector Design for Standard Assembly of BioBricks. Tech. Rep., MIT Synthetic Biology Working Group Technical Reports

-

 Li MZ & SJ Elledge (2007) Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nat Meth 4: 251-256.

-

 Lin L, Y-G Liu, X Xu & B Li (2003) Efficient linking and transfer of multiple genes by a multigene assembly and transformation vector system. Proceedings of the National Academy of Sciences of the United States of America 100: 5962-5967.

-

 Monedero V, J Rodriguez-Diaz, R Viana, J Buesa & G Perez-Martinez (2004) Selection of single-chain antibodies against the VP8 * subunit of rotavirus VP4 outer capsid protein and their expression in Lactobacillus casei. Applied and Environmental Microbiology 70: 6936-6939.

-

 Orzaez D, S Mirabel, WH Wieland & A Granell (2006) Agroinjection of tomato fruits. A tool for rapid functional analysis of transgenes directly in fruit. Plant Physiol 140: 3-11.

-

 Qiu WP, JW Park & HB Scholthof (2002) Tombusvirus p19-mediated suppression of virus-induced gene silencing is controlled by genetic and dosage features that influence pathogenicity. Molecular Plant-Microbe Interactions 15: 269

280.

-

 Quan J&J Tian (2009) Circular Polymerase Extension Cloning of Complex Gene Libraries and Pathways. PLoS ONE 4: e6441.

Weber, E., C. Engler, et al. (2011). "A modular cloning system for standardized assembly of multigene constructs." PLoS One 6 (2): e16765.

Wieland WH, A Lammers, A Schots & DV Orzaez (2006) Plant expression of chicken secretory antibodies derived from combinatorial libraries. J Biotechnol 122: 382-91.

EXAMPLES

The following specific examples provided in this patent document serve to illustrate the nature of the present invention. These examples are included for illustrative purposes only and should not be construed as limitations on the invention claimed herein. In a first example, we developed the domestication of the pDGBs vectors to adapt them to the system object of the invention. In a second example,

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We assemble three fluorescent proteins and the p19 silencing suppressor into a single T-DNA, demonstrating the advantages of multigenic cis-building when tested transiently by agro-infiltration in the plant. In a third example, we show the versatility of the system to test different antibody isotypes in a combinatorial approximation thereof.

Thus, the examples described below illustrate the invention in the field of Plant Synthetic Biology, without limiting its field of application in other fields.

EXAMPLE 1: CONSTRUCTION OF pDGB VECTORS

In this example the embodiment of the invention is described for the specific case of an assembly system intended for the assembly of genetic parts for the genetic transformation of plants. The creation of a system of four binary vectors derived from the binary vector pGreenII, incorporating the scheme described in the invention for the target plasmids (pDGB), is described. The skeleton of any plasmid could be adapted, similar to that proposed in the present example, to the system object of the invention.

The pDGB vectors were constructed by assembly, by a Golden Gate reaction (Engler, Kandzia et al. 2008) with BbsI, of a variable number of parts (Figure 4). Each piece was amplified by PCR incorporating at its ends two BsbI recognition sites and cut sites compatible with the next piece to be assembled, generating the precursors of each piece. Subsequently, each precursor was cloned into a TA cloning vector pGEMT (Promega), generating the so-called input vectors (pENTR) for each piece.

The precursors necessary to assemble a plasmid are generally a negative selection cassette (LacZ, in this case), a positive selection cassette (antibiotic resistance) and a variable number of pieces that make up the vector skeleton and contain the origins of replication and the borders of T-DNA.

To generate the set of vectors of the system object of the invention, four pieces of negative selection (LacZ-A12C, LacZ-C12B, LacZ-1AB3, LacZ-3Ab2) precursors were generated, two antibiotic resistance genes (kanamycin resistance - 2KA and 2KB- and spectinomycin precursors - 2S- precursor) and two pieces (precursor 1 and precursor 3) comprising the pGreenII plasmid skeleton. Thus, the precursors are:

a) Precursor 1 (SEQ.ID.NO:1) contains part 1, which corresponds to nucleotides 1296-2169 of the skeleton of plasmid pGreenII (Hellens, Edwards et al. 2000).

b) The 2KA precursor (SEQ.ID.NO:2) contains part 2AK, which corresponds to nucleotides 21662542 of the pGreenII plasmid skeleton. It includes an A> T mutation in nucleotide 2542 to remove a BsmBI restriction site. It corresponds to the 3 ’fragment of kanamycin resistance.

c) The 2KB precursor (SEQ.ID.NO:3) contains part 2KB, which corresponds to nucleotides 25392989 of the pGreenII plasmid skeleton. It includes an A> T mutation in nucleotide 2542 to remove a BsmBI restriction site. It corresponds to the 5 ’fragment of kanamycin resistance.

d) The 2S precursor (SEQ.ID.NO:4) contains part 2S, which corresponds to nucleotides 248-1392 of the skeleton of plasmid pCR8 (Invitrogen). It corresponds to spectinomycin resistance.

e) Precursor 3 (SEQ.ID.NO:5) contains part 3, which corresponds to nucleotides 2986-565 of the skeleton of plasmid pGreenII.

f) The precursors of the negative selection pieces are amplified from plasmid pUC19 in four different versions. Each piece incorporates, together with the lacZ gene, targets and cut sites of two ERTIIS in different distinct relative positions, as described below. For simplicity, the specific BsaI enzyme cleavage sites are named numerically (1, 2, 3) and for those of BsmBI an alphabetic nomenclature (A, B, C) is used. Concrete nucleotide sequences are also shown: A (CCAC), B (CGTC), C (GGCA), 1 (GGCA), 2 (CGAC), and 3 (GATG). The resulting structure and the resulting nucleotide composition of each of the precursors of the four pieces of negative selection are:

i. The precursor of the LacZ-A12C part (SEQ.ID.NO.6) has the following structure: 5 'BsmBI recognition site - BsmBI cutting site type A-BsaI cutting site type 1- Site recognition of BsaI -LacZ-BsaI recognition site- BsaI cutting site type 2-BsmBI cutting site type C- BsmBI 3 'recognition site. (Figure 5)

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ii. The precursor of the LacZ-C12B part (SEQ.ID.NO.7) has the following structure: 5 'BsmBI recognition site-BsmBI cutting site type C-BsaI cutting site type 1- Site recognition of BsaI-LacZ-BsaI recognition site- BsaI type 2 cutting site-BsmBI type B cutting site- BsmBI 3 'recognition site. (Figure 5)

iii. The precursor of the LacZ-1AB3 part (SEQ.ID.NO.8) has the following structure: 5 'BsaI recognition site -BsaI cutting site type 1-BsmBI cutting site type A- Recognition site BsmBI -LacZ-BsmBI recognition site- BsmBI cutting site B-BsmBI cutting site type 3- BsaI 3 'recognition site. (Figure 5).

iv. The precursor of the LacZ-3AB2 part (SEQ.ID.NO.9) has the following structure: 5 'BsaI recognition site -BsaI cutting site type 3-BsmBI cutting site type A- Recognition site BsmBI-LacZ-BsmBI recognition site- BsmBI cutting site B-BsmBI cutting site type 2- BsaI 3 'recognition site (Figure 5).

All precursors were obtained by PCR amplification and cloned into the pGEMT vector, generating the corresponding pENTR_Pieza entry plasmids. As indicated, the assembly of the pDGBs vectors is performed by a Golden Gate reaction in a single tube (Engler, Kandzia et al. 2008) that contained the different pENTRs, BbsI and T4 ligase in 25 cycles of 37 ° C and 14 ° C. To eliminate false positives, an additional final digestion is performed to that described by Engler et al. (2008) The combinations of pENTRs for each reaction were:

one.

pENTR_1, pENTR_2KA, pENTR_2KB, pENTR_3 and pENTR_LacZ-A12C to construct plasmid pDGB_K_A12C (SEQ.ID.NO:10).

2.

pENTR_1, pENTR_2KA, pENTR_2KB, pENTR_3 and pENTR_LacZ-C12B to construct plasmid pDGB_K_C12B (SEQ.ID.NO:11).

3.

pENTR_1, pENTR_2S, pENTR_3 and pENTR_LacZ-1AB3 to construct plasmid pDGB_S_1AB3 (SEQ.ID.NO:12).

Four.

pENTR_1, pENTR_2S, pENTR_3 and pENTR_LacZ-3AB2 to build plasmid pDGB_S_3AB2 (SEQ.ID.NO:13).

After performing the Golden Gate assembly, one microliter of each reaction was used to transform E. coli. The transformed cells were seeded on LB plates with kanamycin and X-gal in the case of reactions 1 and 2, and on LB plates with spectinomycin and X-gal for the transformations resulting from reactions 3 and 4. From each reaction four blue colonies were selected, grown in liquid medium and the corresponding plasmid was isolated and analyzed by digestion analysis and subsequent sequencing. Both analyzes showed correct assemblies in at least three of the four colonies analyzed for each reaction. Figure 6 shows the restriction analyzes obtained for reactions 1 and 3.

PRACTICAL EXAMPLE 2

The expression of genes transiently in N. benthamiana mediated by Agrobacterium (agroinfiltration) is an efficient technology for the production of recombinant proteins in plants. An interesting point of the system is the high efficiency of co-transformation achieved by combining two or more independent Agrobacterium cultures with different genes of interest (co-transformation in trans). The complicated assembly of multiple transcriptional units in a single T-DNA entails the use of co-transformation in trans when coordinated expression of two or more proteins in the same cell / tissue is pursued. The system object of the invention allows efficient and simple cloning of several transcription units in the same T-DNA (in cis). To test whether this is the case and if the cis co-transformation improves a similar approach in trans, three fluorescent modules are assembled together with a suppressor silencing protein in the same T-DNA and its result is compared with a trans approach.

Thus, in the present example, the functionality of the present invention for the construction of multigenic structures for the genetic transformation of plants is demonstrated. The ordered assembly of a total of 12 consecutive fragments from 7 different basic pieces, including the binary vector, is shown. The standard pieces are grouped three by three (promoter, coding region and terminator), giving rise to four different functional modules (expression cassettes) corresponding to three different fluorescent proteins (green, red and blue) and a p19 silencing suppressor. TBSV (Qiu, Park et al. 2002) to enhance transient expression levels. The construction test by transient transformation of N. benthamiana sheets demonstrates that the resulting multigenic assembly is fully functional.

For the assembly, the corresponding input plasmids (pENTR) are constructed by cloning TA of the different precursors in pGEM-T. The precursors were obtained by PCR amplification of each piece,

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including the corresponding BsaI enzyme cleavage and recognition sites in the sequences of the primers used in the amplification, as detailed below:

a) Precursor of the 35s Promoter (SEQ.ID.NO 14): the 35S promoter is flanked by two BsaI restriction sites being the type 1 cut-off sites at the 5 ’end and type IV (GATG) at 3’. It is amplified from the vector pK2GW, 0 (Karimi, Inze et al. 2002) and includes a C> G mutation in nucleotide 206 to eliminate a BsaI restriction site. b) Tnos Terminator Precursor (SEQ.ID.NO 15): the terminator is flanked by two BsaI restriction sites being type III (TGAG) cutoff sites at the 5 ’end and type 2 at 3’. It is amplified from the pBIN-YFP / GUS vector (Orzaez, Mirabel et al. 2006). c) Precursors of the different CDS: the CDS are flanked by two BsaI restriction sites, these cut-off sites being type IV at the 5 ’end and type III at the 3’ end. The following CDS modules are built:

i. YFP (SEQ.ID.NO 16): amplified from the pBIN-YFP / GUS vector (Orzaez, Mirabel et al. 2006).

ii. DsRed (SEQ.ID.NO 17): Contains the sequence of the DsRed fluorescent protein in which a silent mutation C> A is performed in nucleotide 491 to remove a BsaI restriction site.

iii. BFP (SEQ.ID.NO 18): contains the sequence of the BFP fluorescent protein in which two silent mutations, both G> A, are made in nucleotides 443 and 632 to eliminate both BsmBI and BsaI restriction sites.

iv. P19 (SEQ.ID.NO 19): amplified vector pBINp19. Three silent mutations are made to eliminate two BsaI sites (G> C in nucleotide 386 and G> A in nucleotide 503) and a BsmBI site (G> T in nucleotide 155).

The assembly of the different pieces to generate modules (expression cassettes) was carried out by means of a Golden Gate reaction in a single tube (Engler, Kandzia et al. 2008) that contained the different pENTR_piece, BsaI enzyme and T4 ligase in 25 cycles of 37 ° C and 14 ° C. The combinations of pENTR_piece for assemblies of this strategy are outlined in Figure 7.A and were:

to.

Three-part assembly of pENTR_35S, pENTR_YFP and pENTR_terminator in the target vector pDGB_K_A12C, to give rise to the expression vector pEGB_K_A-YFP-C (SEQ.ID.NO 20)

b.

Three-part assembly of the pENTR_35S, the pENTR_p19 and pENTR_terminator in the destination vector pDGB_K_C12B, to give rise to the expression vector pEGB_K_C-p19-B. (SEQ.ID.NO 21)

C.

Three-part assembly of the pENTR_35S, the pENTR_BFP and the terminator pENTR in the target vector pDGB_K_A12C, to give rise to the expression vector pEGB_K_A-BFP-C. (SEQ.ID.NO 22)

d.

Three-part assembly of the pENTR_35S, the pENTR_DsRed and the pENTR_terminator the target vector pDGB_K_C12B, to give rise to the expression vector pEGB_K_C-DsRed-B. (SEQ.ID.NO 23)

After assembly, one microliter of each reaction was used to transform E. coli. The transformed cells were seeded on LB plates with kanamycin and X-gal. Four white colonies were selected from each reaction, grown in liquid medium and the corresponding plasmid was isolated and analyzed by digestion analysis and subsequent sequencing. Both analyzes showed correct assemblies in at least three of the four colonies analyzed for each reaction.

The four resulting pEGB vectors can in turn function as input plasmids since in them the new genetic modules are flanked by BsmBI restriction sites of type A, B or C. In this way modules two to two can be assembled on the vectors target pDGB level Ω, through Golden Gate reactions with BsmBI as restriction enzyme.

The assembly of the different simple modules to generate higher order modules (double transgenic) was carried out by a Golden Gate reaction in a single tube (Engler, Kandzia et al. 2008) that contained the different pEGB _MODULO, the enzyme BsmBI and ligase T4 in 25 cycles of 37ºC and 14ºC. The combinations of pEGB _MODULO for each reaction were:

to.

Bipartite assembly of pEGB_K_A-YFP-C and pEGB_K_C-p19-B in the target vector pDGB_S_1AB3, to give rise to the expression vector pEGB_S_1-YFP-p19-3 (SEQ.ID.NO 24)

b.

Bipartite assembly of the pEGB_K_A-BFP-C and pEGB_K_C-DsRed-B modules in the target vector pDGB_S_3AB2, to give rise to the expression vector pEGB_S_3-BFP-DsRed-2 (SEQ.ID.NO 25)

After assembly, one microliter of each reaction was used to transform E. coli. The transformed cells were seeded on LB plates with spectinomycin and X-gal. Four white colonies were selected from each reaction, grown in liquid medium and the corresponding plasmid was isolated and analyzed by digestion analysis and subsequent sequencing. Both analyzes showed correct assemblies in at least three of the four colonies analyzed for each reaction.

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The two resulting transgenic double pEGB vectors can function as input plasmids due to the presence of BsaI restriction sites of type 1, 2 or 3. Thus, to assemble a quad-expression cassette pEGB_K_A-YFP-P19-BFP-DsRed -C (SEQ.ID.NO 26), the target vector pDGB_K_A12C was incubated in a single tube, and plasmids pEGB_S_1-YFP-p19-3 and pEGB_S_3-BFP-DsRed-2 in the presence of BsaI and T4 ligase in 50 37ºC and 14ºC cycles

After assembly, one microliter of each reaction was used to transform E. coli. The transformed cells were seeded on LB plates with kanamycin and X-gal. Subsequently, four white colonies were selected, grown in liquid medium and the corresponding plasmid was isolated and analyzed by digestion analysis and subsequent sequencing. Both analyzes showed correct assemblies in the four colonies analyzed (Figure 7B).

One of the correct colonies of the final construction pEGB_K_A-YFP-P19-BFP-DsRed-C was selected and co-transformed with the pSOUP vector in Agrobacterium tumefaciens C58. The integrity of the plasmid isolated from Agrobacterium was checked by restriction analysis of the selected clones on LB plates rifampicin, kanamycin, tetracycline and gentamicin. An Agrobacterium clone containing the correct construct was grown in liquid medium and used to transiently transform Nicotiana benthamiana leaves (Wieland, Lammers et al. 2006). After four days of incubation, the infiltrated leaves in the fluorescence magnifier were observed and, with corresponding filters, the correct expression of the three fluorescent proteins was verified. In parallel, a mixture of Agrobacterium cultures expressing separately the three fluorescent proteins and p19 was agroinfiltrated. As shown in Figure 7C, proteins assembled on the same T-DNA showed a coordinated expression in N. be nthamiana as deduced from the similar fluorescence intensity observed in the three channels. On the other hand, when the fluorescent modules were agroinfiltrated in tra ns, each channel showed a different distribution in terms of intensities, evidencing heterogeneous levels of expression for the different proteins. In addition, we demonstrate how the system is permissive, at least in terms of transient expression, with the introduction of four copies of the 35s promoter into a single T-DNA without affecting the expression of fluorescent proteins. According to the results obtained, an assembly of several transcription units assisted by the present invention improves their expression in cis compared to the result obtained by their agroinfiltration in trans.

PRACTICAL EXAMPLE 3

The production of antibodies for therapeutic use in plants is a field that requires very flexible cloning strategies. In the present example, an embodiment of the invention is shown for the construction of different versions of recombinant antibodies and comparing the results obtained after expressing the proteins in plant. First, basic parts are assembled to form human immunoglobulin chain expression modules. Subsequently, two immunoglobulin modules (light and heavy) are assembled to form a complete immunoglobulin A expression device, assessing which of them all results in better expression. Finally, a kanamycin resistance expression module is assembled as a selection marker to the most efficient heavy and light chain combination. In total 8 basic pieces were assembled including the binary vector. The end result is a functional construction of three genes, ready to be transformed in the plant.

This example has the peculiarity that one of the modules is assembled on a plasmid pDGB_K (level α) and the remaining two are assembled on the plasmids pDGB_S (level Ω), thus demonstrating the versatility of entry points that the object system can have of invention In addition, it shows the versatility and flexibility of the system object of the invention to assemble combinatorial constructions from pre-set pieces.

First, the different input plasmids are constructed, the sequences of the precursors of the modules being amplified by PCR, and cloned into pGEM-T (Promega):

a) KanR Module Precursor (SEQ.ID.NO 27): The Kanamycin Resistance module is flanked by two BsaI restriction sites, being type 1 cut-off sites at the 5 ’end and type III at 3’. It is amplified from the vector pK2GW, 0 (Karimi, Inze et al. 2002). b) Part 35sSP Precursor (SEQ.ID.NO 28): This module comprises the 35s promoter of the vector pK2GW, 0 (Karimi, Inze et al. 2002) together with the signal peptide of Solanum lycopersicum pectate lyase. The module is flanked by two BsmBI restriction sites, with type A cut-off sites at the 5 ’end and type β (TGCC) at the 3’ end. c) CL.Tnos part precursor (SEQ.ID.NO 29): includes the sequences of the constant region of the IgA lambda light chain (Uniprot # P0CG04) and of the Tnos terminator of the vector pBIN-YFP / GUS (Orzaez, Mirabel et al. 2006). The module is flanked by two BsmBI restriction sites, with the γ (GGTC) type cutting sites at the 5 ’end and the B type at the 3’ end. d) Precursor Part CK.Tnos (SEQ.ID.NO 30): includes the sequences of the constant region of the IgA kappa light chain (GenBank AAH62704.1) and of the Tnos terminator of the vector pBIN-YFP / GUS

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(Orzaez, Mirabel et al. 2006). The module is flanked by two BsmBI restriction sites, the type γ (GGTC) cut sites at the 5 ’end and type B at the 3’ end. e) Precursor Piece CHα1.Tnos (SEQ.ID.NO 31): includes the sequences of the constant region of the heavy chain of IgA (Uniprot # P01876) and Tnos terminator of vector pBIN-YFP / GUS (Orzaez, Mirabel et al. 2006). The module is flanked by two BsmBI restriction sites, the sites being type γ ’(GCAT) cut at the 5’ end and type B at the 3 ’end. f) Precursor Part CHα2.Tnos (SEQ.ID.NO 32): includes the sequences of the constant region of the heavy chain of IgA (Uniprot # P01877) and Tnos terminator of the vector pBIN-YFP / GUS (Orzaez, Mirabel et al. 2006). The module is flanked by two BsmBI restriction sites, the sites being type γ ’(GCAT) cut at the 5’ end and type B at the 3 ’end. g) Precursor Part VL (SEQ.ID.NO 33): variable region of the light chain of scFv_2A1 (Wallet, Rodriguez-Diaz et al. 2004). The module is flanked by two BsmBI restriction sites, the β-type cutting sites at the 5 ’end and γ type at the 3’ end. h) Precursor Part VH (SEQ.ID.NO 34): variable region of the heavy chain of scFv_2A1 (Monedero, Rodriguez-Diaz et al. 2004). The module is flanked by two BsmBI restriction sites, the sites of type β being cut at the 5 ’end and γ’ at the 3 ’end.

Once the input plasmids of the different pieces (pENTR_PIEZA) have been constructed, the assemblies schematized in Figure 8A are made to generate the following modules:

1 Expression modules of the human immunoglobulin A heavy chains (IgHα1 and IgHα2) were assembled by means of Golden Gate reactions with BsmBI and T4 ligase, the input plasmids pENTR_35sSP, pENTR_VH and pENTR_CHα1.Tnos or pENTR_CHα2 with the target vector pDGB_S_1 destiny pDGB_S_1 , giving rise to the expression vectors pEGB_S_1-IgHα1-3 (SEQ.ID.NO 35) and pEGB_S_1-IgHα2.-3 (SEQ.ID.NO 36).

2 Expression modules of the human immunoglobulin A light chains (Igλ and Igκ) were assembled by combining in a single tube, in the presence of BsmBI and T4 ligase, the input plasmids pENTR_35sSP, pENTR_VL and pENTR_Cλ.Tnos or pENTR_Cκ.Tnos with the destination vector pDGB_S_3AB2, giving rise to the expression vectors pEGB_S_3-Igλ-2 (SEQ.ID.NO 37) and pEGB_S_3Igκ-2 (SEQ.ID.NO 38).

3 The Kanamycin resistance module was assembled by combining the PENTR_ KanR, PENTR_ Tnos input plasmids in a single tube in the presence of BsaI and T4 ligase with the target vector pDGB_K_A12C, resulting in the vector of pEGB_S_A-KanR-C (SEQ. ID.NO 39)

After performing the assemblies described, one microliter of each reaction was used to transform E. coli. The transformed cells were seeded on LB plates with spectinomycin and X-gal. Four white colonies were selected from each reaction, grown in liquid medium and the corresponding plasmid was isolated and analyzed by digestion analysis and subsequent sequencing. Both analyzes showed correct assemblies in at least three of the four colonies analyzed for each reaction.

Then the four vectors containing the heavy and light chains of the IgA were assembled together in the four possible combinations by Golden Gate reactions with BsaI on the target vector pDGB_K_C12B to give rise to the expression vectors pEGB_K_C-IgHα1-Igλ-B (SEQ.ID.NO 40), pEGB_K_CIgHα2-Igλ-B (SEQ.ID.NO 41), pEGB_K_C-IgHα1-Igκ-B (SEQ.ID.NO 42) and pEGB_K_C-IgHα2-Igκ-B (SEQ.ID .NO 43) with which four isotypes of IgA_2A1 can be expressed.

The four previous vectors, once checked by digestion (two colonies were selected in each case, resulting in the two correct ones - Figure 8B-), were used to transform Agrobacterium tum efaciens C58 cells. The integrity of the plasmid isolated from Agrobacterium was checked by restriction analysis selected on LB plates Rifampicin and Spectinomycin. An Agrobacterium clone containing the correct construct in each case was grown in liquid medium and used to transiently transform Nicotiana benthamiana leaves (Wieland, Lammers et al. 2006). After five days of incubation, the presence of a correctly assembled recombinant IgA was checked by western blotting under reducing and non-reducing conditions (Figure 8C) with the appropriate anti-IgH, anti-Igλ and anti-Igκ antibodies. Additionally, ELISA antigen tests with endpoint titration were performed. Briefly, the wells were upholstered with recombinant VP8 * antigen or BSA as a negative control (10 ug / mL). After blocking the wells, they were incubated with crude extract of plants transformed into serial dilutions and washed 5 times with PBS. They were subsequently incubated with a secondary anti-IgA antibody (SIGMA) conjugated to peroxidase and the result of the colorimetric reaction was quantified on a spectrophotometer. The results of the anti-VP8 activity test of the transgenic extracts can be seen in Figure 8D. From the ELISA and Western Blot antigen test it can be concluded that the four constructs are functional, although the IgHα1-Igλ version shows a better expression in plant.

Since the vector pEGB_K_C-IgHα1-Igλ-B can function as an entry plasmid due to the presence of BsmBI restriction sites of type C and B, the A-KanR-C and C-IgHα1-Igλ-B devices were binary assembled ,

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by a Golden Gate reaction with BsmBI as restriction enzyme, on the target vector pDGB_S_1AB3 to give rise to the expression vector pEGB_S_1-KanR-IgHα1-Igλ-3 (SEQ.ID.NO 44).

After enzymatic assembly, one microliter of each reaction was used to transform E. coli. The transformed cells were seeded on LB plates with spectinomycin and X-gal. From each reaction were selected

5 four white colonies were grown in liquid medium and the corresponding plasmid was isolated and analyzed by digestion analysis and subsequent sequencing. Both analyzes showed correct assemblies in at least three of the four colonies analyzed for each reaction. The resulting vector pEGB_S_1KanR-IgHα1-Igλ-3 is ready to be stably transformed into a plant.

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Claims (13)

1. A target plasmid (pDGB) for in vitro assembly of double stranded DNA pieces, characterized in that it comprises a skeleton of a double stranded DNA plasmid comprising: a) at least one origin of replication, b) at least one positive selection marker, c) at least one cassette that allows negative selection, d) at least 2 recognition sites for a first restriction enzyme of type IIS (E1) each giving rise to a cut-off site of 4 or more nucleotides selected between 1, 2 and 3, being 1, 2 and 3 sequences of any four nucleotides distinct from each other, and e) at least 2 recognition sites for a second restriction enzyme of type IIS (E1) each giving rise to a cut-off site of 4 or more nucleotides selected from A, B and C, A, B and C any four nucleotide sequences distinct from each other so that the cut and recognition sites cited in (d) and the cut and recognition sites cited in (e) flank the selection cassette cited in (c) at their 5 'ends and 3´ with inverted orientations.

2.

 The target plasmid (pDGB) for the in vitro assembly of double stranded DNA pieces according to claim 1, characterized in that the inverted orientations of the cutting and recognition sites cited in (d) and the cutting and recognition sites cited in (e) flanking the selection cassette cited in (c) at its 5 'and 3' ends are selected from: A12C, C12B, 1AB3, and 3AB2.

3.

 The target plasmid (pDGB) for the in vitro assembly of double stranded DNA pieces according to claim 2, characterized in that the orientation of the two cutting and recognition sites mentioned in (d) and the two cutting and recognition sites mentioned in (e) it is A12C, deposited in the Spanish Type Culture Collection as CECT 7900.

Four.

 The target plasmid (pDGB) for the in vitro assembly of double stranded DNA pieces according to claim 2, characterized in that the orientation of the two cutting and recognition sites mentioned in (d) and the two cutting and recognition sites mentioned in (e) it is C12B, deposited in the Spanish Type Culture Collection as CECT 7899.

5.

 The target plasmid (pDGB) for the in vitro assembly of double stranded DNA pieces according to claim 2, characterized in that the orientation of the two cutting and recognition sites mentioned in (d) and the two cutting and recognition sites mentioned in (e) it is 1AB3, deposited in the Spanish Type Culture Collection as CECT 7902.

6.

 The target plasmid (pDGB) for the in vitro assembly of double stranded DNA pieces according to claim 2, characterized in that the orientation of the two cutting and recognition sites mentioned in (d) and the two cutting and recognition sites mentioned in (e) it is 3AB2, deposited in the Spanish Type Culture Collection as CECT 7901.

7.

 An in vitro assembly method of double-stranded DNA pieces characterized in that it comprises an indefinite assembly loop that alternates the α level and the Ω level of quasi-idempotent binary assembly, where the α level comprises the following steps:

i) construction of the α-level input plasmids (pE), each α-level input plasmid (pE) comprises a piece of double stranded DNA selected from (Xi) and / or (Xj) flanked at its ends 5 ´and 3´ for two cut and recognition sites selected between 1, 2 and 3, for a first restriction enzyme of type IIS (E1): pE [1 (Xi) 3] and pE [3 (Xj) 2] ii) construction of the target level plasmids (pDGB) defined in claims 3 and 4: pDGB (A12C) (CECT 7900) and pDGB (C12B) (CECT 7899) iii) enzymatic digestion of the input plasmids obtained in i) and the target plasmids obtained in ii) in the presence of E1, generating at its ends 3 'and 5' single stranded DNA terminal sequences of at least 3 nucleotides, iv) ligation with T4 ligase between the single-chain terminal sequences of the input and target plasmids digested in iii) forming the assembled plasmids of level of closed circular double-stranded DNA α: pE [1 (Xi) 3] + pE [3 (Xj) 2] + pDGB (A12C) (CECT 7900) = pE [A (Xi + Xj) C] 19 pE [1 (Xi) 3] + pE [3 (Xj) 2] + pDGB (C12B) (CECT 7899) = pE [C (Xi + Xj) B]; and where the Ω level comprises the following stages: v) construction of the Ω level input plasmids (pE) each Ω level input plasmid (pE) comprises a piece of double stranded DNA selected from (Xi) and / or (Xj), flanked at its 5 'ends and 3 'by two cut and recognition sites selected from A, B and C, for a second restriction enzyme of type IIS (E2): pE [A (Xi) C] and pE [C (Xj) B] vi) construction of the target Ω level plasmids (pDGB) defined in claims 5 and 6: pDGB (1AB3) (CECT 7902) and pDGB (3AB2) (CECT 7901) vii) enzymatic digestion of the input plasmids obtained in v) and the target plasmids obtained in vi) in the presence of E2, generating at its ends 3´ and 5´ single stranded DNA terminal sequences of at least 3 nucleotides, viii) T4 ligase ligation between the single-chain terminal sequences of the input and target plasmids digested in vii) forming the assembled level A plasmids of closed circular double-stranded DNA: pE [A (Xi) C] + pE [C (Xj) B] + pDGB (1AB3) (CECT 7902) = pE [1 (Xi + Xj) 3] pE [A (Xi) C] + pE [C (Xj) B] + pDGB (3AB2) (CECT 7901) = pE [3 (Xi + Xj) 2]; when the assembly method starts at the α level and continues with the Ω level, the level A input plasmids pE [A (Xi) C] and pE [C (Xj) B] defined in v) are replaced by Level 1 assembled plasmids pE [A (Xj + Xj) C] and pE [C (Xi + Xj) B] obtained in iv); Y when the assembly method starts at the Ω level and continues with the α level, the α level entry plasmids pE [1 (Xi) 3] and pE [3 (Xj) 2] defined in i) are replaced by assembled plasmids of level Ω pE [1 (Xi + Xj) 3] and pE [3 (Xi + Xj) 2] obtained in viii).

8.

 The in vitro assembly method of double stranded DNA pieces according to claim 7 characterized in that (Xi) and / or (Xj) is selected from a promoter, a subcellular localization sequence, a coding sequence, a DNA copy is sequenced of interfering RNA, and a termination sequence, an expression cassette of proteins, enzymes, transcription factors, markers, antibodies, regulatory proteins, or an expression cassette of functional RNAs or chromatin structural elements.

9.

 The in vitro assembly method of double stranded DNA pieces according to claim 7, characterized in that the target plasmids (pDGB) are binary plasmids for the genetic transformation of plants mediated by Agrobacterium tumefaciens.

10.

 The in vitro assembly method of double-stranded DNA pieces according to claim 7, characterized in that (Xi + Xj) is formed by a kanamycin gene, a gene that codes for the light chain of an antibody and a gene that codes for heavy chain of an antibody.

eleven.

 The in vitro assembly method of double stranded DNA pieces according to claim 7, characterized in that (Xi + Xj) is formed by four expression cassettes constituting three genes encoding three fluorescent proteins and a fourth gene encoding the p19 protein.

12.

 A plant transformed by Agrobacterium tumefaciens by the assembly method described in claim 11 and characterized in that said plant expresses a recombinant antibody of type IgA.

13.

 A plant transiently transformed by Agrobacterium tu mefaciens by the assembly method described in claim 12 and characterized in that said plant simultaneously expresses the said three fluorescent proteins.

twenty FIGURE 1  FIGURE 2 (TO) E2 E2 (C) E1 (B) E1E2 E1 E2 (AND) E2 (D) E2 E1 E2 E1 twenty-one FIGURE 3 22 FIGURE 4 FIGURE 5 2. 3 FIGURE 6 FIGURE 7 24 FIGURE 8 25