ES2388255A1 - Transcriptional fusion vectors for uni and bidirectional promoter regions for use in lactic bacteria - Google Patents

Abstract

The invention relates to a recombinant nucleotide sequence that includes the sequence of the mrfp gene that codes for a red autofluorescent protein optimized for expression in lactic bacteria, as well as the vectors that comprise same. The invention likewise relates to the cells that include the sequence or the vector of the invention and the uses thereof. In addition, the invention relates to the use of both the sequence and the vector for the detection and characterization of a unidirectional or bidirectional promoter region, as well as to the use thereof for transcription. The invention further relates to the use of cells that contain the sequence or vector of the invention, for determining the colonizing ability of cells. The invention also relates to the methods associated with the uses of the invention, to a kit that includes the elements of the invention and to the use thereof.

Description

Transcriptional fusion vectors for uni-and bidirectional promoter regions for use in lactic bacteria.

The present invention relates to new transcriptional fusion vectors containing the mrfp gene, which codes for a red fluorescent protein, optimized in codons for expression in lactic bacteria. Such vectors are useful for cloning, detection and characterization of unidirectional or bidirectional promoter regions. In addition, they are also useful for the detection of the expression of polynucleotides of interest and for the determination of the colonizing capacity of the cells that comprise them.

STATE OF THE TECHNIQUE

The genes encoding fluorescent proteins by autocatalytic fluorophore formation are ideal for use as markers in the evaluation of gene expression and for a wide range of molecular biology procedures. There is a wide commercial offer of vectors available for gene expression assays that incorporate as a marker the expression of the green fluorescent protein (GFP) from Aequorea victoria. Other widely spread markers are the genes encoding �-galactosidase (�-gal), luciferase, chloramphenicol acetyltransferase (CAT), secreted alkaline phosphatase (SEAP), among others. The gene that encodes GFP has been used as a marker of biological processes such as gene activity and regulation, protein folding and cell localization and in vitro and in vivo monitoring of labeled cells (Wiedenmann, Oswald and Nienhaus (2009) IUBMB Life 61: 1029-1042).

Among the autofluorescent proteins that have been pursued with greater interest in research, the search for red fluorescent proteins (RFP) stands out, obtaining in the nature the fluorescent protein from Discosoma sp. DsRed. However, the main difficulty in using this protein is that it is a tetramer that matures slowly. In this sense, monomeric fluorescent proteins have been obtained by protein engineering by means of mutagenesis directed in the genetic sequence of the red fluorescent protein DsRed (Shaner, Campbell, Steinbach, Giepmans, Palmer and Tsien (2004) Nature Biotechnol. 22: 1567-1572 ). Of the autofluorescent proteins obtained, the monomeric mCherry protein (mRFP) stands out, which has the advantage of maturing rapidly and completely, possessing high photostability and excellent resistance to acidic pH (pKa <4.5). In addition, the optimal excitation and emission spectra of GFP (488 nm and 511 nm, respectively) and mRFP (587 nm and 610 nm, respectively) do not overlap each other which makes their simultaneous detection possible.

While the commercial supply of vectors containing the gene that codes for GFP (gfp) is very wide, there are few vectors available that code for red fluorescent proteins. The Cherry ™ Express vector, marketed by Evrogen (Moscow, Russia), is composed of a sequence encoding a red 11 kDa polypeptide from the cytochrome heme group binding region. For its part, the Clontech company (Mountain View, California, USA) markets a vector called pmCherry1 that contains the sequence that encodes codon optimized mRFP protein for human gene expression, and that allows the insertion and evaluation of functional promoters . Said vector can be propagated in E. coli and allows expression in eukaryotic cells.

In the scientific literature, the expression of mRFP in different species of Gram-negative bacteria such as Pseudomonas spp., Edwarsiella tard and E. coli (Lagendijk, Validov, Lamers, de Weert, Bloemberg and GV (2010) FEMS Microbiol. Lett. 305: 81-90) and in Gram-positives such as Staphylococcus aureus (Malone, Boles, Lauderdale, Thoendel, Kavanaugh and Horswill (2009) J. Microbiol. Meth. 77: 251-260). Both studies are focused on the use of autofluorescent protein as a marker for the visualization of the communities of these bacteria forming biofilms. In these studies, other fluorescent markers with GFP are also used to differentiate bacterial communities consisting of mixtures of different species or the same species expressing different properties. The gfp gene has also been used as a marker for in vitro and in vivo monitoring of lactic bacteria used as oral vaccine vehicles (Geoffroy, Guyard, Quatannens, Pavan, Lange and Mercenier (2000) Appl. Environ. Microbiol. 66: 383 -391).

The construction of vectors used for the characterization of divergent promoters in Gram-negative bacteria has been described, using as cloning marker genes those that code for enzymatic activities such as 1-glucuronidase and 1-galactosidase (Parry, Sharma and Terzaghi (1994) Gene 150: 105-109; Jiwaji, Matcher and Dorrington (2008) S. Afr. J. Sci. 104: 305-307) and 1-glucuronidase and GFP (Zhang, Gal, Zhu, Chen and Jiang (2010) Front. For China 3: 112-116). The lacZ gene has also been used as a reporter gene to evaluate the activity of divergent promoters in Saccharomyces cerevisiae (Partow, Siewers, Bjorn, Nielsen and Maury (2010) Yeast 27: 955-964). However, the verification of cloning and the detection of promoter expression in most of these examples require the evaluation of the enzymatic activity employed as a marker discontinuously and not simultaneously with growth.

Lactic bacteria have described vectors that comprise the gene that codes for GFP as well as cloning marker genes that code for enzymatic activities, such as the pAK80 vector and its derivatives that include the 1-galactosidase gene (Margolles, Moreno, Ruiz, Marelli, Magni, de los Reyes Gavilán, Ruas Madiedo (2010) J. Biosci. Bioeng. 109: 322-324). However, the GFP protein has a low acidity resistance (pKa> 6.6), so its use in lactic bacteria represents a difficulty since these bacteria tend to acidify the medium and, therefore, the medium must necessarily contain buffers to maintain the pH at values close to neutrality.

Lactococcus lactis is a Gram-positive bacterium of the order Lactobacillales and constitutes one of the most representative species within lactic bacteria. In addition, it is characterized by having been safely consumed in food, especially in cheeses, throughout the history of mankind. Together, L. lactis is the most widely used lactic bacterium in physiological and expression studies due to the possibilities of being manipulated by genetic engineering. In recent decades, this species has demonstrated a valuable role as a safe vehicle for the expression of therapeutic proteins and vaccines and for their potential application in the treatment of inflammatory diseases and allergies (Rottiers, De Smedt and Steidler (2009) Int. Rev. Immunol. 28: 465-486).

Like L. lactis, Enterococcus faecalis is a Gram-positive bacterium of the order Lactobacillales, however, this species is emerging in recent times as a nosocomial pathogen in immunocompromised patients, clones adapted to hospitals due to its multiple resistance characteristics to antibiotics and the acquisition of virulence genes (Sava, Heikens and Huebner (2010) Clin. Microbiol. Infect. 16: 533-540).

For everything described here, there is currently a need for vectors that contain genes that code for red fluorescence and are useful in lactic bacteria (Lactobacillales order) of agri-food and health interest. Vectors that combine green fluorescence (through the use of the gfp gene) with genes encoding enzymatic activities have the disadvantage of not being able to be used for simultaneous detection of expression from two divergent promoters nor can they be used for simultaneous detection of expression and cell growth. It is necessary, therefore, the development of a new tool that combines two genes that code for fluorescent proteins that allow the rapid and simultaneous detection of bi-directional promoter regions in lactic bacteria.

DESCRIPTION OF THE INVENTION

The present invention relates to vectors that allow rapid and simultaneous detection of bi-directional promoter regions in lactic bacteria, by expression of fluorescence with two autofluorescent proteins used as markers and placed divergently.

The vectors of the invention increase the molecular tools available for gene expression and regulation studies of lactic bacteria of the L. lactis and E. faecalis species. In addition, the vectors of the invention are functional in E. coli, which greatly facilitates the cloning procedures.

The vectors of the present invention contain the gene encoding the red autofluorescent protein mCherry (mRFP) whose nucleotide sequence has been optimized in the present invention for heterologous expression in Gram-positive bacteria high in adenine (A) and thymine (T ) (A + T), as is the case with lactic bacteria. For this reason, the first aspect of the invention relates to a nucleotide sequence consisting of the sequence of the mrfp gene encoding a red fluorescent protein, wherein said sequence is SEQ ID NO: 1. In the present invention, "the sequence of the mrfp gene ”refers to the sequence of the gene that encodes the mRFP protein that contains four more codons at the 5 'end of the optimization product in lactic bacteria and also includes at the ends the restriction sites of the SmaI and SalI enzymes .

Hereinafter we will refer to SEQ ID NO: 1 as the "sequence of the invention" and the protein encoding the sequence of the invention as the "protein of the invention".

Optimization is understood as the use of codons that favor an increase in the production yield of the fluorescent protein. In some cases, such optimization is necessary for the expression of the heterologous protein to exist. In the present invention it refers to the use of codons high in adenine and thymine (A + T) that allow the expression of the protein of the invention in lactic bacteria. Therefore, "optimized sequence" in the present invention refers to the mrfp gene sequence that contains codons high in A + T.

Codons means nucleotide triplets that encode, according to the genetic code known to any person skilled in the art, for a given amino acid or for a translation termination signal.

The term "high content in A + T" refers to rich in adenine and thymine. In the present invention it refers to the optimized mrfp gene referred to in SEQ ID NO: 1 contains a greater proportion of adenine and thymine than cytosine (C) and guanine (G) than the unimproved mrfp gene.

"Heterologous expression" means that expression of a gene sequence that does not belong to the same organism as that used for its expression. In the present invention the mRFP is native to an organism other than that used for its expression, whereby the expression of the mrfp gene in lactic bacteria is a heterologous expression.

Fluorescent protein means that protein that, after being excited with a certain wavelength, emits light of a longer wavelength. In the present invention the terms "fluorescent" and "autofluorescent" are used interchangeably.

The red fluorescent protein mCherry (mRFP; GenBank accession number AAV52164) is a monomeric protein derived from the red fluorescent protein from Discosoma sp. (coral DsRed protein, GenBank accession number Q9U6Y8) (Shaner, Campbell, Steinbach, Giepmans, Palmer and Tsien (2004) Nature Biotechnol. 22: 1567-1572). The mRFP has excellent resistance to acidic pH (pKa <4.5) which makes it potentially useful for use in lactic bacteria. The mRFP protein has an optimum excitation wavelength at 587 nm and an optimal emission wavelength at 610 nm, for which the fluorescence intensity is maximum. mRFP emits light in the red zone of the visible spectrum. The protein of the invention is the mRFP that due to the optimization process contains four extra amino acids at its amino terminal end, Methionine-Asparagine Serine-Aspartate (MNSD) (SEQ ID NO: 4) that does not significantly affect its characteristics such as pKa or excitation and emission spectra. The protein of the invention has a maximum excitation wavelength of 587 nm and an emission of 612 nm.

A second aspect of the present invention relates to a recombinant nucleotide sequence comprising the sequence of the invention.

By "recombinant nucleotide sequence" is meant a genetic construction of DNA encoding the sequence of the invention and comprising genetic material of different organisms, for example genetic material of Discosoma sp. and from L. lactis. The present invention also relates to the genetic construction of RNA comprising the RNA that is transcribed from the sequence of the invention. The recombinant nucleotide sequence may be comprised in a vector where said vector is a plasmid.

In the present invention, molecular tools known to any person skilled in the art have been used to generate the vectors of the invention, including cloning and the use of restriction enzymes. For this reason, a preferred embodiment of the second aspect of the invention relates to a sequence that further comprises at least one sequence recognized by at least one restriction enzyme.

A sequence recognized by a restriction enzyme is understood to refer to a site or site that recognizes an endonuclease that is capable of cutting the phosphodiester bonds of the deoxyribonucleotide chain generating a cut in the deoxyribonucleic acid (DNA) chain. For example sequences recognized by the restriction enzymes SmaI, SalI, BglII, XhoI, BamHI, PstI and EcoRI

The present invention also relates to nucleotide sequences comprising, in addition to the sequence of the invention, the gfp gene sequence, which encodes the green fluorescent protein (GFP). In the present invention, the orientation of both autofluorescent proteins is divergent, that is in the opposite direction, the 5 ′ end of both nucleotide sequences being closer to each other than the 3 ′ end.

The green fluorescent protein (GFP) is an isolated protein from Aquorea victoria that emits light in the green area of the visible spectrum. The excitation and emission spectrum of GFP is 488 nm and 511 nm, respectively. The gfp gene used in the present invention refers to the sequence SEQ ID NO: 8.

For this reason, an even more preferred embodiment of the present invention relates to the nucleotide sequence of the invention which further comprises the gfp gene, which encodes the green fluorescent protein (GFP), wherein said sequence is SEQ ID NO: 8. In Hereinafter we will refer to the sequence of this preferred embodiment as the "second nucleotide sequence of the invention".

The nucleotide sequence of the second aspect of the invention can also comprise at least one sequence recognized by at least one restriction enzyme between the mrfp and gfp genes.

A third aspect of the invention relates to a vector comprising the nucleotide sequence of the first or second aspect of the invention. Hereinafter we will refer to this vector as the "first vector of the invention".

A preferred embodiment of the third aspect of the invention relates to a vector that further comprises a functional replicon in L. lactis. Hereinafter we will refer to this vector as the "second vector of the invention".

In the present invention, replicon is understood as the minimum DNA sequence required for the replication of the vector or plasmid in an organism. Said replicon may be, for example, the replicon of plasmid pCT1138 which is functional in both L. lactis and E. faecalis.

A preferred embodiment of the third aspect of the invention relates to a vector that further comprises the plasmid p15A replicon that is functional in Escherichia coli. Hereinafter we will refer to this vector as the "third vector of the invention".

Hereinafter we will refer to "vector of the invention" as the first, second or third vector of the present invention.

A vector is understood to mean a DNA sequence corresponding to a gene expression system comprising the sequence coding for the sequence of the invention, operably linked to at least cut sites or a promoter that directs the transcription of said sequence, and with other sequences to regulate its transcription such as, for example, start signals or secondary structures. Preferably the vector is a plasmid.

For these reasons, a preferred embodiment of both the first vector, the second vector and the third vector of the invention relates to a vector where the nucleotide sequence is operatively linked to at least one gene expression regulatory sequence.

The regulatory sequence of gene expression can be selected from:

a) a promoter, or

b) a transcription initiation signal.

A promoter, or promoter region, is a nucleotide sequence that controls the transcription of a particular gene (nucleotide sequences). In the present invention it refers to a nucleotide sequence that controls the transcription of at least the nucleotide sequences of the first or second aspect of the invention. Promoter sequences can be unidirectional or bidirectional. A unidirectional promoter is one that controls the transcription of one gene or more genes that are placed in tandem with the first one, for example the wild promoter PX (SEQ ID NO: 10) of S. pneumoniae (Nieto, Espinosa and Puyet (1997 ) J. Biol. Chem. 272: 30860-30865). "In tandem" means that the 3 ′ end of the first gene is followed, either consecutively or separated by a particular nucleotide sequence, by the 5 ’end of the second gene. A bidirectional promoter refers to the promoter region that controls transcription in two opposite directions, for example the sequence that precedes the kivD gene of L. lactis IFPL730 that acts as a bidirectional promoter (De la Plaza, Peláez and Requena (2009) J. Mol. Microbiol. Biotechnol. 17: 96-100). That is, a bidirectional promoter directs the transcription of two genes located divergently, that is in the opposite direction, the 5 ′ end of both nucleotide sequences being closer to each other than the 3 ′ end. In the present invention the terms "promoter" and "promoter region" are used interchangeably.

In addition, the promoters in the present invention can be constitutive or inducible. The term "inducible", as used in the present description, refers to the possibility that the promoter has a control element that allows activating or deactivating (repressing) the transcription of the gene that regulates, in the presence of a factor external to the promoter.

The promoters referred to in the present invention control at least the transcription of one of the fluorescent proteins. Therefore, another preferred embodiment of the third aspect of the invention relates to a vector where the promoter controls at least the transcription of one of the fluorescent proteins.

For all that is described herein, another preferred embodiment of the third aspect of the invention relates to a vector where the promoter is a unidirectional promoter.

Another even more preferred embodiment of the third aspect of the invention relates to a vector where the unidirectional promoter is the Streptococcus pneumoniae PX promoter, whose sequence is SEQ ID NO: 10.

Another even more preferred embodiment of the third aspect of the invention relates to a vector where the vector is the pAK80 derived vector that lacks the gene encoding for -galactosidase, is the vector of the invention called pAKmRFP-PX.

pAK80 is a plasmid that contains as a marker -galactosidase, has as a selection marker the resistance to erythromycin and contains two functional replicons respectively in Gram-positive (L. lactis and E. faecalis) and Gram-negative (E. coli) bacteria ( Israelsen, Madsen, Vrang, Hansen and Johansen (1995) Appl. Environ. Microbiol. 61: 2540-2547).

Another preferred embodiment of the third aspect of the invention relates to a vector where the promoter is bidirectional.

Another even more preferred embodiment of the third aspect of the invention relates to a vector where the bidirectional promoter is the promoter region comprising the divergent promoters PKivD and PRmaF-RlrC (PKivD-PRmaF-RlrC) of

L. lactis, whose sequence is SEQ ID NO: 13.

Another even more preferred embodiment of the third aspect of the invention relates to a vector where the vector is the pAK80 derived vector that lacks the gene encoding for -galactosidase, is the vector of the invention called pAKmRFP-PKivD-PRmaF-RlrC.

A fourth aspect of the present invention relates to a cell comprising the nucleotide sequence of the first and second aspects of the invention or the vector according to the third aspect of the invention. Hereinafter we will refer to this cell as the "cell of the invention". The cell of the invention is preferably a bacterium. The introduction of said gene constructs can be carried out with the methods known in the state of the art.

A preferred embodiment of the fourth aspect of the invention relates to the cell where said cell is a Gram-positive bacterium. Preferably it is a bacterium of the order Lactobacillales. More preferably the cell is selected from L. lactis or E. faecalis.

The term "Gram-positive" refers to those bacteria that are stained dark blue or violet by Gram staining. This characteristic is closely linked to the structure of the cell envelope. The cell envelope of Gram-positive bacteria comprises the cytoplasmic membrane and a cell wall composed of a thick layer of peptidoglycan, which surrounds the previous one and is responsible for retaining the dye during Gram staining. Unlike Gram-negative, Gram-positive do not have a second lipid membrane external to the cell wall and this wall is much thicker.

Lactic bacteria are Gram-positive bacteria of the order Lactobacillales that produce lactic acid from sugars. The L. lactis species belongs to the Streptococcaceae family, genus Lactococcus. The present invention also relates to the subspecies of L. lactis, for example L. lactis subsp. lactis, L. lactis subsp. lactis biovar. diacetylactis, L. lactis subsp. cremoris and L. lactis subsp. horniae

Through biotechnology strategies known to those skilled in the art, E. coli can be used for the propagation of the third vector of the invention, whereby the cell of the invention can also refer to E. coli.

Another preferred embodiment of the fourth aspect of the invention refers to the cell of the invention which, when comprising the protein of the invention, emits red fluorescence; green fluorescence by expression of the GFP protein, which would happen if mRFP was not expressed; or brown fluorescence by simultaneous expression of both proteins.

In the case of the simultaneous use of the mrfp and gfp genes, in the present invention the pH of the culture medium is controlled so as to maintain a suitable pH for the expression of both proteins so that the possible differences found in The expression is not due to a condition that disadvantages the expression of either of the two fluorescent proteins.

This invention also refers to the cell population comprising the cell of the invention. Hereinafter we will refer to this as the "cell population of the invention".

The applications of both the sequences of the invention, as of the vectors of the invention or of the cells of the invention could encompass different functions related to both biotechnological characteristics of agri-food interest and probiotic functionality, as well as for food and pharmaceutical safety studies.

The vectors of the invention are usable for the cloning and characterization of a promoter by detecting the fluorescence of the mRFP or two divergent promoters by detecting two autofluorescent proteins, mRFP and GFP. In addition, vectors also allow quantitative evaluation of gene expression regulation from the cloned promoter (s) in them. Thus, the generation of transcriptional fusions using the aforementioned expression vectors also allows the in situ evaluation of gene expression from the promoter or promoters during the growth of the recombinant carrier bacteria as fluorescence and optical density can be simultaneously evaluated by spectrofluorimetry and spectrophotometry. . This fact involves the detection of promoter cloning and expression evaluation not only specifically and quantitatively, but also dynamically over time.

As described herein, another aspect of the present invention relates to the use of the nucleic acid of the first or second aspect of the invention or of the vector of the third aspect of the invention for the detection and characterization of a promoter. The promoter region can be unidirectional or bidirectional.

Another aspect of the present invention relates to the use of the nucleic acid of the first or second aspect of the invention or of the vector of the third aspect of the invention for the transcription of at least one polynucleotide (also called "polynucleotide of interest"). That is, its use for heterologous transcription. The polynucleotides could be polynucleotides of interest in the food or health sector related to lactic bacteria. For example, in the case of using the vectors of the invention in L. lactis, the polynucleotides would be those that code for proteins of agri-food interest such as enzymes, nutraceuticals, bacteriocins, vitamins, exopolysaccharides, etc. In the case of using the vectors of the invention in E. faecalis, the polynucleotides of interest could be, for example, those that encode resistance to antibiotics, hemolysins, aggregation and adhesion proteins, cytolysins, etc.

Another aspect of the present invention relates to the use of the cells of the invention or of the cell populations comprising the cell of the invention for the determination of the colonizing capacity of said cells in a mixed bacterial culture or in cell co-cultures. eukaryotes isolated or in culture of isolated biological tissue or in an animal model. For example, the animal model could refer to zebrafish or Caenorhabditis elegans.

The methods of fluorescence detection of the cells of the invention are also described in the present invention. From visual procedures of the transforming colonies that allow the direct selection of the clones that have acquired the promoter region, to the evaluation by means of spectrofluorimetry and spectrophotometry of the conditions of regulation of promoters during the expression in vivo and in function of variation of environmental conditions and physiological The present invention also describes the process by fluorescence microscopy that allows differentiating labeled bacterial species with different fluorescence in mixed cultures and in interaction studies with cultures of human cell lines.

For these reasons, another aspect of the invention relates to a method of detecting the cell of the fourth aspect of the invention which comprises detecting the fluorescence of said cells. A preferred embodiment relates to the method where detection is carried out by means of spectrofluorimetry or by fluorescence microscopy, techniques widely known to any person skilled in the art. Another even more preferred embodiment relates to the method where fluorescence is detected in colonies of said cells.

For all of the foregoing, another aspect of the present invention relates to a method of detection and characterization of promoters comprising:

to.

transform a host cell with the vector of the invention,

b.

cultivate the cell in step (a) under appropriate conditions,

C.

determine the fluorescence levels of the fluorescent protein,

d.

compare the fluorescence obtained in step (c) with the fluorescence emitted by a control cell.

The transformation of the host cell, the cell of the invention, with the vector of the invention is carried out using the tools known to any person skilled in the art, for example, by electroporation.

"Suitable conditions" means those conditions that are necessary for the growth of the cell of the invention and for the expression of the nucleotide sequences comprised in the vectors of the invention. The suitable culture medium of the transformed host cell referred to in any of the methods described in the present invention, as well as the culture conditions (such as, but not limited to, nutrients, pH or temperature) are selected according to the nature of the host cell.

"Control cell" means a cell that has been transformed with a control vector, this being a vector that lacks the differential characteristics of the vector used in the test.

The term "compare" refers to conducting a study to determine the significant statistical differences between the cell of the invention and the control cell. In the case of a statistical study, it is intended to collect data that shows a significant deviation between the results of both cells.

In the present invention "statistically significant difference" refers to the fact that there is statistical difference between the values compared, the statistical probability being at least greater than or less than 0.05 (p> 0.05 op> 0.05) and this being obtained according to the test statistic applicable to each case.

The present invention also relates to a method of detecting the expression of polynucleotides, such as enzymes, bacteriocins, membrane proteins, while detecting the growth of the cell of the invention. As another aspect of the present invention relates to method of detecting polynucleotide expression comprising:

a) inserting into the vector of the invention a polynucleotide that is controlled by the same promoter that regulates the transcription of the gene encoding the fluorescent protein,

b) transform a host cell with the vector resulting from step (a),

c) cultivate the cell of step (b) under the appropriate conditions and

d) determine the fluorescence levels of the fluorescent protein simultaneously with the determination of cell growth.

On the other hand, for everything described above, another aspect of the invention relates to a method of determining the colonizing capacity of the cells of the invention or of the cell population of the invention comprising:

a) cultivate the cells in the right conditions,

b) contact the cells with bacteria, tissues or an animal model,

c) determine the fluorescence levels of the fluorescent protein by spectrofluorimetry or by fluorescence microscopy and

d) compare the fluorescence determined in step (c) with the fluorescence emitted by a cell,

tissue or control animal that do not have the same characteristics as the cell, tissue, or animal

of step (b)

Another aspect of the invention relates to a kit comprising:

a) at least one recombinant nucleotide sequence, which could be the sequence of the first aspect of the invention or that of the second aspect of the invention,

b) at least one vector of the invention, or

c) at least one cell of the invention

Said kit could be used for the detection and characterization of promoters, or for the transcription of a polynucleotide of interest or for the determination of the colonizing capacity in mixed bacterial cultures or in co-cultures with isolated eukaryotic cells or in isolated biological tissue culture. or in an animal model, whereby the invention also relates to the uses described herein of the kit.

The terms "polynucleotide" and "nucleic acid" refer to a polymeric form of nucleotides of any length, both ribonucleotides and deoxyribonucleotides.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following figures and examples are provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES

Figure 1. Construction scheme of the vectors containing the mrfp gene. The construction scheme of plasmids pAKmRFP, pAKmRFP-Px, pAKmRFPGFP and pAKmRFPGFP-PKivD-PRmaF-RlrC from plasmid pAK80 is shown. The location of the sequences recognized by useful restriction enzymes present in the plasmids, SmaI, SalI, BglII, XhoI, BamHI, PstI and EcoRI is shown. Replicon pCT1138, functional replicon in L. lactis and E. faecalis. Replicon P15A, functional replicon in E. coli. ermC, erythromycin resistance gene. -gal, gene that codes for –galactosidase. mrfp, sequence of the invention. gfp, gene that codes for the GFP protein used in the invention. Px, Px promoter. PKivD-PRmaF-RlrC, PKivD-PRmaF-RlrC promoter.

Figure 2. Detection of red fluorescence in bacteria containing the vector pAKmRFP-PX. Red fluorescence emission (UFR, 0) of bacteria during growth (DO480, 0) of E. coli DH5a (E.coli) (A), L. lactis MG1363 (L. lactis) (B) and E. faecalis JH2 -2 (E. faecalis) (C) containing the vector pAKmRFP-PX. T, time; h, hours

Figure 3. Detection of red and green fluorescence in bacteria containing the vector pAKmRFPGFP-PKivDPRmaF-RlrC. Emission of red fluorescence (UFR, 0) and green fluorescence (UFV, 0) during growth (00480, 0) of

E. coli DH5a (E.coli) (A), L. lactis MG1363 (L. lactis) (C) and E. faecalis JH2-2 (E. faecalis) (E) containing the vector pAKmRFPGFP-PKivD-PRmaF- RlrC Fluorescence microscopy images of fluorescent cells of E. coli DH5a (B), L. lactis MG1363 (D) and E. faecalis JH2-2 (F) expressing the red fluorescent protein mRFP and the green fluorescent protein GFP of the invention . T, time; h, hours

Figure 4. Detection of inducible and repressive expression of PKivD-PRmaF-RlrC. Emission of red fluorescence (UFR, 0) and green fluorescence (UFV, 0) of L. lactis MG1363 (L. lactis) containing the vector pAKmRFPGFP-PKivDPRmaF-RlrC during growth (DO480, 0) in (A) chemically medium defined (CDM) containing casitone (repressive conditions) (CDM-r) and in (B) CDM in the absence of isoleucine (inducing conditions) (CDM-i). T, time; h, hours

Figure 5. Detection of the expression of mRFP and GFP proteins by fluorescence microscopy. Fluorescence microscopy images of E. coli DH5a fluorescent cells (pGreenTIRGFP) (A), which express the green GFP fluorescent protein used in the invention, and L. lactis MG1363 (pAKmRFP-Px) (B) and E. faecalis JH2 -2 (pAKmRFP-Px) (C), which express the mRFP red fluorescent protein of the invention, which are attached to human Caco-2 cells.

ILLUSTRATIVE EXAMPLES OF THE INVENTION

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. Therefore, the examples described below illustrate the invention without limiting its scope of application.

EXAMPLE 1: Construction of plasmids comprising the mrfp gene.

The construction scheme is shown in Figure 1. For the construction of the transcriptional fusion vectors, the vector pAK80 was started (Israelsen, Madsen, Vrang, Hansen and Johansen (1995) Appl. Environ. Microbiol. 61: 2540-2547 ). The vector pAK80 contains an origin of replication from plasmid pCT1138 of L. lactis subsp. lactis biovar. CRL264 diacetylactis which is also functional in E. faecalis (Marelli and Magni (2010) World J. Microbiol. Biotechnol. 26: 999-1007). In addition, pAK80 contains the origin of replication from plasmid p15A of E. coli, which gives it a shuttle vector character so that it can also be propagated in E. coli. As a marker for the selection of transformants, pAK80 contains the ermC gene, which confers resistance to erythromycin in E. coli and L. lactis and E. faecalis.

The pAK80 vector was digested with the restriction enzymes SmaI and SalI. One of the resulting 7 kb DNA fragments from pAK80, which contains the 2 origins of replication and the determinant of resistance to erythromycin, was separated on a 0.8% agarose gel. Subsequently, the fragment was purified (Qiagen) to be used as a base in the construction of the recombinant plasmids.

The gene encoding the monomeric protein with red fluorescence mCherry (mRFP), optimized in codons for expression in Gram-positive bacteria high in A + T (SEQ ID NO: 1), was chemically synthesized including at the ends the sites restriction of SmaI and SalI enzymes and was amplified by PCR. The specific primers, sense primer (SEQ ID NO: 2) and antisense primer (SEQ ID NO: 3), containing, respectively, the SmaI and SalI restriction sites and which are designed to generate an amplicon that, preceded by a ribosome binding site necessary to initiate the translation of the protein, comprises the gene (SEQ ID NO: 1) that codes for the mRFP protein that is characterized by four extra amino acids (MNSD) at its amino terminal end (SEQ ID NO: 4). Phusion Hot Start High Fidelity Polymerase (Finnzymes) enzyme was used for amplification to obtain a reliable copy of the original fragment. The resulting 0.8 kb reaction product was ligated using bacteriophage T4 DNA ligase (Fermentas) with the 7 kb fragment from pAK80 to obtain the 7.8 kb plasmid called pAKmRFP that was established by transformation into E Coli DH5a and selection with 250 µg / ml of erythromycin in Luria broth (LB) -agar plates. The fusion of the gene encoding mRFP in pAKmRFP is located following a multiple cloning site (SCM) (SEQ ID NO: 5). The correct nucleotide sequence of the optimized mRFP gene and its ribosome binding site present in pAKmRFP was verified by plasmid sequencing using the sense primers (SEQ ID NO: 2) and antisense (SEQ ID NO: 3).

The plasmid pAKmRFP therefore contains the optimized mRFP expression gene in bacteria with high A + T DNA preceded by an SCM containing the restriction zones for BglII, XhoI, PstI, BamHI, and SmaI (Fig. one). Plasmid pAKmRFP is thus available for use as a transcriptional fusion vector of functional promoters in E. coli, L. lactis and E. faecalis.

To construct the pAKmRFPGFP vector, the gfp gene encoding the GFP autofluorescent protein was amplified by PCR from the pGreenTIR plasmid, where the gene had been optimized in codons for expression in prokaryotes (Miller and Lindow (1997) Gene 191: 149- 153), by amplification with the specific sense primers (SEQ ID NO: 6) and antisense (SEQ ID NO: 7), designed to include in the cloning the gfp gene (SEQ ID NO: 8) preceded by its binding site to the ribosomes and to incorporate at the 5 'and 3' ends of the amplicon, respectively, the restriction sites for PstI and XhoI. The amplification product obtained with the 0.77 kb Phusion Hot Start High Fidelity Polymerase enzyme was ligated by T4 DNA ligase with the digested linearized pAKmRFP plasmid also with the 8.6 kb PstI and XhoI enzymes to generate the 8.6 kb pAKmRFPGFP plasmid . The correct nucleotide sequence of the gfp gene inserted into pAKmRFPGFP was verified by plasmid sequencing using the sense primer (SEQ ID NO: 6) and antisense (SEQ ID NO: 7). The plasmid pAKmRFPGFP therefore contains the gfp gene and the optimized mRFP expression gene in divergent orientation and on both sides of a SCM (multiple cloning site) (SEQ ID NO: 9) containing the restriction sites for PstI, BamHI, and SmaI (Fig. 1). Plasmid pAKmRFPGFP is thus available for use as a transcriptional fusion vector for divergent functional promoters in E. coli, L. lactis and E. faecalis.

EXAMPLE 2: Identification of strains of E. coli, L. lactis and E. faecalis that express the gene encoding mRFP by cloning under the PX promoter. Construction of the pAKmRFP-PX vector.

To evaluate the expression of mRFP during the growth of lactic bacteria, the wild promoter PX (SEQ ID NO: 10) of S. pneumoniae (Nieto, Espinosa and Puyet (1997) J. Biol. Chem. 272: 30860-30865 was cloned ) in the pAKmRFP vector to obtain the pAKmRFP-PX vector (Fig. 1). The promoter region of S. pneumoniae was obtained by amplification with the enzyme Phusion Hot Start High Fidelity Polimerase and the specific sense primers (SEQ ID NO: 11) and antisense (SEQ ID NO: 12), which contain the PstIy restriction sites BamHI, respectively. The 0.5 kb promoter region and pAKmRFP were digested with both PstI and BamHI enzymes and ligated using T4 DNA ligase. The resulting 8.3 kb vector plasmid was used to transform E. coli 0H5a. Colonies of erythromycin resistant transformants were obtained with an intense reddish coloration that verified the expression of mRFP under the control of PX (Fig. 2A). The correct fusion of PX with the gene encoding mRFP was confirmed by sequencing the plasmid pAKmRFP-PX with the ForPM and RevmRFP primers. To verify the usefulness of mRFP as a marker of cloning and expression in lactic bacteria, L. lactis MG1363 and E. faecalis JH2-2 were transformed with pAKmRFP-PX. The cloning of promoters and expression of mRFP in these species was observed visually by the coloration of the transforming colonies with an intense red color in L. lactis MG1363 (pAKmRFP-PX) and asalmonado in E. faecalis JH2-2 (pAKmRFP-PX) .

The determination of mRFP fluorescence expression under the control of the PX promoter during cell growth was monitored in the L. lactis MG1363 (pAKmRFP-PX), E. faecalis JH2-2 (pAKmRFP-PX) and E. coli DH5a (pAKmRFP) strains -PX) simultaneously by spectrofluorimetry (excitation measurements at 587 nm and emission at 612 nm) and spectrophotometry (optical density measurements at 480 nm, DO480) at 1-hour incubation intervals at 37 ° C for E. coli and E. faecalis and at 30 ° C for L. lactis in chemically defined medium (CDM) (Otto, ten Brink, Veldkamp and Konings (1983) FEMS Microbiol. Lett. 16: 69-74; Poolman and Konings (1988) J. Bacterial. 170 : 700-707). In the CDM, riboflavin was removed by interfering with the fluorescence reading and 0.5% glucose and MOPS buffer (3- (N-morpholino) propanesulfonic acid) 0.19 M were added to keep the pH of the medium neutral during the incubation of the cultures. Fig. 2 shows the red fluorescence (UFR) values corresponding to the fluorescence emission results of the E. coli 0H5a (pAKmRFP-PX) (2A), L. lactis MG1363 (pAKmRFP-PX) (2B) and E. faecalis JH2-2 (pAKmRFP-PX) (2C) corrected with the values obtained from non-carrier isogenic strains of pAKmRFP-PX. In all three cases, it was observed that the fluorescence expression evolved in parallel with the growth of the lines.

EXAMPLE 3: Identification of E. coli, L. lactis and E. faecalis lines that simultaneously express the genes encoding mRFP and GFP by cloning under a bidirectional promoter region. Construction of plasmid pAKmRFPGFP-PKivD-PRmaF-RlrC.

To evaluate the simultaneous expression of the fluorescence of the GFP and mRFP proteins as cloning markers of regions containing divergent promoters, the SMC of the pAKmRFPGFP vector of the region preceding the kivD gene of L. lactis IFPL730 (De La Plaza, Peláez and Requena (2009) J. Mol. Microbiol. Biotechnol. 17: 96-100). This region has two presumed consensus sequences of promoters with divergent polarity, one in the direction of the kivD gene and the other in the opposite direction in front of an operon composed of the rmaF and rlrC genes belonging to the families of MarR and LysR regulators, respectively.

The region called PKivD-PRmaF-RlrC containing the divergent promoters PKivD and PRmaF-RlrC (SEQ ID NO: 13) was amplified using L. lactis IFPL730 chromosomal DNA and the specific sense primers (SEQ ID NO: 14) and antisense ( SEQ ID NO: 15), in which the restriction site for BamHI was incorporated. The 0.28 kb PKivD-PRmaF-RlrC promoter region fragment and pAKmRFPGFP were digested with BamHI and ligated using T4 DNA ligase. The resulting 8.9 kb vector plasmid, pAKmRFPGFP-PKivD-PRmaF-RlrC, was used to transform E. coli 0H5a. Erythromycin resistant transformants were obtained with a brown coloration that evidenced the simultaneous expression of mRFP and GFP under the control of PKivD and PRmaF-RlrC, respectively, thus validating the functionality of the two promoters, and which clearly differed from the expression only of mRFP or GFP (results not shown). The correct fusion of PKivD-PRmaF-RlrC with the genes encoding mRFP and GFP was confirmed by sequencing plasmid pAKmRFPGFP-PKivD-PRmaF-RlrC with primers ForPKivD, RevPKivD, RevmRFP and RevGFP. The plasmid was used to transform L. lactis MG1363 and E. faecalis JH2-2 where the functionality of the vector was verified as a cloning and expression marker in lactic bacteria of bidirectional promoter regions. The regulating nature of the promoter region in L. lactis (see example 4) resulted in a marked expression from PKivD and, therefore, in a higher expression of mRFP that is evidenced in a redder coloration of the colonies of L. lactis MG1363 (pAKmRFPGFP-PKivD-PRmaF-RlrC) compared to those of

E. faecalis JH2-2 (pAKmRFPGFP-PKivD-PRmaF-RlrC) and E. coli DH5a (pAKmRFPGFP-PKivD-PRmaF-RlrC).

The different strength of the PKivD and PRmaF-RlrC promoters, and the consequent expression of GFP and mRFP, was quantified by fluorescence measurement during cell growth at 37 ° C for E. coli DH5a (pAKmRFPGFP-PKivD-PRmaF-RlrC) (Fig 3A) and E. faecalis JH2-2 (pAKmRFPGFP-PKivD-PRmaF-RlrC) (Fig. 3C) and at 30 ° C for L. lactis MG1363 (pAKmRFPGFP-PKivD-PRmaF-RlrC) (Fig. 3E) in CDM without riboflavin. The incorporation of 0.5% glucose and 0.19 M MOPS buffer, which allows to maintain the culture pH around 7.0, prevents the loss of fluorescence of the fluorophore from GFP that occurs at acidic pH values. Fluorescence measurements of GFP (488 nm excitation and 511 nm emission) and mRFP (587 nm excitation and 612 nm emission) were taken simultaneously, as well as optical density (DO480 measurements), at intervals of 1 h during 42 h of incubation (Fig. 3). In the same way that it had been observed in plaque-grown colonies, mRFP expression was higher in L. lactis in relation to that observed in the other two species and continued to increase during the stationary phase (Fig. 3C). The hypothesis that the higher expression of mRFP in L. lactis MG1363 (pAKmRFPGFP-PKivD-PRmaF-RlrC) was due to the strength of the PKivD promoter, as opposed to the fact that the differences in fluorescence emission could be due to the greater sensitivity to GFP pH that mRFP (although the culture pH remained stable between 6.7-6.9 during the entire incubation time), was demonstrated by cloning the PKivD-PRmaF-RlrC promoter region in the plasmid pAKmRFPGFP in the in the opposite direction, in such a way that the expressions of .mRFP and GFP will be controlled by the PRmaF-RlrC and PKivD promoters, respectively. The results showed that the expression of GFP was higher than that of mRFP (results not shown), confirming that the differences in fluorescence expression were due to the greater strength of the PKivD promoter.

The expression of fluorescence by GFP and mRFP was equivalent in E. coli DH5a (pAKmRFPGFP-PKivD-PRmaF-RlrC) (Fig. 3A), registering high values in both cases from the beginning of growth. This characteristic shows the brown color of the colonies of this strain of E: coli DH5a. On the other hand, E. faecalis JH2-2 (pAKmRFPGFP-PKivD-PRmaF-RlrC) showed higher GFP expression values than mRFP (Fig. 3E), thus observing a more greenish coloration of plaque-grown colonies (results not shown). In both cases it was observed that the fluorescence of GFP and mRFP evolved in parallel with the growth of the lineages.

EXAMPLE 4: Detection of inducible and repressive expression of PKivD-PRmaF-RlrC by simultaneous evaluation of fluorescence from GFP and mRFP in real time during the growth of L. lactis MG1363 (pAKmRFPGFP-PKivD-PRmaF-RlrC).

The strain of L. lactis MG1363 carrying the pAKmRFPGFP-PKivD-PRmaF-RlrC vector was grown in M17 medium supplemented with 0.5% glucose and 5 µg / ml of erythromycin to an OD 600 of 1.0. The cells were pelleted by centrifugation, washed twice with saline (0.85%) and finally resuspended in CDM without isoleucine (inducing medium) or CDM with casitone (1.5%). It had previously been shown that expression from PKivD and PRmaF-RlrC promoters in L. lactis IFPL730 is affected by the presence of branched amino acids and by peptide content of the culture medium, probably due to CodY-mediated regulation (De La Plaza, Peláez and Requena (2009) J. Mol. Microbiol. Biotechnol. 17: 96-100). In that work, the results showed that isoleucine is the main effector of regulation, such that the absence of isoleucine prevents CodY's repressive action on promoters.

To determine the levels of fluorescence and absorbance in real time, the same procedure was described as in Example 2, except in the growth medium (CDM) where the isoleucine content (inducing conditions) was eliminated or added casitone (enzymatic casein hydrolyzate; repressive conditions and growth equivalent in M17). The results of green (GFP) and red (mRFP) fluorescence during the growth of L. lactis MG1363 (pAKmRFPGFP-PKivD-PRmaF-RlrC) in the inductor and repressor media, as well as the growth results are shown in Fig. 4. expressed by measure of DO480. As indicated in Example 2, the proportion of mRFP expression increased during the stationary phase, an increase that proved to be exponential under inductive conditions (Fig. 4B). Under repressive conditions the increase in mRFP fluorescence was markedly lower (Fig. 4A). On the other hand, GFP expression was also superior under inductive conditions, although no significant variation was observed depending on the growth phase. As detailed in Example 3, cloning of the PKivD-PRmaF-RlrC promoter region in the opposite direction, fusion of PKivD with GFP and expression under inductive conditions, resulted in equivalent logarithmic increases in GFP expression during stationary phase of growth and resulted in obtaining colonies of L. lactis of an intense green color (results not shown). The results obtained demonstrate the usefulness of the pAKmRFPGFP vector to quantitatively and in real time monitor the simultaneous expression of mRFP and GFP fluorescence under inductive or repressive conditions and its usefulness to perform gene expression studies in L. lactis.

EXAMPLE 5. Detection of mRFP and GFP protein expression by fluorescence microscopy

To determine the expression of the mRFP and GFP fluorescent proteins in individual cells of the bacterial populations, they were detected by fluorescence microscopy.

To detect the expression of mRFP encoded by plasmid pAKmRFP-PX, the following cultures were analyzed.

L. lactis MG1363 (pAKmRFP-PX) and E. faecalis JH2-2 (pAKmRFP-PX) grown at 30 ° C in M17 medium supplemented with 0.5% glucose to an OD620 of 0.6. E. coli 0H5a (pAKmRFP-PX) was grown at 37 ° C with agitation of 300 revolutions per minute in LB medium to an OD620 of 0.6. Before performing the analysis, 1 ml samples of the cultures were centrifuged and the bacteria settled after being washed with PBS buffer (10 mM Na2HPO4, 1mM KH2PO4, 140 mM NaCl, 3 mM KCl), pH 8, were resuspended in 0 , 5 ml of the same buffer. These samples were analyzed without prior fixation using a multi-invivo optics microscope, Leica AF6000 LX model DMI6000B (Leica Microsystems GmbH, Wetzlar, Germany) equipped with a monochrome camera Hamamatsu CCD C9100-02 (Leica Microsystems GmbH, Wetzlar, Germany) with a objective × 100 and a numerical aperture of 1.6. The lighting was done with a HG 100 W fluorescent light mercury lamp, using an emission filter to let the red light through. Image processing was performed using the FRET program (Leica Microsystems GmbH, Wetzlar, Germany).

The fluorescence of the mRFP protein allowed the detection of L. lactis, E. faecalis and E. coli cells carrying pAKmRFP-PX by fluorescence microscopy. The results obtained indicated that mRFP protein expression from pAKmRFP-PX could be used to conduct competition studies of L. lactis, E. faecalis and E. coli in different habitats. Therefore, the usefulness of the plasmid was evaluated in studies of bacterial adhesion to human epithelial cells (Caco-2). For this, exponential cultures of L. lactis MG1363 (pAKmRFP-Px), E. faecalis JH2-2 (pAKmRFP-Px) and E. coli DH5a (pGreenTIRGFP), grown as previously indicated, were independently incubated or mixed with cells Intestinal Caco-2 for 1 h at 37 ° C in a 5% CO2 atmosphere. After the incubation time, the non-adherent bacteria were removed by three washes with PBS buffer, pH 8. The samples were observed after washing using the inverted multi-invivo microscope described above. The results obtained with appear in Fig. 5. The fluorescence of the GFP and mRFP proteins allowed to detect by fluorescence microscopy the cells of E. coli carrying GFP and L. lactis and E. faecalis carrying mRFP. The results obtained demonstrate the usefulness of plasmid pAKmRFP-Px to perform interaction studies of the lactic bacteria of L. lactis and E. faecalis with eukaryotic cells.

To detect the expression of the mRFP and GFP proteins encoded by pAKmRFPGFP-PKivD-PRmaF-RlrC, carrier bacteria of said plasmid grown in CDM without riboflavin were used and supplemented with 0.5% glucose to an OD620 of 0.6. Cultures of E. coli 0H5a (pAKmRFPGFP-PKivD-PRmaF-RlrC) were grown at 37 ° C with agitation of 300 rpm, while cultures of E. faecalis JH2-2 (pAKmRFPGFP-PKivD-PRmaF-RlrC) and L Lactis MG1363 (pAKmRFPGFP-PKivD-PRmaF-RlrC) were grown at 30 ° C in static. One milliliter of each of the cultures was sedimented by centrifugation and the bacteria were resuspended in 0.5 ml of PBS. These preparations were analyzed by phase contrast and fluorescence microscopy using an inverted multi-invivo microscope. The superposition of the images obtained with phase contrast and fluorescent illumination to detect the two fluorophores revealed that in both E. coli and L. lactis and E. faecalis cultures, most bacteria showed fluorescence due to mRFP and GFP protein expression (results not shown). In the case of L. lactis, very low fluorescence emitted by mRFP and GFP was observed, an expected result since the bacteria were in non-optimal conditions for induction of expression from the PKivD and PRmaF-RlrC promoters (exponential growth phase and composition of the culture medium). As a result of this result, the expression of mRFP and GFP in cultures of L. lactis MG1363 (pAKmRFPGFPPKivD-PRmaF-RlrC) grown under repression conditions and induction of fluorescent protein expression was compared.

Thus, L. lactis MG1363 (pAKmRFPGFP-PKivD-PRmaF-RlrC) was grown at 30 ° C in CDM medium supplemented with 1.5% casitone (repression conditions) to an OD620 of 0.6. The culture was divided into two subcultures: the first was processed for analysis by microscopy as previously described; the second was centrifuged and the cells were resuspended in CDM medium without isoleucine and after 12 hours of incubation at 30 ° C (induction conditions), samples were taken and processed for analysis by microscopy. In the superposition of the images obtained by phase contrast and fluorescence microscopy to detect mRFP and mGFP it was seen that bacteria grown in repression conditions showed only low levels of mRFP expression, while after 12 h of induction a prevalence of mRFP expression was observed detecting a brown coloration of the bacteria (results not shown).

Claims (43)

one.

Nucleotide sequence consisting of the sequence of the mrfp gene encoding a red fluorescent protein, where said sequence is SEQ ID NO: 1.

2.

Recombinant nucleotide sequence comprising the nucleotide sequence according to claim 1.

3.

Nucleotide sequence according to claim 2 further comprising at least one sequence recognized by at least one restriction enzyme.

Four.

Nucleotide sequence according to any of claims 2 to 3 which further comprises the gfp gene, which encodes the green fluorescent protein (GFP), wherein said sequence is SEQ ID NO: 8.

5.

Nucleotide sequence according to any of claims 2 to 4 which further comprises at least one sequence recognized by at least one restriction enzyme between the mrfp and gfp genes.

6.

Vector comprising the nucleotide sequence according to any one of claims 1 to 5.

7.

Vector according to claim 6 further comprising a functional replicon in Lactococcus lactis.

8.

Vector according to claim 7 further comprising a functional replicon in Escherichia coli.

9.

Vector according to any one of claims 6 to 8, wherein the nucleotide sequence is operatively linked to at least one gene expression regulatory sequence.

10.

Vector according to claim 9 wherein the gene expression regulatory sequence is selected from: a) a promoter, or b) a transcription initiation signal.

eleven.

Vector according to claim 10 wherein said promoter controls at least the transcription of one of the fluorescent proteins.

12.

Vector according to claim 11 wherein the promoter is a unidirectional promoter.

13.

Vector according to claim 12 wherein the unidirectional promoter is the Streptococcus pneumoniae PX promoter, whose sequence is SEQ ID NO: 10.

14.

Vector according to claim 13 wherein the vector is the vector derived from pAK80 that lacks the gene encoding �-galactosidase.

fifteen.

Vector according to claim 11 wherein the promoter is bidirectional.

16.

Vector according to claim 15 wherein the bidirectional promoter is the promoter region comprising the PKivD and PRmaF-RlrC divergent promoters of Lactococcus lactis, whose sequence is SEQ ID NO: 13.

17.

Vector according to claim 16 wherein the vector is the vector derived from pAK80 lacking the gene encoding �-galactosidase.

18.

Cell comprising the nucleotide sequence according to any one of claims 1 to 5, or the vector according to any of claims 6 to 17.

19.

Cell according to claim 18 wherein said cell is a Gram-positive bacterium.

twenty.

Cell according to claim 19 wherein said cell is selected from Lactobacillus lactis or Enterococcus faecalis.

twenty-one.

Cell according to claim 18 wherein the cell is Escherichia coli

22

Cell according to any of claims 18 to 21, wherein said cell emits red fluorescence by expression of the mRFP protein, green fluorescence by expression of the GFP protein or brown fluorescence by simultaneous expression of both proteins.

2. 3.

Cellular population comprising a cell according to any of claims 18 to 22.

24.

Use of the nucleic acid according to claims 1 to 5 or of the vector according to claims 6 to 17 for the detection and characterization of a promoter.

25.

Use according to claim 24 wherein the promoter is unidirectional.

26.

Use according to claim 24 wherein the promoter is bidirectional.

27.

Use of the nucleic acid according to claims 1 to 5 or of the vector according to claims 6 to 17 for the transcription of at least one polynucleotide.

28.

Use of the cells according to any of claims 18 to 22 or of the cell population according to claim 23 for the determination of the colonizing capacity of said cells in a mixed bacterial culture, in co-cultures with isolated eukaryotic cells, in tissue culture Biological isolated or in an animal model.

29.

Method of detecting the cell according to any of claims 18 to 22 which comprises detecting the fluorescence of said cells.

30

Method according to claim 29, wherein the fluorescence detection is carried out by means of spectrofluorimetry or by fluorescence microscopy.

31.

Method according to any of claims 29 or 30, wherein the fluorescence is detected in colonies of said cells.

32

Method of detection and characterization of promoters comprising: a) transforming a host cell with the vector according to any of claims 6 to 17, b) culturing the cell of step (a) under suitable conditions, c) determining the fluorescence levels of the fluorescent protein, d) compare the fluorescence obtained in step (c) with the fluorescence emitted by a control cell.

33.

Method for detecting the expression of polynucleotides comprising: a) inserting into the vector according to any of claims 6 to 17 a polynucleotide that is controlled by the same promoter that regulates the transcription of the gene encoding the protein

fluorescent, b) transforming a host cell with the vector resulting from step (a), c) culturing the cell of step (b) under the appropriate conditions and d) determining the fluorescence levels of the fluorescent protein simultaneously with the Determination of cell growth. 34. Method for determining the colonizing capacity of the cells according to any of claims 18 to 22 or of the cell population according to claim 23 comprising: a) cultivate the cells in the right conditions, b) contact the cells with bacteria, tissues or an animal model, c) determine the fluorescence levels of the fluorescent protein by spectrofluorimetry or by fluorescence microscopy and d) compare the fluorescence determined in step (c) with the fluorescence emitted by a cell, tissue or control animal that does not have the same characteristics as the cell, tissue, or animal of step (b)

35

Kit comprising: a) at least one recombinant nucleotide sequence according to any one of claims 1 to 5, b) at least one vector according to any one of claims 6 to 17, or c) at least one cell according to any of features 18 to 21 .

36.

Use of the kit according to claim 35 for the detection and characterization of promoters, for the transcription of a polynucleotide of interest or for the determination of the colonizing capacity in mixed bacterial cultures, in co-cultures with isolated eukaryotic cells, in biological tissue culture isolated or in an animal model.

SPANISH OFFICE OF THE PATENTS AND BRAND Application no .: 201130356 SPAIN Date of submission of the application: 03.15.2011 Priority Date: REPORT ON THE STATE OF THE TECHNIQUE 51 Int. Cl.: C07K14 / 435 (2006.01) C12N15 / 74 (2006.01) RELEVANT DOCUMENTS

Category

Documents cited Claims Affected

Y

SHANER, N.C. et al., ‘Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein ’, NATURE BIOTECHNOLOGY, 2004, Vol. 22, No. 12, pages 1567-1572, ISSN: 1087-0156, the whole document, in particular, Table 1, Figure 2. 1-36

Y

FUGLSANG, A., ctic Lactic acid bacteria as prime candidates for codon optimization ’, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 2003, Vol. 312, No. 2, pages 285-291, ISSN: 0006-291X, Results, Table 1. 1-36

TO

MÜLLER-TAUBENBERGER, A. et al., 'Monomeric red fluorescent protein variants used for imaging studies in different species', EUROPEAN JOURNAL OF CELL BIOLOGY, 2006, Vol. 85, Nos. 9-10, pages 1119-1129, doi: 10.1016 / j.ejcb.2006.05.006, the whole document. 1-36

TO

FISCHER, M. et al., 'Visualizing cytoskeleton dynamics in mammalian cells using a humanized variant of monomeric red fluorescent protein', FEBS LETTERS, 2006, Vol. 580, No.10, pages 2495-2502, ISSN: 0014-5793, whole document. 1-36

TO

MÜLLER-TAUBENBERGER, A. et al., 'Recent advances using green and red fluorescent protein variants', APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 2007, Vol. 77, No. 1, pages 1-12, ISSN: 0175-7598, all document. 1-36

TO

SHANER, N.C. et al., van Advances in fluorescent protein technology ’, JOURNAL OF CELL SCIENCE, 2007, Vol. 120, No. 24, pages 4247-4260, ISSN: 0021-9533, the entire document. 1-36

TO

WO 2009059305 A2 (THE UNIVERSITY OF CHICAGO) 07.05.2009, the whole document. 1-36

Category of the documents cited X: of particular relevance Y: of particular relevance combined with other / s of the same category A: reflects the state of the art O: refers to unwritten disclosure P: published between the priority date and the date of priority submission of the application E: previous document, but published after the date of submission of the application

This report has been prepared • for all claims • for claims no:

Date of completion of report 31.10.2011

Examiner J. L. Vizán Arroyo Page 1/5

REPORT OF THE STATE OF THE TECHNIQUE Application number: 201130356 Minimum documentation sought (classification system followed by classification symbols) C07K, C12N Electronic databases consulted during the search (name of the database and, if possible, terms of search used) INVENES, EPODOC, BIOSIS, MEDLINE, EMBASE, EBI State of the Art Report Page 2/5  WRITTEN OPINION Application number: 201130356 Date of Completion of Written Opinion: 10.31.2011 Statement

Novelty (Art. 6.1 LP 11/1986)

Claims Claims 1-36 IF NOT

Inventive activity (Art. 8.1 LP11 / 1986)

Claims Claims 1-36 IF NOT

The application is considered to comply with the industrial application requirement. This requirement was evaluated during the formal and technical examination phase of the application (Article 31.2 Law 11/1986).  Opinion Base.- This opinion has been made on the basis of the patent application as published. State of the Art Report Page 3/5  WRITTEN OPINION Application number: 201130356  1. Documents considered.- The documents belonging to the state of the art taken into consideration for the realization of this opinion are listed below.

Document

Publication or Identification Number publication date

D01

SHANER, N.C. et al., Nat. Biotechnol., (2004), 22 (12): 1567-72. 2004

D02

FUGLSANG, A., Biochem. Biophys Res. Commun., (2003), 312 (2): 285-91. 2003

D03

MÜLLER-TAUBENBERGER, A. et al., Eur. J. Cell. Biol., (2006), 85 (9-10): 1119-29. 2006

D04

FISCHER, M. et al., FEBS Lett., (2006), 580 (10): 2495-502. 2006

D05

MÜLLER-TAUBENBERGER, A. et al., Appl. Microbiol Biotechnol., (2007), 77 (1): 1-12 2007

D06

SHANER, N.C. et al., J. Cell Sci., (2007), 120 (Pt 24): 4247-60. 2007

D07

WO 2009059305 A2 05.07.2009

Different variants of the red fluorescent monomeric protein (mRFP) are described in D1. In D2 the most frequent codons in lactic bacteria (Gram-positias) are analyzed. Different technological applications of the mRFP protein are disclosed in D3-D7.  2. Statement motivated according to articles 29.6 and 29.7 of the Regulations for the execution of Law 11/1986, of March 20, on Patents on novelty and inventive activity; quotes and explanations in support of this statement 1. NEW (Art. 4.1. And Art. 6.1. Of the Patent Law). 1.1. Independent claim 1. 1.1.1. The object of claim 1 is the nucleotide sequence SEQ ID NO: 1 consisting of a variant of the sequence of the mrfp gene encoding the red fluorescent protein (mRFP) (SEQ ID NO: 4). According to the description, SEQ ID NO: 1 is an optimized sequence of the gene coding for the autofluorescent protein mCherry (mRFP) for its heterologous expression in Gram-positive bacteria high in A + T (cf page 5, lines 17-24) . In the closest state of the art, constituted by documents D1-D7, no sequence has been disclosed with the same technical characteristics as the sequence claimed in the patent application. Accordingly, the object of protection of independent claim 1 and that of dependent claims 2-36 is considered to be new on the basis of documents D1-D7.

1.2.

 The present invention satisfies the criteria established in Art. 4.1. of the Patent Law because the object of the invention, defined in claims 1-36, is new in accordance with Art. 6.1. of the Patent Law.

2.

 INVENTIVE ACTIVITY (Art. 4.1. And Art. 8.1. Of the Patent Law).

2.1. Independent claim 1. 2.1.1. Documents D1 and D2 constitute the closest state of the art. In D1, different monomeric fluorescent proteins derived from the DsRed red fluorescent protein of Discosoma sp. Specifically, one of the variants described is the red fluorescent protein mCherry and its coding sequence (cf. D1: Table 1, Figure 2). D2 describes the predominant use of codons with high A + T content in lactic (Gram-positive) bacteria and their application in the design of heterologous sequence expression systems in lactic bacteria as producing strains (cf. D2: Results, Table 1). 2.1.2. The technical problem to be solved by the object of claim 1 can therefore be considered as the provision of a new coding sequence of the mRFP red fluorescent monomeric protein. 2.1.3. The proposed solution is the nucleotide sequence SEQ ID NO: 1 encoding the mCherri variant of the red fluorescent protein mRFP. According to the description, the sequence of the invention SEQ ID NO: 1 has been optimized according to the predominant use of codons of lactic (Gram-positive) bacteria (see page 5, lines 17-24). This technical characteristic is the basic difference of SEQ ID NO: 1 with respect to the sequence disclosed in D1 (cf. D1: Figure 2). The list of codons most commonly used in lactic bacteria and their application in the design of heterologous gene expression systems in said microorganisms has been disclosed in the state of the art (cf. D2: Results, Table 2). On the other hand, optimized mRFP coding sequences have been disclosed according to the predominant use of codons of different species for expression in the corresponding species (c.D3). Therefore, it is considered that, on the basis of documents D1 and D2, the solution proposed by the patent application to the technical problem raised would be evident to the person skilled in the art. Therefore, the object of independent claim 1 can be considered non-inventive. As set forth above, the object of dependent claims 2-36 is also considered to be non-inventive. State of the Art Report Page 4/5  WRITTEN OPINION Application number: 201130356 2.2. The present invention does not meet the criteria established in Art. 4.1. of the Patent Law because the object of the invention, defined in claims 1-36 does not imply inventive activity in accordance with Art. 8.1. of the Patent Law. State of the Art Report Page 5/5