ES2939335B2 - ESTROGEN BIOSENSOR - Google Patents

Description

DESCRIPTION

ESTROGEN BIOSENSOR

The present invention relates to a biosensor to detect estrogens in the environment, mainly in aquatic environments, based on a bacterial regulator of the TetR family called EdcR encoded by the gene EGO55_13520 (edcR) of the bacterium Caenibius Tardaugens (formerly Novosphingobium Tardaugens). Therefore, the present invention is included within the technical field of environmental biotechnology, specifically, in the detection of environmental contamination.

BACKGROUND OF THE INVENTION

Estrogens are hormones produced by the ovaries and in lesser quantities by the adrenal glands, adipose tissue and placenta, and are responsible for female sexual characteristics and many metabolic processes. Three estrogens are produced in humans, estradiol (E2, 17β-estradiol, or oestradiol), estrone (E1), and estriol (E3). Estradiol is the most powerful estrogen, and its functions include regulating the development of secondary female characteristics, in addition to intervening in the regulation of different metabolic processes.

When estrogens are free in the environment, they generate great health concern, since they are part of a group of toxic substances called endocrine disruptors, because they can affect the development of living beings. Examples of these estrogens that act as endocrine disruptors (called EDCs) are 17p-estradiol (E2) and its main metabolites: estriol (E3) and estrone (E1).

Currently, different pharmaceutical formulations contain these compounds and their high consumption by humans or animals means that significant quantities of these compounds are finally released into the environment through excretion - both through urine and feces - and through the discharge of the drug. to municipal waste or directly through liquid discharges from home or farm sewers. Estrogens released into the environment, and especially those released into the aquatic environment, can reach continental waters (lakes, rivers, aquifers) or marine waters directly, and indirectly through inadequate treatments at municipal wastewater treatment plants.

The presence of low concentrations of these compounds in water can affect not only people but also animals, and especially fish. Among the main effects of exposing different species of fish to estrogenic compounds include the alteration of sexual development, the generation of intersex species (those that, being males, suffer feminization and the opposite, masculinization of female fish), as well as changes in behavior. of mating, reduced reproductive success and alterations of the endocrine system, which leads, in exposed animals, to alterations in growth, development and reproduction. These changes can manifest later in the fish's life cycle or even be maintained throughout generations. Although the adverse effects caused by the presence of estrogenic compounds in contaminated water consumed by humans is a question open to debate; However, some studies describe among its effects the deterioration of reproductive health in men, due to the generation of a low amount of sperm, and the appearance of breast cancer in women, as a consequence of the increasing exposure to these compounds.

For all these reasons, as environmental contamination by estrogens is of utmost importance for health, there is increasing interest in their early detection and elimination.

Currently, the detection of estrogenic EDCs in environmental or clinical samples is carried out mainly by chromatographic analysis, having described analytical methods based on the use of an automated solid phase extraction line coupled to high pressure liquid chromatography with detectors of different types (e.g., DAD, MS/MS, UV, ESI, or MS).

Several biosensors have also been developed to detect estrogens, including: i) immunosensors, based on the binding of antibodies to a specific antigen (Monerris et al., 2015, Sensors Actuators, B Chem.208, 525-531; ii ) aptasensors, based on the use of short chains of DNA or RNA (15 to 100 nucleotides) that can bind to a specific target molecule (aptamers) (Fan et al., 2015, Biosens. Bioelectron.68, 303-309; Zhu et al., 2015, Biosens. Bioelectron.70, 398-403; iii) biosensors based on isolated animal estrogen receptors a (ERa) or p (ERP), either as electrogenic biosensors or as cellular biosensors integrated into the receptor in bacteria. , in fungi, in yeast, in eukaryotic cell lines, in plants or in transgenic animals (Fine et al., 2006, Biosens Bioelectron21, 2263-2269).

A steroid biosensor has also been developed based on the system for regulating the expression of the hsdA gene, which encodes the enzyme 3a-hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD-CR) in Comamonas testosteroni. To do this, the hsdA gene was replaced in this strain by gene that encodes the green fluorescent protein GFP so that the resulting strain responded poorly to various steroids such as testosterone, estradiol and cholesterol (Xiong et al., 2009, Chem Biol Interact 178, 215-220).

However, the biosensors mentioned above present the following drawbacks: Biosensors based on aptamers or antibodies require additional laboratory equipment and equipment to perform the measurements, with complex portability, making their use in the field very difficult. Furthermore, the measurement protocols are complex and require highly specialized personnel in analytical laboratory techniques. All of this makes these biosensors expensive and complicated to use.

- Biosensors based on the Era or Erp estrogen receptors respond non-specifically to compounds with agonist/antagonist activity of these receptors, which includes a wide range of different molecules, from molecules with a steroidal structure, such as EE2, to others that are not. steroids, such as 4-nonylphenol, for example.

- The biosensor developed inC. Testosteronin distinguishes between compounds as different as testosterone (androgen), estradiol (estrogen) and cholesterol (without androgenic or estrogenic activity) and therefore its usefulness to specifically detect estrogens is null.

Therefore, in view of the above, there is a need in the state of the art to provide alternative biosensors for the detection of estrogens to those described above that do not present the aforementioned disadvantages.

DESCRIPTION OF THE INVENTION

The authors of the present invention have developed a system for the detection of estrogens based on the EdcR regulatory system that regulates the expression of the edcde Caenibius Tardaugens NBRC 16725 gene cluster (previously named Novosphingobium Tardaugens ARI-1). The inventors observed that the genesedcdeC cluster. tardaugens, the cluster responsible for the degradation of estrogens, is regulated by the protein EdcR, which acts as a repressor of the expression of the rest of the genes that are part of the clusteredc. Thus, in the absence of estrogens in the culture medium, the expression of The cluster genes are repressed by the EdcR protein, while, in the presence of estrogens, they bind to the EdcR repressor protein and block its binding to DNA, facilitating the expression of the cluster genes responsible for their degradation.

The advantages of the biosensor developed by the inventors compared to state-of-the-art biosensors are, among others, the following:

- It presents great versatility since it allows it to be transferred to different host cells depending on the needs;

- The results can be viewed with the naked eye without requiring additional equipment and, in addition, it can be used by people without high laboratory specialization.

Furthermore, the inventors observed that this regulation system used in the biosensor responds to the presence/absence of both estradiol (E2) and estrone (E1), which allows the development of a new cellular biosensor type tool for the detection of the presence of these estrogens in the medium.

Thus, in one aspect, the present invention relates to a cell, hereinafter "cell of the invention" comprising

(a) a first gene construct comprising (i) the nucleotide sequence of a promoter operatively linked to (ii) the nucleotide sequence encoding the EdcR regulatory protein of the edcdeC genes cluster. tardaugens; and

(b) a second gene construct comprising (iii) the nucleotide sequence of the promoter of Operon A, or Operon B, of the genesedcdeC cluster. tardaugensoperatively linked to (iv) a nucleotide sequence encoding a reporter protein.

Due to the ability of the cell of the invention to detect the presence of estrogens in the medium, said cell acts as a sensor of the presence of estrogens. Therefore, in the present description, the cell of the invention is called a biosensor, hereinafter also called "biosensor of the invention." Both terms “cell of the invention” and “biosensor of the invention” are equivalent and can be used interchangeably throughout the present description. In the present invention, "biosensor" is understood as an analytical device that transforms a biological recognition event into another type of signal, for example, optical, chemical, electrical or physical, which can be measured and quantified in real time.

Any cell comprising the gene constructs (a) and (b) defined above can be used as a biosensor of the invention. Cells (or host cells) suitable to be the biosensor of the invention include, but are not limited to, insect, fungal and bacterial cells. Bacterial cells include, but are not limited to, cells of Gram-positive bacteria such as species of the genus Bacillus, Streptomyces, Mycolicibacterium, Corynebacterium, Arthrobacter, Rhodococcus, Lactococcus and Lactobacillus, and cells of Gram-negative bacteria such as species of the genus Escherichia (e.g. E. . coliamong others), Comamonas (e.g. C. testosteroniamong others) and Pseudomonas (e.g. P. putidaamong others). In a preferred embodiment, the biosensor of the invention is an Escherichia coli cell. Fungal cells preferably include yeast cells such as Saccharomyces (e.g. S. cerevisiae among others), Pichia pastoris and Hansenula polymorpha, and filamentous fungal cells such as Aspergillus and Penicillium. Insect cells include, but are not limited to, Drosophila cells and Sf9 cells from Spodoptera frugiperda. However, in a preferred embodiment, the biosensor of the invention is not a C cell. tardaugens.

The biosensor of the invention comprises a first and a second genetic construction. In the present invention, “genetic construction” or “gene construction” is understood as the grouping in a single sequence of two or more nucleotide sequences not naturally associated. The sequences that are part of the gene construction can be found in the same or different reading frame.

The first genetic construct (a) comprises (i) the nucleotide sequence of a promoter operatively linked to (ii) the nucleotide sequence encoding the regulatory protein EdcR of the genesedcdeC cluster. tardaugens.

The first gene construct (a) comprises two nucleotide sequences that are operatively linked. The term "operably linked" or "operably linked" is defined herein as a configuration in which a regulatory or control sequence, in this particular case the promoter, is appropriately placed in a position relative to a second nucleotide sequence. such that the control sequence directs the expression of the second sequence.

In the present invention, the term "promoter" or "promoter sequence" refers to a nucleotide sequence located in the 5' direction of the transcriptional start of a gene, and involved in the recognition and binding of RNA polymerase and other proteins responsible for the transcription of a gene in the 3' direction. In general, the promoter will be suitable for the host cell in which the target gene is expressed which, in the first gene construct is the nucleotide sequence encoding the EdcR regulatory protein of the edcdeC genes cluster. tardaugens(item (ii)).

The promoter can control the expression of the nucleotide sequence in different ways, and can be a constitutive or inducible promoter. A "constitutive promoter" refers to a promoter that is transcriptionally active during most, but not necessarily all, phases of growth and development of a cell. Alternatively, an “inducible promoter” is one that has initiated or increased the initiation of transcription in response to a chemical or physical stimulus. Examples of inducible promoters include, but are not limited to, the operonlacdeE promoter. coli (Pac), so said expression can be achieved by adding lactose and/or IPTG (isopropyl-beta-D-thiogalactopyranoside) as an inducer to the culture medium. In a preferred embodiment, the constitutive promoter of the first gene construct is the PexA promoter. PiexA is a promoter derived from the plasmid pSEVA237PlexA (Fernández et al., 2014, PLoS One 9, e110771), where PexA is the native promoter of the LexA repressor of E. colique controls the response to DNA damage. In another even more preferred embodiment, the PexA promoter comprises, or consists of, a nucleotide sequence that has a sequence identity of at least 75, 80, 85, 90, 95, 96, 97, 98 or 99% with the sequence SEQ ID NO: 1. In another even more preferred embodiment, the PexA promoter comprises, or consists of, the nucleotide sequence SEQ ID NO: 1.

SEQ ID NO: 1.

In the first gene construct, a promoter (either constitutive or inducible) controls the expression of the nucleotide sequence encoding the regulatory protein EdcR of the genesedcdeC cluster. tardaugens.

In a preferred embodiment, the EdcR regulatory protein of the edcdeC gene cluster. tardaugens comprises, or consists of, an amino acid sequence that has a sequence identity of at least 75, 80, 85, 90, 95, 96, 97, 98, or 99% with the sequence SEQ ID NO: 2.

In the present invention, "identity", "percentage identity" or "sequence identity" is understood as the degree of similarity between two nucleotide (or amino acid) sequences obtained by optimal alignment of the two sequences. Depending on the number of common residues between the aligned sequences, a degree of identity expressed as a percentage will be obtained. By “best alignment” or “optimal alignment” is meant the alignment for which the percent identity determined as described below is the highest. The degree of identity between two nucleotide (or amino acid) sequences can be determined by conventional methods, for example, by standard sequence alignment algorithms known in the state of the art, such as BLAST (Altschul S.F. et al. Basic local alignment search tool. J Mol Biol. 1990 Oct 5; 215(3):403-10). BLAST programs, for example, BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN, are in the public domain on the National Center for Biotechnology Information (NCBI) website.

In another even more preferred embodiment, the EdcR regulatory protein of the edcdeC gene cluster. tardaugens comprises, or consists of, the amino acid sequence SEQ ID NO: 2.

SEQ ID NO: 2

In another preferred embodiment, the nucleotide sequence encoding the EdcR regulatory protein of the edcdeC gene cluster. tardaugens comprises, or consists of, the sequence SEQ ID NO: 3.

SEQ ID NO: 3

On the other hand, the second gene construct of the biosensor of the invention comprises (iii) the nucleotide sequence of the promoter of operon A, or operon B, of the genesedcdeC cluster. Tardaugens, operably linked to (iv) a nucleotide sequence encoding a reporter protein.

The terms “promoter” and “operationally united” have been defined above.

In a preferred embodiment, the nucleotide sequence of the operon A promoter of the genesedcdeC cluster. tardaugens comprises, or consists of, a nucleotide sequence with a sequence identity of at least 75, 80, 85, 90, 95, 96, 97, 98 or 99% with the sequence SEQ ID NO: 4. In another embodiment Even more preferred, the nucleotide sequence of the operon A promoter of the genesedcdeC cluster. tardaugens comprises, or consists of, the sequence SEQ ID NO: 4.

SEQ ID NO: 4

In another preferred embodiment, the nucleotide sequence of the operon B promoter of the genesedcdeC cluster. Tardaugens comprises, or consists of, a nucleotide sequence with a sequence identity of at least 75, 80, 85, 90, 95, 96, 97, 98, or 99% with the sequence SEQ ID NO: 5. In another Even more preferred embodiment, the nucleotide sequence of the operon B promoter of the genesedcdeC cluster. tardaugens comprises, or consists of, the sequence SEQ ID NO: 5.

SEQ ID NO: 5

The second construct (b) comprises the nucleotide sequence that encodes a reporter protein. In the present invention, “reporter protein” is understood as that protein encoded by a gene (reporter gene) whose expression is indicative that the promoter that regulates its expression is active. In general, the expression of these reporter proteins is detectable through a change in color, the appearance of fluorescence, the appearance of light, or the production of an enzyme or a metabolite in the culture of the organism that can be detected by some physical or chemical. There are many reporter proteins in the state of the art and any of them can be used in the present invention. Examples of suitable fluorescent reporter proteins include, but are not limited to, EGFP and its variants, such as YFP, Cyan, and dEGFP; DS-Red, mKO, the far-red fluorescent protein "Katushka", or variants of any of the above. In other embodiments, the reporter protein is an enzyme that converts a substrate that, in the process, produces a detectable signal, for example, a fluorescent or luminescent signal in the presence of a fluorogenic or luminogenic substrate, respectively. For example, in some embodiments, the selectable marker enzyme comprises the amino acid sequence of a luciferase, for example, a firefly luciferase, spring beetle luciferase, or Renilla luciferase. Luciferase activity can be detected by providing an appropriate luminogenic substrate, for example, firefly luciferin for firefly luciferase or coelenterazine for Renilla luciferase. Luciferase activity in the presence of an appropriate substrate can be quantified by luminometry to assess the total luciferase activity of whole cell populations in the wells of the culture plate, or, alternatively, the luciferase activity of the cells. or individual colonies can be detected by using a microscope in combination with a photonic counting camera. In other embodiments, the reporter protein comprises the amino acid sequence of an enzyme such as modified beta-lactamase, the expression of which can be detected and quantified in living cells using a ratiometric fluorescence assay for the breakdown of fluorogenic lactam substrates.

In a preferred embodiment, the reporter protein is selected from the group consisting of green fluorescent protein (GFP), or the enzymes beta-glucuronidase, betagalactosidase and luciferase. In another even more preferred embodiment, the green fluorescent protein (GFP) is the variant of the monomeric superfolder green fluorescent protein (msf gfp)).

Both the gene construct (a) and (b) may comprise other regulatory elements that help or enhance the expression (transcription and translation) of the nucleotide sequences. Examples of these regulatory elements include, but are not limited to, activator and enhancer sequences for the binding of transcription factors to assist RNA polymerase binding and promote expression, operator or silencing sequences to which repressor proteins bind to block RNA polymerase binding and preventing expression, ribosome binding sequences (RBS), transcription terminator sequences, etc.

In a particular embodiment, the second gene construction of the biosensor of the invention comprises between elements (iii) and (iv), the nucleotide sequence of a synthetic bicistronic RBS operatively linked to said elements (iii) and (iv). In another even more particular embodiment, the nucleotide sequence of said synthetic bicistronic RBS comprises, or consists of, a nucleotide sequence with a sequence identity of at least 75, 80, 85, 90, 95, 96, 97, 98, or 99% with the sequence SEQ ID NO: 6. In another even more particular embodiment, the nucleotide sequence of said synthetic bicistronic RBS is the sequence SEQ ID NO: 6.

SEQ ID NO: 6

As understood by those skilled in the art, the expression regulatory sequences will be those appropriate to the cell chosen to be the biosensor of the invention, that is, they will be those that are recognized by the cellular machinery of the host cell.

Constructs (a) and (b) of the biosensor of the invention can be part of a vector or can be integrated into the genome of the cell. If they are part of a vector, constructs (a) and (b) can be part of the same vector or, thanks to the capacity of the regulatory protein EdcR of the genesedcdeC cluster. tardaugens (element (ii)) to act enters, they can be in different vectors. Thus, in a preferred embodiment of the biosensor of the invention, the first and second gene constructs are located in different vectors.

In the present invention, "vector" is any vehicle, often a virus or plasmid, that is used to transport a DNA sequence to a desired host cell, as part of a molecular cloning procedure. The vector may be a biologically functional vector or plasmid, such as a cloning vector or an expression vector. Any vector can be used as long as it is replicable and viable in the cells into which it is introduced.

In the present invention, "expression vector" is understood as a plasmid that is used to introduce and express a specific gene in a cell of interest. Expression vectors allow the production of large amounts of stable messenger RNA (mRNA). The proteins encoded by the nucleotide sequence of the gene constructs are produced by the transcription and translation machinery of the cell once the vector is inside it. The plasmid is designed to contain a highly active promoter, which causes the production of large amounts of mRNA. A plasmid is an extrachromosomal DNA molecule, separated from the cell's chromosomal DNA and replicating independently of it. Plasmids are generally circular and have two strands of nucleic acid. Alternatively, the plasmid can be designed so that its contents are inserted into the genomic DNA by genetic recombination. A large number of suitable vectors are known to those skilled in the art, and these are commercially available. In general, the nucleotide sequence to be expressed is inserted into the vector at an appropriate restriction endonuclease site or sites by standard procedures. These and related subcloning procedures are considered to be within the knowledge of those skilled in the art. Examples of vectors that can be used in the context of the present invention include, but are not limited to, viral and non-viral vectors. Among non-viral vectors, we can mention bacterial vectors, of which there are numerous examples, including any plasmid (expression or not) described for bacteria (E. coli, Bacillus sp., etc.) or its modifications, etc. Additionally, said vectors can be derived from commercial expression vectors, such as those marketed by Promega (e.g., PinPointXa plasmids, etc.), Invitrogen (e.g., Gateway system plasmids, etc.), Amersham (e.g., pGEX plasmids, etc.). ), Ingenius (e.g., pINK vector, etc.), Sigma (e.g., BICEP vectors, etc.), etc. Alternatively, the vector of the invention can be obtained by conventional methods known to those skilled in the art described.

In the context of the present invention, the vector may be a plasmid with a broad host spectrum. In the context of the present invention, “broad host spectrum plasmid” is understood to mean a plasmid that comprises the genetic elements necessary to be functional in multiple host cells.

Other elements that the vector or vectors used in the present invention may comprise are origins of replication, restriction enzyme recognition sequences and marker genes (or selection markers) that allow the identification of the cells that have incorporated the vector. which comprises the gene constructions.

As explained previously, a "selection marker", "selection marker gene" or "reporter gene" includes any gene that confers a phenotype to a cell in which it is expressed to facilitate the identification and/or selection of the cells. cells that are transfected or transformed with a vector. However, as understood by the person skilled in the art, the reporter gene included within construction (b) of the biosensor of the invention will have to be different from the selection marker used to detect/select/identify those cells that have incorporated the (the ) vector/vectors. In general, suitable selection markers may be markers that confer antibiotic resistance, that introduce a new metabolic trait, or that allow visual selection. Examples of selection marker genes include genes that confer resistance to antibiotics (such as nptl, which phosphorylates neomycin and kanamycin, ohpt, which phosphorylates hygromycin, or genes that confer resistance to, for example, bleomycin, streptomycin, tetracycline, chloramphenicol, ampicillin, gentamicin, geneticin (G418), spectinomycin or blasticidin), or genes that provide a metabolic trait. The expression of visual marker genes results in the formation of color (e.g., beta-glucuronidase (GUS) or beta-galactosidase (beta-Gal) with their color substrates, e.g., X-Gal), luminescence (such as the called luciferin/luciferase) or fluorescence. This list only represents a small number of possible markers. The expert is familiar with said markers. Depending on the organism and the selection procedure, different markers are preferred. If desired, the genetic markers can be removed from the host cell at a later stage.

The vectors and genetic constructions have to be introduced into the cell that is going to act as a biosensor of the invention. Methods for introducing gene constructs or vectors into a cell are widely known to those skilled in the art and their use is common practice in the laboratory. The term "introduce" in the context of the present invention refers to the incorporation of a nucleotide sequence into a cell by "transfection" or "transformation" or "transduction", where the nucleotide sequence can be incorporated into the genome of the cell (e.g., chromosome, plasmid, mitochondrial DNA), or be an autonomous replicon, or be transiently expressed (e.g., transfected mRNA). As used herein, the terms "transformed", "stably transformed" or "transgenic", with reference to a cell, mean that the cell has a non-native (heterologous) nucleotide sequence integrated into its genome, or in an episomal plasmid that is maintained through multiple generations. Examples of methods for introducing gene constructs or vectors into host cells include, but are not limited to, electroporation; nuclear microinjection or direct microinjection into single cells; fusion of bacterial protoplasts with intact cells; use of polycations, for example, polybrene or polyornithine; membrane fusion with liposomes, lipofectamine-mediated transfection, or lipofection; high-speed bombardment with DNA-coated microprojectiles; incubation with DNA-calcium phosphate precipitation; DEAE-dextran-mediated transfection; infection with modified viral nucleic acids; DNA transfer mediated by Agrobacterium and the like.

In the present invention, constructs (a) and (b) of the biosensor of the invention comprise elements of the genesedcdeC cluster. tardaugens.In a preferred embodiment of the biosensor of the invention, C. TardaugensesC. tardaugensNBRC 16725 (Ibero, et al., 2020, FrontMicrobiol 11, 588300).

In another aspect, the invention relates to a cell population, hereinafter "cell population of the invention", comprising the cell of the invention. In the context of the present invention, said cell population can be essentially pure, that is, at least 95% of the cells that make up the population belong to the cell of the invention, or a combination of cells in which the cells come from of different microorganisms and at least 1 of them belongs to the same microorganism as the cell of the invention.

Additionally, within the present invention, compositions comprising the cell of the invention or the cell population of the invention are also contemplated. Thus, in another aspect, the invention relates to a composition, hereinafter "composition of the invention" comprising the cell of the invention or the cell population of the invention. As understood by those skilled in the art, the composition of the invention must comprise all those elements that allow the viability of the cell or the biosensor of the invention, including conditions of pH, temperature, salinity, carbon source, etc. The conditions suitable for maintaining the cell of the invention are widely known in the state of the art and are routine practice for those skilled in the art.

As explained at the beginning of the present description, the invention is based on the capacity of the EdcR protein of the edcdeC gene cluster. Tardaugens acts as a repressor protein of estrogen degradation genes in the absence of estrogen, while in the presence of estrogen the DNA binding of the repressor protein is blocked and the degradation genes can be expressed. Therefore, said system, and the present invention insofar as it is based on it, can be used as a biosensor of estrogens in the environment, which has great environmental applications since said estrogens act as contaminants in many aquatic environments such as rivers, lakes, etc. , estuaries, aquifers, oceans, etc. due to anthropogenic activity.

Thus, in another aspect, the present invention relates to the use of the cell or biosensor of the invention, the cell population of the invention, or the composition of the invention, hereinafter "use of the invention", for the detection of estrogen in a sample.

As previously explained, estrogens are hormones produced by different organs and tissues, being responsible for female sexual characteristics and many metabolic processes. Three estrogens are produced in humans, estradiol, estrone and estriol. Estradiol is the most potent estrogen, followed by estrone and estriol. Thus, in a preferred embodiment of the use of the invention, the estrogens detected are estradiol and/or estrone.

As previously explained, currently, different pharmaceutical formulations contain estrogens and their high consumption means that significant quantities of these compounds can finally be released into the environment through different forms of discharges and contaminate the aquatic environment. Thus, in a preferred embodiment of the use of the invention, the sample is an environmental sample, which, in another even more preferred embodiment, the environmental sample is a water sample, more preferably, a wastewater sample or a water sample. coming from a river, a lake, an estuary, an aquifer, or the ocean.

Analogously to the use of the invention, the present invention also relates to an in vitro method for detecting estrogens in a sample, hereinafter "method of the invention", comprising

(a) contact the sample with the cell or biosensor of the invention, the cell population of the invention, or the composition of the invention, and

(b) determine the expression of the reporter gene,

wherein an expression of the reporter gene is indicative that the sample contains estrogens.

The terms used in the method of the invention have already been defined and explained in previous paragraphs for other inventive aspects of the present invention.

Thus, in a preferred embodiment of the method of the invention, estrogens are selected from the group consisting of estradiol (E2) and estrone (E1).

In another preferred embodiment of the method of the invention, the sample is an environmental sample, more preferably, the environmental sample is a water sample, even more preferably, a wastewater sample or a water sample from a river, a lake , an estuary, an aquifer, or the ocean.

As understood by those skilled in the art, in order to put into practice the use or method of the invention, it is necessary to have a device or kit in which the necessary elements are incorporated to carry out the detection of estrogens.

Therefore, in another aspect, the present invention relates to a kit, hereinafter "kit of the invention" comprising the cell or biosensor of the invention, the cell population of the invention, or the composition of the invention . Additionally, other components that can be part of the kit are tubes or test tubes for sample collection, reagents, etc.

In another aspect, the present invention relates to the use of the kit of the invention for the detection of estrogens in a sample. In a preferred embodiment, the estrogens are selected from the group consisting of E1, and E2.

In another preferred embodiment of the use of the kit of the invention, the sample is an environmental sample, more preferably, the environmental sample is a water sample, even more preferably, a wastewater sample or a water sample from a river, a lake, an estuary, an aquifer, or the ocean.

DESCRIPTION OF THE FIGURES

Figure 1. Schematic representation of the estrogen degradation cluster of C. tardaugensNBRC 16725. The regions corresponding to operons A (OpA) and B (OpB) have been represented with a solid and dotted line, respectively.

Figure 2. Schematic representation of the expression of clusteredcdeC. tardaugensNBRC 16725 grown in E2 compared to PIR. (a) Mapping against the clustered sequence of raw reads obtained by RNA-seq under the PIR and E2 conditions. (b) Scheme of the clusteredc. (c) Induction of the clusteredc genes measured in fold induction of gene expression (CF).

Figure 3. Fluorescence signal of E. cultures. co liDH10B (pSEVA23PlexA),E. co liDH10B (pSEVA237-Pa),E. co liDH10B (pSEVA237-Pb) and E. co liDH10B (pSEVA237-Pt). The values represented correspond to the average of three biological replicates.

Figure 4. Growth curves (DOeoo) of (a)C. tardaugensNBRC 16725 wt yC. tardaugensAedcR and (b)C. tardaugensAedcR (pSEVA23PlexA), AedcR-v, andC. tardaugensAedcR (pSEVA23edcR), AedcR-c, in minimal medium supplemented with 2mM E2. The values represented correspond to the average of three independent biological replicates.

Figure 5. RT-PCR analysis of the expression of clusteredcenC genes. tardaugensNBRC 16725 yC. tardaugensAedcR grown in E2 and TES. C-is the control without RNA, gDNA is the control with genomic DNA and M is the 100 bp molecular weight marker.

Figure 6. Fluorescence signal of E. co liDH10B (pSEVA237-Pb) (pSEVA651) and E. co liDH10B (pSEVA237-Pb) (pSEVA65-edcR). The values represented correspond to the average of three independent biological replicates.

Figure 7. Fluorescence signal of transcriptional fusions of the promoter regionsPayPbmeasured in cultures ofC. tardaugensNBRC 16725 (a) andC. tardaugensAedcR (b) in the presence of CDX and E1.

Figure 8. Proposed estrogen degradation pathway in C. tardaugensNBRC 16725. The names of the compounds are indicated with an abbreviation: (E2) estradiol; (E1) estrone; (4-OHE1) 4-hydroxyestrone; (M5) 4-norestrogen-5(10)-en-3-oyl-CoA; (le/lia) 3aa-H-4a-[3'-propanoic acid]-5p-[4'-but-3-enoic acid]-7ap-methyl-1-indanone; (Id/la) 3aa-H-4a-[3'-propanoic acid]-5p-[2'-ketopropyl]-7ap-methyl-1-indanone and (HIP) 3-a-H-4a(3'-propanoate) -7ap-methylhexahydro-1,5-indanedione. The names of the enzymes are: (17p-hsd) 3p,17p-hydroxysteroid dehydrogenase;(edcA)E14-hydroxylase;(edcB)4-OHE1 4,5-dioxygenase; y(edcC)decarboxylase. The catalytic genes of C. are indicated in italics, with the nomenclature EGO55_xxxxx. tardaugensproposed. Metabolites identified by mass spectroscopy are marked with an asterisk, while the rest have been confirmed by NMR or authentic standards. Hypothetical metabolites are boxed in dashed squares.

Figure 9. RT-PCR analysis of the expression of clusteredcenC genes. tardaugensAedcA growing on E2 and TES. C- is the control without RNA, gDNA is the control with genomic DNA and M e s e l 100 bp molecular weight marker.

Figure 10. Fluorescence signal of the transcriptional fusion of the Pb promoter region. The signal was determined in cultures of E. coliDH10B (pSEVA237-Pb) (pSEVA651) and E. coliDH10B (pSEVA237-Pb) (pSEVA65-edcR), in the graph, Pb and Pb+EdcR, respectively. Cultures grown in the presence of the solvent (solv) as a control and the inducers, 5 pM E1 and 5 pM E2. The values represented correspond to the average of three independent biological replicates.

Figure 11. Fluorescence signal of the transcriptional fusion of the Pb promoter region. The signal was determined in cultures of E. coliDH10B (pSEVA237-Pb) (pSEVA65-edcR) when grown in the presence of the solvent (solv) as a control, 10 pM E1, 10 pM E2, 10 pM E3, 10 pM 40H-E1, 10 pM EE2 and 10 pM TES. The values represented correspond to the average of three independent biological replicates.

Figure 12. Schematic representation of the vector constructed for the pSEVA237M-Pb estrogen sensor system. The main elements of the plasmid have been drawn: Pb, Pb\BCD2 promoter region, bicistronic RBS; msf gfp, monomeric superfolder green fluorescent protein, T0 and T1, transcription terminators; KmR, kanamycin resistance gene; OriT, origin of transfer and OriV(pBBR1), origin of replication. The restriction enzymes used in cloning are indicated.

Figure 13. Analysis of the estrogen sensor system built with the E strains. co liDH10B (pSEVA237M-Pb) (pSEVA651) and E. co liDH10B (pSEVA237M-Pb) (pSEVA65-edcR), in the graph, Pb and Pb+EdcR, respectively, (a) Fluorescence signal of the transcriptional fusion of the promoter regionPbmeasured in the cultures grown in the presence of the solvent (solv) , 5 pM E1 and 5 pM E2. The values represented correspond to the average of three independent biological replicates. Visual comparison of the cultures grown in the presence of the solvent (solv) as a control and 15 pM E (b) and of the 1.5 ml pellets of said cultures (c).

EXAMPLES

Next, the invention will be illustrated through tests carried out by the inventors, which demonstrate the effectiveness of the biosensor of the invention.

EXAMPLE 1. Bacterial strains, plasmids and oligonucleotides used

The bacterial strains used in this work are detailed in Table 1 along with their genotypes and relevant characteristics.

Table 1. Bacterial strains used in this invention.

The plasmids used are shown in Table 2.

Table2. Plasmids used in this invention.

The oligonucleotides used in this work were synthesized by Sigma-Aldrich and are shown in Table 3, where their sequence and their application in the present work are indicated.

Table 3. Oligonucleotides used in this invention. The targets used for cloning are indicated in parentheses.

EXAMPLE 2. Culture media and conditions

The culture solutions and media used in this work were sterilized by moist heat in an autoclave at 121 °C and 1 atm pressure, or by filtration using 0.2 pm Millipore sterile filters. Antibiotics were prepared in aqueous solutions at 1000 mg mL-1, except for rifampicin, which was prepared at 50 mg mL'1 in dimethyl sulfoxide (DMSO). The prepared solutions were sterilized by filtration and stored at −20 °C.

1. Media and culture conditions used for Escherichia coli.

The medium used to culture the cells of E. coliwas Luria-Bertani (Lysogeny Broth, LB). Cultures on solid medium were carried out in LB medium to which Bacto Agar (Pronadisa) was added at 1.5% (w/v). If necessary, antibiotics were added at the following final concentrations: kanamycin (Km) (50 pg mL'1), gentamicin (Gm) (10 pg mL'1) and chloramphenicol (Cm) (34 pg mL'1) ( Sigma). The cells of E. colise were grown at 37 °C. Cultures in liquid medium were carried out on an orbital shaker at 200 rpm. Growth in liquid medium was monitored by turbidimetry at 600 nm (OD600) using a UVMini-1240 spectrophotometer (Shimadzu).

2. Media and culture conditions used for Caenibius Tardaugens.

C. tardaugensNBRC 16725 (strain ARI-1) was obtained from the type culture collection of the Leibniz-lnstitut DSMZ. This strain was grown at 300C on an orbital shaker at 200 rpm. The rich Nutrient Broth (NB) medium (Difeo) was used as a liquid medium at 16 g L'1 (m/v), to which 1.5% agar was added to prepare the solid Nutrient Broth Agar (NA) medium. The minimal medium M63 [KH2P04 (13.6 g L'1), (NH4)2S 04 (20 g L'1), FeS04-7H20 (0.5 mg L'1), MgS04 (1 mM) was used (pH 7.0 )], to which was added 20.39 mM CaCl and the appropriate concentration of the appropriate carbon source for each experiment. Carbon sources were used at the following concentrations: pyruvate (PIR) 12 mM, estradiol (E2) 2 mM, testosterone (TES) 1.89 mM. If necessary, antibiotics were added at the following final concentrations: kanamycin (Km) (10 pg mL'1) and gentamicin (Gm) (10 pg mL'1). Growth in liquid medium was followed by turbidimetry at 600 nm (OD600) using a UVMini-1240 spectrophotometer (Shimadzu).

3. Steroid dissolution

Steroids are highly hydrophobic molecules that are not soluble in water, so it is necessary to dissolve them in a special way to be able to follow the growth of cultures in liquid medium by turbidimetry.

To carry out the cultures of C. Tardaugens steroids were dissolved in 70 mM methyl-pcyclodextrin (CDX) (TRMB-T Randomly Methylated-Beta-Cyclodextrin, CTD Inc.). Steroid stock solutions were prepared in PBS buffer with 70 mM CDX at the following concentrations: 10.5 mM E2, E1, and E3 and 10 mM TES. Thus, in the cultures, the final concentration was 13.33 mM CDX and equimolar in carbon number.

4. Conservation of bacterial strains.

For periods of less than one month, the strains were preserved at 4 ° C on LB or NB plates. For long-term conservation, strains of E. coli were grown in LB with the corresponding antibiotics and frozen at -80 °C with 20% (v/v) glycerol. The strains of C. Tardaugens were cultured in NB or M63 minimal medium with the corresponding antibiotic and frozen at -80 °C with 20% (v/v) glycerol.

EXAMPLE 3. Genetic transformation methods for the construction of recombinant strains

The methods used for genetic transformations are described below.

1. Transformation of E cells. coli.

The cells of E. coli were genetically modified by transformation using the RbCl method and heat shock.

2. Transformation of C cells. tardaugens.

A) Isolation of a spontaneous mutant resistant to rifampicin.

The wild strain ofC. Tardaugens, sensitive to rifampicin (Rf), was cultured in NB medium supplemented with rifampicin until stationary phase ("48 hours). The cells were then plated in NA in the presence of Rf and cultured for 24 hours. A single colony was isolated and subsequently used for conjugation.

B) Transformation of C.tardaugens cells by triparental conjugation.

For transformation by triparental conjugation, strain C was used. tardaugensRfR as a recipient, the strain E. coliDH10B carrying the plasmid to be transferred as a donor and the strain E. coliHB101 (pRK600) as an auxiliary according to the method described by De Lorenzo and Timmis, 1994, Methods Enzymol 235, 386-405, with some modifications. The recipient strain was grown in 65 mL of NB with rifampicin in a 250 mL flask until the late exponential phase (~48 hours), at which time it was centrifuged at 4000 rpm for 15 minutes at 40 C and the pellet was washed with a volume of sterile NaCl solution in H20mq at 0.85% (w/v)). The cells were centrifuged again and the pellet was resuspended in a final volume of 800 pL of sterile NaCl solution in 0.85% (w/v) H2Omq). The donor and helper strains were inoculated with 2 mL of LB (with the corresponding antibiotics), in 10 mL plastic tubes, with 50 pL of stationary phase culture and cultured until they reached the late exponential phase (~6 hours). The cultures were centrifuged at room temperature at 13,000 rpm for 1 minute and the pellet was resuspended in one volume of sterile NaCl solution in 0.85% (w/v) H2Omq). The washing was repeated once more and the pellet was resuspended in 100 pL of sterile 0.85% (w/v) NaCl H2Omq solution). 50 pL of each strain was mixed and pipetted onto a 0.22 pm filter disk placed on a NA plate. The plate was incubated for « 20 hours at 30 ° C. The next day, the cells were separated from the filters in a 1.5 mL tube with 1 mL of sterile NaCl solution in 0.85% (w/ v)) by vortex stirring. Transconjugants were selected by plating 100 pL and the rest of the cells on NA plates containing rifampicin (50 pg/mL) and the corresponding antibiotic whose resistance gene is located on the plasmid. This protocol was used for the transfer of replicative plasmids and suicide plasmids.

EXAMPLE 4. DNA manipulation techniques

The molecular biology techniques used in this work were applied essentially as described previously. Restriction endonucleases were supplied by New England Biolabs and Takara. T4 DNA ligase enzyme, Instant Sticky-end Ligase Master Mix kit, and Phusion High-Fidelity polymerase were supplied by New England Biolabs. DNA polymerase I and Pfu polymerase were supplied by Biotools B. M. Labs. All enzymes were used according to the specifications of the different commercial companies. The DNA fragments were purified using the QIAquick PCR Purification Kit (Qiagen) or the QIAquick Gel Extraction Kit (Qiagen).1

1. DNA electrophoresis in aqarose gels

To visualize the DNA fragments, 0.7% or 1.5% (w/v) agarose gels in TAE buffer (40 mM Tris-HCI, 20 mM acetic acid, 2 mM EDTA (pH 8.1)) were used. , using the same buffer as electrolyte. One sixth of their volume of loading buffer (30% (w/v) Ficol 400, 0.2% (w/v) bromophenol blue, 0.2% (w/v) ) of xylencianol and 40 mM EDTA (pH 8.0)). Electrophoresis was performed at 135 V for 15-20 minutes and, once completed, the gels were stained with Gel Red Nucleid Acid Gel Stain (Biotium) and the DNA fragments were visualized with ultraviolet radiation in a transilluminator. DNA from phage A digested with the restriction enzyme BstEII (Amersham) and the replicative form of phage 0X174 digested with Haelll (New England Biolabs) were used as size markers.

2. Isolation of plasmid DNA in E. colivC. tardaugens.

The extraction of plasmid DNA from E cells. coliyC. Tardaugens was carried out using the High Puré Plasmid Purification Kit system (Roche), according to the manufacturer's specifications.

3. Amplification chain reaction with thermoresistant DNA polymerase (PCR) The DNA amplification was carried out in a Mastercycler Gradient equipment (Eppendorf) and the enzymes used were DNA polymerase I (Biotools) and the NZYTaq II 2x Green Master Mix ( NZYTech) for the check PCRs and Phusion High-Fidelity polymerase (New England Biolabs) for the cloning PCRs. The reaction mixtures contained 1.5 mM MgCl2, 0.5 pM oligonucleotides, and 0.25 mM dNTPs. The amplified products were purified with the QIAquick PCR Purification Kit (Qiagen) or QIAquick Gel Extraction Kit (Qiagen) system. Oligonucleotides were provided by Sigma-Aldrich or IDT. Standard PCR conditions were as follows: a first cycle of 5 minutes at 950C; 30 cycles of 1 minute at 950C, 1 minute at 600C (or at the appropriate oligonucleotide hybridization temperature, optimized by temperature gradient), 1 minute for every 1,000 bp of the fragment to be amplified at 720C; a final cycle of 5 min at 720C.

4. Construction of enC deletion mutants. tardaugensNBRC 16725 using the pK18mobsacB double homologous recombination system.

The construction of the different mutant strains (see Table 1) was carried out by deletion by double homologous recombination using the suicide plasmid pK18mobsacB (Schafer et al., 1994, Gene 145, 69-73). This non-replicative vector inC. Tardaugens presents two selection markers: a kanamycin resistance gene and a gensacB, which encodes the levansucrase of B. subtilis, whose expression is lethal for the bacteria in the presence of sucrose.

First, two fragments of approximately 500 bp were amplified by PCR, one in position 5' to the gene to be mutated (UP fragment, using the corresponding oligonucleotides UPf and UPr, see Table 3) and another in position 3' (DOWN fragment , using the corresponding oligonucleotides DOWNf and DOWNr, see Table 3). The UP and DOWN fragments were digested with the corresponding restriction enzymes to make their ends compatible and subsequently incubated with the Instant Sticky-end Ligase Master Mix ligation kit for 5 minutes, so that both fragments were fused into a single fragment of DNA (UP+DOWN), which was used to carry out the gene deletion. The UP+DOWN fragments of each gene were cloned into the plasmid pK18mobsacB in the same ligation reaction of the UP and DOWN fragments. The ligation mixture was transformed into E. coliDH10B, where colonies carrying the recombinant plasmid were selected by POR.

The resulting plasmids (see Table 2) were transformed into C cells. tardaugensNBRC 16725 by triparental conjugation and the transformants were selected on NA medium plates with Km (10 pg mL'1) and Rf (50 pg mL'1). In the Km-resistant colonies, the insertion of the plasmid was verified after the first recombination event by PCR using an oligonucleotide (F24 or R24) that flanks the MCS (Multiple Cloning Site) of the plasmid and an oligonucleotide external to the fragment used to carry out the recombination. homologous (Table 3). The transformants selected in this way were grown in liquid NB medium until stationary phase (~48 hours) to force the second recombination event, the biomass produced was diluted 2 times and cultured on NA plates with 5% sucrose. The sucrose-resistant colonies were replicated on NA plates with Km (10 pg mL'1), and in those resistant to sucrose and sensitive to Km, the loss of the plasmid was verified after the second homologous recombination event by PCR using the oligonucleotides. external to the fragments used to perform homologous recombination (Table 3).

EXAMPLE 5. RNA manipulation techniques

1. Extraction of total RNA from C. tardaugens.

RNA purification was carried out starting from cells from 12 mL of culture in the exponential phase of growth (OD600 of 0.5). The cells were resuspended in a solution of 50 mg mL'1 lysozyme (Sigma) in TE buffer (Tris-HC110 mM pH 8.0, 1 mM EDTA). Next, total RNA from the culture was isolated using the High Puré RNA Isolation kit (Roche), following the manufacturer's recommendations. To eliminate contaminating DNA, the DNAse and Removal treatment kit (Ambion) was used, following the manufacturer's specifications. The concentration of the obtained RNA was analyzed using a nanodrop Nanophotometer Pearl, IMPLEN).

2. Retrotranscription followed by PCR reaction (semiquantitative RT-PCR).

Complementary DNA (cDNA) was generated using the Transcriptor First Strand cDNA Synthesis kit (Roche). The reactions were carried out in 20 pL total containing 1 pg of RNA, 10 U of reverse transcriptase, 20 U of RNases inhibitor, 1 mM dNTPs and 60 pM random hexamer primer. The reaction mixtures were incubated for 10 minutes at 250C, with a cycle of 30 minutes at 550C and a final incubation of 5 minutes at 85 0C to inactivate the enzyme, using a Mastercycler Gradient equipment (Eppendorf). The cDNA obtained was used as a template (1 pL) for the subsequent RT-PCR using the oligonucleotides required in each case (Table 3) at a final concentration of 0.5 pM, and 1 U of DNA polymerase I (Biotools). To rule out the presence of genomic DNA, a control with 1 pL of the RT reaction in which reverse transcriptase was not used was included in each of the PCR reactions. The total volume of the PCR reaction was 50 pL and 25 cycles of amplification were carried out following the program described in section 4.4.

3. Massive RNA sequencing (RNA-seq).

For the sequencing of the complete transcriptome, lllumina technology was used and both the construction of the libraries and their sequencing were carried out by Macrogen (Korea). The integrity of the extracted total RNA was analyzed on an Agilent Technologies 2100 bioanalyzer and those samples whose RNA Integrity Number (RIN) value was equal to or greater than 7 were analyzed. Ribosomal RNA was removed from the total RNA using the Ribo-Zero rRNA Removal lllumina kit to later build a library with the 100bp mRNA, paired, using the TruSeq RNA Sample Prep Kit v2. The quality of the libraries was verified on an Agilent Technologies 2100 bioanalyzer. Sequencing was performed on a HiSeqTM 3000 4000 instrument (lllumina) using TruSeq 30004000 SBS Kit v3 as reagent. The subsequent analysis of the data was carried out in the Bioinformatics and Biostatistics service of the Margarita Salas Biological Research Center (CIB-MS) using the default options in all the programs used. Raw reads were quality analyzed with FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and filtered with Trimmomatic (Bolger et al., 2014, Bioinformatics 30, 2114-2120) . The filtered reads were mapped against the genome sequence of C. tardaugens(accession number CP034179.1) using Bowtie2 (Langmead et al., 2012, Nat Methods9, 357-359) and expression quantification was done with HTSeq-count (Anders, et al., 2010, Genome Biol 11 , R106). Normalization of quantified reads and differential expression analysis was performed with DESeq2 (Love et al., 2014, Genome Biol 15, 550). To prepare the heat map, the similarity matrix shown was obtained using the Euclidean distance and the cluster analysis was performed with the minimum variance Ward method.

EXAMPLE 6. Crop florescence measurement in a plate reader.

The fluorescence signal of the GFP (Green Fluorescent Protein) in the cultures of the strains carrying the transcriptional fusions was measured using a Variouskan Flash microplate reader (Thermo scientific), in which the excitation and emission wavelengths were of 485 nm and 511 nm, respectively. The measurement was made in 96-well plates and 3 technical replicas of each sample were taken. The measurement was carried out by loading 200 pL of cells into each well at an OD600 of 1, previously washed with a 0.85% (w/v) NaCl solution in H20mq.

EXAMPLE 7. Bioinformatic analysis.

The analysis of nucleotide sequences, of chromatograms from sequencing reactions, the design of genetic engineering experiments, the design and analysis of oligonucleotides, the alignment of the C genome sequences. Tardaugensen 54 contigs against the unique sequence obtained with PacBio, as well as the mapping of the reads obtained in the transcriptome sequencing against the genome of C. tardaugens, were made with the Geneious R11.1.5 program (https://www.geneious.com). The identification of conserved sequences of promoters was carried out using the BPPOM tools (Solovyev and Salamov 2011, Metagenomics its Appl Agrie Biomed Environ Stud., 62-78) of the Softberry server (https://www.softberry.com) and SAPPHIRE ( Coppens and Lavigne, 2020, BMC Bioinformatics21, 1-7.) (https://www.biosapphire.com).

The amino acid sequences of specific proteins were compared with those existing in databases by using the BLASTp program (Altschul et al., 1990, J Mol Biol 215, 403-410) from the National Center for Biotechnology Information (NCBI) server. ; https://blast.ncbi.nlm.nih.gov/Blast.cgi). Multiple alignments of the nucleotide and amino acid sequences were performed with ClustalOmega (Abio Madeira et al., 2019, Web Serv issue Publ online 47) from the European Bioinformatics Institute (EMBL-EBI; http://www.ebi) server. .ac.uk/Tools/msa/clustalo/).

EXAMPLE 8. In silico analysis of the regulatory sequences of the clusteredc. The transcriptome analysis revealed a high level of induction of the genes of the clusteredc in the presence of E2. The structure of the cluster suggests the existence of a promoter region in the intergenic region between the divergent operons OpA and OpB, and another promoter region in front of the gene EGO55_13520 (edcR), which encodes the regulatory protein EdcR, which is transcribed in a divergent manner to the OpA operon genes (Figure 1). Furthermore, between the genes EGO55_13595 and EGO55_13600, there is a region of 85 bp, unlike the rest of the cluster, where the genes overlap or are separated by a maximum of 9 bp, which suggests the presence of another promoter region in front of EGO55_13600.

The greater abundance of reads from transcriptome sequencing that align in a specific area of the genome indicates a greater number of transcripts produced from said sequence, indicating the existence of a promoter region. In a first qualitative analysis of the expression of the clusteredc, the raw reads obtained were mapped to its sequence. The mapping reflected a greater number of reads in the presence of E2, which corresponds to the highest level of induction (Figure 2a). There is also an area with a lower number of aligned reads, between the genes EGO55_13565 and EGO55_13570, coinciding with the intergenic region (Figure 2a). The alignment of the reads also shows a greater number of reads at the beginning of the gene EGO55_13600 (edcT) in the presence of E2 (Figure 2a). The opposite is observed in the gene EGO55_13520 (edcR), where the reads are more abundant at the beginning of the gene in the presence of PIR, while in the presence of E2 they are distributed homogeneously throughout the entire gene (Figure 2a).

On the other hand, the distribution of the increase in expression in the E2 condition, calculated as fold induction (FC) from the data collected in transcriptomics, revealed a greater induction of the genes of the OpA operon compared to those of OpB. (Figure 2c). Furthermore, a large difference in expression is observed between the EGO55_13595 and EGO55_13600 genes, which would indicate the influence of an additional regulatory element on the expression of the EGO55_13600 gene (Figure 2c). This comparison of the FC along the cluster genes would also support the existence of 4 promoter regions in the edc cluster. In this way, Pr, Pa, PbyPt would be the promoters of the regulatory gene edcR, of the OpA and OpB operons and of the transporter edcT, respectively. (Figure 2b).

In parallel, the expression of the regulatory gene edcR was compared to the rest of the genes in the cluster edc using the number of normalized reads, which allows comparison between genes of different sequence length and between conditions with different depth of total reads. In this way, it was observed that the average of normalized reads corresponding to EGO55_13520(edcR) in the presence of pyruvate (PIR) (1553) is greater than that of the rest of the genes in the cluster (523). Meanwhile, in the presence of E2 the opposite happened, fewer reads correspond to aedcR (9390) than to the rest of the genes in the clusteredc (18035). These data indicate a higher level of expression of this genedcRen in relation to the others in the PIR condition. The increase in expression of edcRe in the PIR condition suggests a possible repressive function of EdcR on the clusteredcya since in this condition the genes are repressed.

1. In silico analysis of the promoter regions of the edc cluster

Next, the sequences of the proposed promoter regions of the clusteredc\ Pr, Pa, PbyPt were analyzed in order to search for RNA polymerase binding consensus sequences (boxes -10 and -35) as well as transcriptional factor binding operator sequences. In the 4 regions, possible RNA polymerase recognition sequences -10 and -35 were found. Being, in the case of Pb, the most similar to the consensus sequences of boxes -10 (TATAAT) and -35 (TTGACA) described in E. coli (Rosenberg and Court, 1979, Annu Rev Genet 13, 319-353). Furthermore, in the <promoters Pa>, Pb, Pt<and P>r<conserved palindromic sequences were found that>would overlap in both cases with the possible -10 box. Analyzing the conserved regions in more detail, a palindromic sequence TnnCnnTACnAnTnGTAnnGnnA (SEQ ID NO: 35) was found, which is located in Pa,yPbdeC. tardaugens.This conserved sequence could correspond to the binding operator region of a transcription regulatory protein that would direct the transcription of the OpB operon and the possible transporter of the pathway, EdcT.

In parallel, the promoter regions of C. tardaugensNBRC 16725 with those of other estrogen-degrading bacteria such as Altererythrobacter estronivorus MHB5 and Sphingomonas sp. KC8 that have a gene cluster very similar to clusteredc (Chen et al., 2017, Cell Chem Bio. 24, 712-724.e7). Thus, conserved sequences were found in the PayPbdeC regions. Tardaugens and the intergenic region of the cluster of A. estronivorusMHB5. However, when comparing the promoter regions of C. tardaugens with the corresponding estrogen degradation cluster II of Sphingomonas sp. KC8 (Chen et al., 2017 cited supra), these do not share conserved regions. The palindromic sequence TnnCnnTACnAnTnGTAnnGnnA (SEQ ID NO: 35) is also found in the intergenic region of A. estronivorusMHB5.

2. In silico analysis of the edcR regulatory gene

The genedcR (EGO55_13520) is annotated as a transcriptional regulator of the TetR family and presents the two characteristic domains of this family (Ramos et al., 2005, Microbiol Mol Biol Rev 69, 326-356): a DNA binding domain (helix turn helixHTH, accession number PF00440 Pfam database, EMBL-EBI) and another for binding to the inducer or oligomerization (TetR_C_11 domain, accession number PF16859 Pfam database, EMBL-EBI). This protein shares 71.3% identity with that encoded by the MB02_RS00150deA gene. estronivorusMHB5 that has a cluster with a gene organization identical to clusteredc (Chen et al., 2017 cited above). Comparing its domains, it was observed that the DNA binding domain presents 95.0% identity, while the binding to the inducer or oligomerization shares 76.0%. EdcR shares 37.7% identity with the protein encoded by the Sphingomonassp geneKC8_05330. KC8. Specifically, it has 53.3% identity in the DNA binding domain and 34.6% in the inducer binding or oligomerization domain.

The identity between the DNA binding domains of their respective regulatory proteins and the existence of possible conserved operator regions suggests the presence of a similar regulatory system in C. tardaugensNBRC 16725 yA. estronivorusMHB5. Continuing with this approach, the lower similarity of the DNA binding domain of the regulatory protein of Sphingomonassp. KC8 would be consistent with the lack of these operator regions in its estrogen degradation cluster, since said domain could recognize different sequences than those of C. tardaugensNBRC 16725 yA. estronivorusMHB5. It is also worth highlighting the low percentage of identity of the effector binding domain among the regulatory proteins of C. tardaugens NBRC 16725 and Sphingomonas sp. KC8, which could indicate that the effector with which the regulatory protein interacts is different in these strains.

EXAMPLE 9. Functional analysis of the Pa, PbyPt promoter regions

The transcriptomic analysis (Figure 2) showed that the genedcTy genes of the OpA, OpB operons are inducible in the presence of E2. To monitor gene expression from the promoters Pa, Pb, and P under different conditions, transcriptional fusions were performed with the marker gene gfp. To do this, the promoter sequences Pa, Pb, and P were cloned in the plasmid pSEVA237 (Table 2) and the resulting plasmids, pSEVA237- Pa, pSEVA237-Pb and pSEVA237-Pt were transformed into E. coliDH10B. The cloned Pa, Pb, and Pt sequences comprise, in each case, about 300 bp upstream from the ribosome binding site (RBS) of the EGO55_13565, EG55_13570, and EGO55_13600 genes, respectively. The strains obtainedE. coliDH10B (pSEVA237-PlexA), E. coliDH10B (pSEVA237-Pa),E. coliDH10B (pSEVA237-Pb) and E. coliDH10B (pSEVA237-Pt) were grown in LB and the fluorescence of the cultures was determined after 5 hours to verify the functionality of the different enE promoters. coli (see example 6). As can be seen in Figure 3, only the fluorescence signal was obtained for strain E. coliDH10B (pSEVA237-Pb), suggesting that only the Pbes region recognized as a promoter by the RNA polymerase of E. coli(Figure 3).

EXAMPLE 10. Functional analysis of the transcriptional regulator EdcR.

To know the role of EdcR in estrogen metabolism in C. tardaugensNBRC 16725, said gene was deleted and the resulting strain, AedcR, was grown in minimal medium supplemented with E2. The AedcR strain was able to grow on E2 as the sole carbon source (Figure 4a), suggesting that EdcR is not an essential transcription activator for E2 degradation. Furthermore, the AedcR culture presents a lower initial latency phase than the wild strain (Figure 4a), suggesting a possible repressive function of the regulator. In order to complement the mutation in the AedcR strain, the genedcRe was cloned into the plasmid pSEVA23PlexA (Table 2), and the resulting plasmid, pSEVA23ecdR, was transformed into the AedcR strain. The AedcR-c strain was grown in minimal medium with E2 as the sole carbon and energy source and its growth curve was compared with that of the control strain AedcR-v carrying the empty plasmid. Entransdel genedc complementation increased the duration of the initial dormancy phase in the AedcR-c strain (Figure 4b). The fact that the strain's ability to efficiently metabolize E2 is slowed supports the possible repressive function of EdcR in estrogen metabolism in C. tardaugensNBRC 16725 and would indicate that in the AedcR-c strain the expression of this gene would be increased compared to the wild strain, since edcR is expressed from a multicopy plasmid and under the control of a strong constitutive promoter {PiexÁ)-

Taking into account these results, together with the data provided by the transcriptome analysis described in the previous section, it is proposed that EdcR is a specific regulator of the E2 degradation pathway whose function is to repress the expression of the clusteredc genes. .

Subsequently, in order to determine the influence of EdcR on the expression of the clusteredc genes, total RNA was extracted from the cells of the wild strain and the AedcR strain cultured in minimal medium with E2 or TES as the only carbon sources and energy. RT-PCRs were performed (see section 5.2 of materials and methods) to determine the expression level of several genes using specific primers that hybridized to the representative genes of each of the operons of the clusteredc, EGO55_13555(OpA operon), EGO55_13570( OpB operon) and EGO55_13600(EdcT) (Table 3) (Figure 2). GenrecÁ (EGO55_01665) was used as an internal control.

The expression of all the edc genes tested was detected in the wild strain (wt) and in the AedcR strain in the presence of E2. However, in the presence of TES the expression in the wild strain is greatly decreased while the levels in the AedcR strain were similar to those detected in the presence of E2 (Figure 5). This result indicates that EdcR negatively regulates the expression of genesedc and that the inducing molecule is E2 or some intermediate of the degradation pathway.

To confirm the repressive function of EdcR, its effect on transcription was studied in a heterologous host. To do this, the genedcRen plasmid pSEVA651 (Table 2) was cloned under the control of the PiexA promoter, generating the vector pSEVA65-edcR, which was transformed into the E strain. coliDH10B (pSEVA237-Pb). The generated strain,E. coliDH10B (pSEVA237-Pb) (pSEVA65-edcR) was grown in LB and the fluorescence signal emitted was measured after 5 hours. The presence of the regulator showed a decrease in fluorescence intensity indicating that EdcR is a transcription repressor (Figure 6).

EXAMPLE 11. Analysis of molecules that induce the transcriptional expression of the clusteredc.

Transcriptomics data show that OpA and OpB operon genes are inducible in the presence of E2 enC. tardaugensNBRC 16725. To study the expression mediated by the PayPbenC promoters. Tardaugens PayPb promoter sequences were cloned into plasmid pSEVA237 (Table 2) and the resulting plasmids, pSEVA237-Pa and pSEVA237-Pb, were transformed into the wild-type strain of C. tardaugensNBRC 16725 and in the AedcR mutant strain. The strains generated,C. tardaugensNBRC 16725 (pSEVA237-Pa),C. tardaugensNBRC 16725 (pSEVA237-Pb), C.tardaugensAedcR (pSEVA237-Pa) andC. tardaugensAedcR (pSEVA237-Pb) were cultured in rich NB medium supplemented with 2 mM E1 and fluorescence was measured at 36 hours. In this case, E1 was used since, from the information provided by transcriptomics, it is inferred that E2 would be capable of inducing the expression of the system, either directly or through a metabolite produced from it. However, it was unknown if E1 would be capable of inducing it, which is why it was used in this experiment to restrict the range of possible inducers to a single metabolite. The results show that in the wild strain there is an increase in the fluorescence signal in the presence of E1 (5.9 times in Pay and 2.9 times in Pb), while in the mutant strain the signal does not vary, suggesting that the expression in the wild strain it is inducible by E1. This result is consistent with that obtained from the transcriptomic analysis, where the average induction fold (FC) is greater for the genes of the OpA operon (57.7) than for the genes of the OpB operon (32.2). Two fundamental conclusions can be obtained from this result. On the one hand, the EdcR regulator has a repressive function on the activity of Pay PbenC promoters. Tardaugensy, on the other hand, E1, the second metabolite of the pathway, is able to induce expression through the EdcR regulator.

Figure 7 shows that E1 induces the expression of Pay Pben the wild strain. However, given that E1 can be transformed into downstream intermediates of the E2 degradation pathway in the strains tested, it is not possible, at this point, to unequivocally identify the possible inducer(s). To confirm the ability of E1 to act as an inducer, the AedcA strain (clusteredc cytochrome mutant) was used, in which the estrogen degradation pathway stops in the E1 metabolite (Figure 8). Total RNA was extracted from cells of the AedcA strain cultured in minimal medium supplemented with TES as a carbon and energy source to which 2 mM E1 was added as an inducer.

RT-PCRs were performed (see section 5.2 of materials and methods) to determine the expression level of several genes using specific primers that hybridize to the representative genes of each of the operons of the clusteredc, EGO55_13555(OpA operon), EGO55_13570( OpB operon) and EGO55_13600(EdcT) (Table 3) (Figure 2). genrecA (EGO55_01665) was used as an internal control. Figure 9 shows the induction in the presence of E1 of all the selected genes in the AedcA strain (Figure 9). This result indicates that E1 would act as an inducer of the clusteredc,enC genes. tardaugensNBRC 16725.

Next, in order to confirm induction with E1 in a heterologous host, the transcriptional fusions system with GFP enE was used. coli. For this purpose, the strain E. coliDH10B (pSEVA237-Pb) (pSEVA65-edcR), which carries the transcriptional fusion with the Pby promoter, expresses the regulator EdcR, in the presence of E1 and E2 and the fluorescence emission was measured at 5 hours. When adding 5 pM of E1 or E2, a partial recovery of the fluorescence signal was observed, which was not observed in the control to which only the solvent used to add the steroid was added, which indicates that both metabolites are inducing expression. of GFP, by counteracting the repressive effect of the EdcR protein (Figure 10).

Subsequently, the experiment was repeated using other steroids such as E3, 4-OHE1, EE2 and TES. The data collected in the experiment show that only E1 and E2 functioned as transcription inducers (Figure 11).

EXAMPLE 12. Application of the regulation system in the design of a biosensor for the detection of estrogens.

Having a system that responds specifically to the presence of estrogens, the possibility of building a biosensor and applying it to the detection of estrogens in the environment was raised. With this objective, a new transcriptional fusion of the Pb promoter was performed using the plasmid pSEVA237M-BCD2-14g (Table 2). This plasmid includes a synthetic bicistronic RBS, BCD2, which increases translation by possessing two tandem ribosome-binding Shine-Dalgarno motifs (Mutalik et al., 2013, Nat Methods 10, 354-360); and a modified version of GFP, the so-called monomeric superfolder green fluorescent protein, MsfGfp, whose emission signal is more intense (Landgraf, D., 2012, PhD Thesis. Harvard University, Cambridge.). The synthetic promoter of plasmid pSEVA237M-BCD2-14g was replaced by the Pb promoter and the resulting plasmid, pSEVA237M-Pb (Figure 12), was transformed into E. coliDH10B, obtaining the strain E. coliDH10B (pSEVA237M-Pb). This strain was in turn transformed with the vector pSEVA65-edcR and with the empty plasmid pSEVA651 as a control, obtaining strains DH10B (pSEVA237M-Pb) (pSEVA65-edcR) and DH10B (pSEVA237M-Pb) (pSEVA651). Both were grown in LB in the presence of the estrogens E1 and E2, and the fluorescence signal emitted was measured. It was possible to verify that the reporter system responded to the presence of E1 and E2 (Figure 13a), and the signal collected was an order of magnitude higher than that obtained with the previously constructed reporter system (Figure 10). Furthermore, this emitted signal is intense enough to be able to distinguish the induction visually in both the cultures and the cell pellets (Figures 13b and 13c). Thus, the generated strain serves as proof of concept for the construction of a biosensor to determine the presence of these estrogens in a sample.

Claims (29)

1. A cell that comprises (a) a first gene construct comprising (i) the nucleotide sequence of a promoter operatively linked to (ii) the nucleotide sequence encoding the EdcR regulatory protein of the edcdeC genes cluster. tardaugens]y (b) a second gene construct comprising (iii) the nucleotide sequence of the promoter of Operon A or Operon B of the genesedcdeC cluster. tardaugensoperatively linked to (iv) a nucleotide sequence encoding a reporter protein. 2. Cell according to claim 1, wherein the cell is selected from the list consisting of Escherichia coli, Comamonas testosteroni and Pseudomonas putida. 3. Cell according to claim 1 or 2, wherein the first and second gene constructs are located in different vectors. 4. Cell according to any one of claims 1 to 3, wherein the promoter of the first gene construct is a constitutive promoter or an inducible promoter. 5. Cell according to claim 4, wherein the constitutive promoter of the first gene construction is the PlexA promoter. 6. Cell according to any one of claims 1 to 5, wherein the EdcR regulatory protein of the edcdeC gene cluster. tardaugens comprises, or consists of, an amino acid sequence that has a sequence identity of at least 75, 80, 85, 90, 95, 96, 97, 98 or 99% with the SEO sequence ID NO: 2. 7. Cell according to any one of claims 1 to 6, wherein the nucleotide sequence that encodes the EdcR regulatory protein of the edcdeC gene cluster. tardaugens comprises, or consists of, the sequence SEO ID NO: 3. 8. Cell according to any one of claims 1 to 7, wherein the second gene construction comprises between elements (iii) and (iv), the nucleotide sequence of a synthetic bicistronic RBS operatively linked to said elements (i) and V). 9. Cell according to any one of claims 1 to 8, wherein the nucleotide sequence of the promoter of Operon A of the genesedcdeC cluster. tardaugens comprises, or consists of, the sequence SEQ ID NO: 4. 10. Cell according to any one of claims 1 to 8, wherein the nucleotide sequence of the promoter of Operon B of the genesedcdeC cluster. tardaugens comprises, or consists of, the sequence SEQ ID NO: 5. 11. Cell according to any one of claims 1 to 10, wherein the reporter protein is selected from the group consisting of green fluorescent protein, beta-glucuronidase, beta-galactosidase and luciferase. 12. Cell according to claim 11, wherein the green fluorescent protein is the monomeric superfolder green fluorescent protein (msf gfp)). 13. Cell according to any one of claims 1 to 12, wherein C. TardaugensesC. tardaugensNBRC 16725. 14. A cell population comprising a cell according to any one of claims 1 to 13. 15. A composition comprising a cell according to any one of claims 1 to 13, or a cell population according to claim 14. 16. Use of a cell according to any one of claims 1 to 13, a cell population according to claim 14, or a composition according to claim 15, for the detection of estrogens in a sample. 17. Use according to claim 16, wherein the sample is an environmental sample. 18. Use according to claim 17, wherein the environmental sample is a water sample. 19. Use according to any one of claims 16 to 18, wherein the estrogens are selected from the group consisting of estradiol (E2) and estrone (E1). 20. In vitro method to detect estrogens in a sample comprising (a) contacting the sample with a cell according to any one of claims 1 to 13, a cell population according to claim 14, or a composition according to claim 15, and (b) determine the expression of the reporter gene, wherein an expression of the reporter gene is indicative that the sample contains estrogens. 21. In vitro method according to claim 20, wherein the method comprises an intermediate step between steps (a) and (b) that comprises the addition of an inducer of the promoter of the first gene construction. 22. Method according to claim 20 or 21, wherein the estrogens are selected from the group consisting of estradiol (E2) and estrone (E1). 23. Method according to any one of claims 20 to 22, wherein the sample is an environmental sample. 24. Method according to claim 23, wherein the environmental sample is a water sample. 25. A kit comprising a cell according to any one of claims 1 to 13, a cell population according to claim 14, or a composition according to claim 15. 26. Use of a kit according to claim 25 for the detection of estrogens in a sample. 27. Use according to claim 26, wherein the estrogens are selected from the group consisting of estradiol (E2) and estrone (E1). 28. Use according to claim 26 or 27, wherein the sample is an environmental sample. 29. Use according to claim 28, wherein the environmental sample is a water sample.