Reporter gene construct sensitive to queuosine levels

Abstract

The present invention relates to a genetic construct sensitive to queuosine (Q)-tRNA levels within a cell. It is based on the regulation mechanism of the histidine (His) operon of Escherichia coli, which depends on the queuosine modification of tRNA. The construct includes a promoter, such as the promoter of the His operon (Phis), and the His leader gene (hisL). In addition to Phis and hisL, the genetic construct of the invention comprises a gene encoding for a reporter protein, wherein the gene encoding for the reporter protein lacks NAU codons (i.e., all NAU codons in the gene encoding for the reporter protein have been modified to NAC codons). In this way, the translation of the reporter gene itself is independent on Q-tRNA levels. Moreover, the genetic construct comprises a sequence with at least an 80% homology to SEQ ID NO: 1. The construct can be used as a marker for successful detection of Q-tRNA(His) within the cell and it can also be used to quantify queuosine and / or queuosine precursor in a sample.

Description

[0001] REPORTER GENE CONSTRUCT SENSITIVE TO QUEUOSINE LEVELS

[0002] DESCRIPTION

[0003] FIELD OF THE INVENTION

[0004] The present invention lies within the technical field of biotechnology. More specifically, the invention relates to a reporter gene construct sensitive to Q-tRNA levels within a cell, which may be used to measure queuosine (Q) or queuosine precursors in a sample.

[0005] BACKGROUND OF THE INVENTION

[0006] Translation of genetic information into proteins requires an efficient and accurate decoding of the messenger RNA (mRNA) by ribosomes and transfer RNAs (tRNAs). Among all RNA species, tRNAs undergo the greatest number of chemically diverse modifications. Growing evidence indicates that tRNA modifications are critical for many aspects of tRNA functions such as folding, stability and decoding, representing an additional level of gene regulation. Despite their essential role in translation, the physiological function of several tRNA modifications is not very well understood.

[0007] One such modification is queuosine. Queuosine (Q) is a nucleoside that can replace guanosine at position 34 in the anticodon of the tyrosine, histidine, asparagine, and aspartic acid tRNAs to fine tune protein translation. The codons coding for these amino acids are UAU / UAC (Tyr), CAU / CAC (His), AAU / AAC (Asn) and GAU / GAC (Asp), which are collectively represented as NAU / NAC. Q is found in Bacteria and Eukarya, although its de novo biosynthesis only occurs in Bacteria. Q biosynthesis pathway starts with five sequential modifications of GTP catalysed by the enzymes FolE, QueD, QueE, QueC and QueF to obtain the preQi precursor. This precursor is incorporated to tRNA by the tRNA guanine transglycosylase (TGT) and finally transformed into Q by QueA and QueG / QueH. Although many species can synthesize Q de novo, salvage of Q precursors also occurs. Some species use the YhhQ transporter for importing preQo and preQi precursors. Certain bacteria capture queuine (q), the Q nucleobase, and transform it into preQi. Other bacteria and eukaryotes directly replace the guanine at position 34 of the tRNAs with q by using a TGT homolog or a eukaryotic TGT (eTGT), respectively. Bacteria that cannot produce Q de novo and eukaryotes need to salvage Q precursors through the microbiome and / or nutrient sources. Given that Q modification is in the wobble anticodon position critical for codon recognition, one suggested purpose of tRNA Q-modification is modulating NAU codon translation rate. However, its physiological role remains elusive. Multiple studies conducted in eukaryotes have revealed the involvement of tRNA Q-modification in very diverse processes, including pupae maturation in Drosophila melanogaster, cell aggregation in Dictyostelium discoideum, and antioxidant defence system, hypoxia, cancer, and proliferation in mammals. In bacteria, it has been reported that tRNA Q-modification is involved in resistance to several stressors, such as heat shock, low acidic pH, UV-radiation, perchlorate, and arsenic, as well as in the regulation of the virulence of the pathogen Shigella flexneri and the nodule cell infection efficiency of Sinorhizobium meliloti. The molecular mechanism underlying these widely diverse and spread phenotypes is not well understood. Previous studies suggest that availability of Q would especially affect the translation of genes enriched in NAU codons (known as “Q-genes”), leading to a variation in the expression levels of the proteins they encode depending on the degree of Q- modification of tRNAs. In this respect, this general mechanism of regulation could be responsible for the wide variety of reported Q-related phenotypes, which may vary depending on the roles of the specific NAU codon-enriched genes in each organism. Moreover, as Q modification is fully dependent on diet or on gut microbiome in multicellular organisms, and Q controls biofilm formation and virulence in bacteria, it is thought that higher levels of Q are associated with certain pathologies related to microbial dysbiosis, such as chronic intestinal diseases.

[0008] Therefore, there is an obvious interest in the field for detecting and controlling queuosine levels, not only to shed light on the physiological role of tRNA Q-modifications, but also to help treat bacterial infections and other complex pathologies related to the microbiome. In these regards, there is currently no specific technique for the measurement of queuosine or its precursors. Theoretically, it would be possible to make these measurements using the general techniques of chromatography or mass spectrometry. However, there is no standardised protocol that would allow measuring queuosine using these techniques, which in addition are expensive and require specialised personnel due to their high complexity.

[0009] DESCRIPTION OF THE FIGURES

[0010] Figure 1 shows (A) the nucleotide sequence of the essential elements of the queuosine responding portion of the Q-reporter gene construct of the invention and (B) the nucleotide sequence of a preferred embodiment of the promoter and the queuosine responding portion of the Q-reporter gene construct of the invention. Both panels A and B additionally show the position and a brief description of each element within the queuosine responding portion of the construct.

[0011] Figure 2 shows a preferred embodiment of the Q-reporter gene construct of the invention, Phis-hisL- hisG egfp(NAC). Panels show (A) a schematic representation of the gene construct and (B) the nucleotide sequence of the gene construct, together with the position and a brief description of each element within the construct.

[0012] Figure 3 shows a scheme of the queuosine (Q) biosynthetic pathway.

[0013] Figure 4 shows the linear fittings of preQi concentration vs corrected fluorescence (signal to noise) measurements of M63 medium samples (control; represented as a scatter plot with circular markers) and of the biological samples (10 nM preQi in M63 medium; represented as a scatter plot with squared markers). The fluorescence measured in the presence of a high concentration of extracellular preQi (250 nM) is considered as noise, as tRNA would be Q-modified at a maximum degree in these conditions.

[0014] Figure 5 shows the preQi concentration obtained for a sample containing M63 medium alone, M63 medium plus 10 nM of preQi, culture supernatant of an E. coli DH10B queF mutant strain that does not produce preQi (E. coli DH10B AqueF), and the same supernatant plus 10 nM of preQi, incubated in M63 at 37 °C and 200 rpm for 16 h.

[0015] Figure 6 shows the preQi concentration measured in supernatants (SB) of different E. coli strains (ST131 , DH10B), in Staphylococcus aureus and, in sera from foetal bovine, mouse and human, human urine, and in filtered LB medium.

[0016] Figure 7 shows a quantitative relationship between preQi concentration and corrected fluorescence of E. coli DH10B AqueF PhiS-hisL-AhisG egfp(NAC) strain (A) and linearised (B).

[0017] Figure 8 shows linearized quantitative relationship between extracellular preQi concentration and fluorescence of E. coli DH10B AqueF PhiS-hisL-AhisG egfp(NAC) strain incubated with different concentrations of indole. Figure 9 shows the calculated values of Vmax(app) and KM(aPP) when E. coli DH10B AqueF Pms- hisL-AhisG egfp(NAC) strain was incubated with different concentrations of indole.

[0018] Figure 10 shows linearized quantitative relationship between extracellular preQi concentration and fluorescence of E. coli DH10B AqueF PhiS-hisL-AhisG egfp(NAC) strain transformed with plasmid 84 or the two genes harboured by plasmid 84 cloned individually.

[0019] Figure 11 shows the calculated values of Vmax(aPP) and KM(aPP) when E. coli DH10B AqueF Phis-hisL-AhisG egfp(NAC) strain was transformed with plasmid 84 or the two genes harboured by plasmid 84 cloned individually.

[0020] DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention provides a novel artificial gene construction sensitive to Q-tRNA levels in a cell, that can be used to measure queuosine and / or queuosine precursors in a sample. The inventors have originally developed a construction that comprises EGFP as a reporter gene under the control of the histidine (His) operon of Escherichia coli. This construct is sensitive to queuosine because of the regulatory mechanisms of the histidine operon. However, all NAU codons in the egfp reporter gene have been modified to NAC codons such that expression of EGFP itself is independent of queuosine levels. The modified EGFP reporter protein is termed EGFP(NAC). The invention allows to correlate EGFP(NAC) fluorescence levels with the levels of queuosine or a queuosine precursor in a sample, and the technology makes it possible to measure queuosine at very low concentrations in the nanomolar range. This invention represents a cheap, simple and straightforward tool, and does not require specialised technical personnel or complex or expensive machinery. Instead, it can be used anywhere with minimal laboratory equipment.

[0022] Thus, in a first aspect, the invention refers to a genetic construct, wherein the genetic construct comprises: a promoter; a histidine leader gene (hisEy histidine operon regulatory elements between hisL and the following gene of the histidine operon (hisGy a gene encoding for a reporter protein replacing hisG, wherein the gene encoding for the reporter protein lacks NAU codons; and a transcriptional terminator, wherein the genetic construct comprises a sequence with at least an 80% homology to SEQ ID NO: 1.

[0023] The construct of the invention is based on the regulation mechanism of the histidine (His) operon of Escherichia coli, which involves the promoter of the His operon (PA / S), the His leader gene (hisL.) and the histidine operon regulatory elements between hisL and the following gene of the histidine operon (hlsG). The histidine operon regulatory elements are elements A, B, C, D, E and F, capable of forming alternative secondary structures for the histidine leader mRNA. The histidine leader gene (hisL) is composed of 17 codons, seven of them encoding for His. Four out of these seven His codons are CAU, and the other three are CAC. When the His levels are high, tRNA(His) are charged with His, the translation of the seven His codons is fast, and this causes the formation of a transcription termination fork called E-F. On the other hand, when the His levels are low, tRNA(His) is poorly charged with His, the translation of the seven His codons is slow, and this prevents the formation of the transcription termination fork E-F (a stem-loop structure formed with regulatory elements E and F), allowing for the histidine operon transcription (see Johnson et al., 1980. Model for regulation of the histidine operon of Salmonella. Proc. Natl. Acad. Sci. USA, 77, 508-512; Carlomagno et al., 1988. Structure and function of the Salmonella typhimurium and Escherichia coli K-12 histidine operons. J. Mol. Biol., 203, 585-606). It is known that the presence of queuosine (Q) in certain tRNAs alters the translation efficiency of some codons. More precisely, it has been demonstrated that Q enhances codon translation of NAU codons so that when Q is absent NAU codons are not well translated. This mechanism regulates the translation levels of genes containing a high percentage of NAU codons. Therefore, as hisL contains a high percentage of CAU codons, its translation depends on the levels of Q-tRNA(His). It follows that the genetic construct of the invention is sensitive to queuosine (Q)-tRNA levels within the cell.

[0024] As explained above, the genetic construct comprises a sequence with at least an 80% homology to SEQ ID NO: 1. That is, the genetic construct comprises in a portion of its entire sequence, a sequence with at least an 80% homology to SEQ ID NO: 1. This sequence corresponds to the queuosine responding portion of the Q-reporter gene construct of the invention. In particular embodiments, the genetic construct of the invention comprises a sequence with at least an 80% homology to SEQ ID NO:1. In particular embodiments, the genetic construct of the invention comprises a sequence with at least an 80%, 85%, 90%, 95%, 98%, 99% or with 100% homology to SEQ ID NO:1. In a particular embodiment, the genetic construct of the invention comprises the sequence of SEQ ID NO:1 . As used herein, the term "percent homology" is used to mean "sequence similarity”, in the sense of the percentage of identical residues (percent identity). In embodiments of the invention, the queuosine responding portion of the Q-reporter gene construct of the invention is derived from the histidine operon of Escherichia coli, of Salmonella typhimurium, of Klebsiella pneumoniae, or of Yersinia pestis.

[0025] In the context of the invention the term “promoter” refers to a sequence of nucleotides that can be recognised by RNA polymerases, which can then initiate gene transcription. In the context of the instant invention, any promoter capable of operating in the host organism where the gene construct is inserted may be used. In certain embodiments, the promoter of the genetic construct of the invention is the promoter of the histidine operon (P S) of Escherichia coli, of Salmonella typhimurium, of Klebsiella pneumoniae, or of Yersinia pestis.

[0026] In a particular embodiment of the invention, the genetic construct comprises: the promoter of the histidine operon (Pws); the histidine leader gene (hisEy the histidine operon regulatory elements between hisL and the following gene of the histidine operon ( / 7 / sG); a gene encoding for a reporter protein replacing hisG, wherein the gene encoding for the reporter protein lacks NAU codons; and a transcriptional terminator, wherein the genetic construct comprises a sequence with at least an 80% homology to SEQ ID NO: 2. In this embodiment, the genetic construct comprises a sequence with at least an 80% homology to SEQ ID NO: 2. That is, the genetic construct comprises, in a portion of its entire sequence, a sequence with at least an 80% homology to SEQ ID NO: 2. This sequence corresponds to the promoter of the histidine operon (P S) and the queuosine responding portion of the Q-reporter gene construct of the invention. In particular embodiments, the genetic construct of the invention comprises a sequence with at least an 80% homology to SEQ ID NO:2. In particular embodiments, the genetic construct of the invention comprises a sequence with at least an 80%, 85%, 90%, 95%, 98%, 99% or with 100% homology to SEQ ID NO:2. In a particular embodiment, the genetic construct of the invention comprises the sequence of SEQ ID NO:2.

[0027] In the context of the invention, the term “gene encoding for a reporter protein”, or “reporter gene”, is a gene that can be attached to the regulatory sequence of the histidine operon in the construct of the invention such that, when introduced into a suitable host cell, expression of the said reporter gene can confer the host cell certain characteristics that are easily identified and / or measured. Detection of expression of the reporter gene may be carried out by any suitable method, according to the specific gene. Suitable methods may include enzymatic assays, histochemistry, fluorescence, microscopy, spectrophotometry, bioluminescence and the like. The gene product of the reporter gene may be, but is not limited to, p-galactosidase, chloramphenicol acetyltransferase, green fluorescent protein, red fluorescent protein, luciferase, etc. As indicated above, the gene encoding for the reporter protein lacks NAU codons. In an embodiment of the invention, the gene encoding for the reporter protein lacks NAU codons when said gene contains no codons encoding for tyrosine (Tyr), histidine (His), Asparagine (Asn) or Aspartic Acid (Asp), or when said gene only contains naturally occurring NAC codons encoding for tyrosine (Tyr), histidine (His), Asparagine (Asn) or Aspartic Acid (Asp). In another embodiment of the invention, the gene encoding for the reporter protein lacks NAU codons when all naturally occurring NAU codons in said gene encoding for the reporter protein have been modified to NAC codons. In both types of embodiments where the gene encoding for the reporter protein lacks NAU codons, translation of the reporter gene itself is not depending on Q-tRNA levels. In particular embodiments of the invention, the reporter protein is selected from the group consisting of NAC-p-galactosidase, NAC-chloramphenicol acetyltransferase, NAC- luciferase, RFP (NAC), GFP(NAC), EGFP(NAC), or combinations thereof. In a particular embodiment of the invention, the reporter protein is the Enhanced Green Fluoresce Protein (EGFP). The gene encoding for EGFP is modified to change all the NAU codons by NAC codons [EGFP(NAC)], thus translation of EGFP itself does not depend on Q-tRNA levels. However, EGFP(NAC) in the construct of the invention is under the control of the histidine operon. Therefore, the levels of EGFP fluorescence can be correlated with the levels of Q- tRNA(His).

[0028] In the context of the instant invention, any transcription terminator capable of operating in the host organism where the gene construct is inserted may be used. Non limiting examples are the transcriptional terminator TO from phage lambda, the transcriptional terminator T7, or the transcriptional region of the rrnB gene from E. coli. Thus, in embodiments of the invention, the transcriptional terminator may be selected from the group consisting of the transcriptional terminator TO from phage lambda, the transcriptional terminator T7, or the transcriptional region of the rrnB gene from E. coli. In a particular embodiment, the transcriptional terminator is the transcriptional terminator TO from phage lambda.

[0029] In a particular embodiment, the genetic construct of the invention has a sequence with at least an 80% homology to SEQ ID NO:3. In particular embodiments, the genetic construct of the invention has a sequence with at least an 80%, 85%, 90%, 95%, 98%, 99% or with 100% homology to SEQ ID NO:3. In a particular embodiment, the genetic construct of the invention has the sequence of SEQ ID NO:3 (i.e., the genetic construct of the invention is SEQ ID NO: 3).

[0030] In another aspect, the invention relates to a host cell, wherein the host cell can only use exogenous queuosine or an exogenous queuosine precursor selected from the group consisting of 7-cyano-7-deazaguanine (preQo), 7-aminomethyl-7-deazaguanine (preQi), and queuine (q), and wherein the host cell comprises a genetic construct according to the instant invention. This limitation of the host cell implies that the cell will necessarily produce Q-tRNA using the exogenous queuosine or queuosine precursors. In a particular embodiment, the host cell is a AfolE, AqueD, AqueE y AqueC mutant. In a particular embodiment, the host cell is a AqueA y AqueG / H mutant. In a particular embodiment, the host cell is a AqueF mutant; that is, a host cell wherein the queF gene was deleted to prevent the biosynthesis de novo of preQi. This way, all the Q-tRNA is formed from the exogenous preQi of a certain sample from which the concentration of preQi wants to be measured. The gene queF codifies for the protein NADPH-dependent 7-cyano-7- deazaguanine reductase, wherein the protein catalyses the NADPH-dependent reduction of 7-cyano-7-deazaguanine (preQo) to 7-aminomethyl-7-deazaguanine (preQi), a late step in the queuosine pathway. Its UniProt entry in respect of organism Bacillus subtilis (strain 168) is 031678. In a particular embodiment, the host cell is a cell wherein the genes queL (queuine lyase), queK (queuosine hydroxylase) and the transporter yhhQCt have been cloned. The yhhQCt transporter is a homologue of E. coli yhhQ, but with the difference that yhhQCt can import free queuine and queuosine. QueL and QueK are two enzymes that catalyse the transformation of free queuosine into queuine (QueK), and queuine into preQi (QueL). With these mutations, the host cell can use free Q or q to produce Q-tRNA. Suitable cells to be used as a host cell in the context of the invention irrespective of the specific mutation or mutations may include, without limitation, bacterial cells. In particular embodiments, the host cell is a bacterium of a genus selected from the group consisting of Escherichia, Bacillus, Pseudomonas, Streptococcus, Streptomyces, Staphylococcus and Lactobacillus. In a particular embodiment, the host cell is a bacterium of a species selected from the group consisting of Escherichia coli, and Bacillus subtilis. In a particular embodiment, the host cell is a AqueF mutant strain of Escherichia coli or a AqueF mutant strain of Bacillus subtilis. In another aspect, the invention refers to the use of a genetic construct according to the invention, or of a host cell according to the invention, for measuring the degree of queuosine modification of tRNA in a cell, for detecting queuosine and / or a queuosine precursor in a sample, preferably in a biological sample, or for measuring queuosine and / or a queuosine precursor in a sample, preferably in a biological sample.

[0031] In the context of the invention, the degree of queuosine modification may be expressed as a relative value with respect to a measurement used as a reference. Once the reference value is established, the degree of queuosine modification can be compared with this reference value, and thus be assigned a level of “increased” or “decreased”. For example, an increase in the degree of queuosine modification above the reference value of at least 1.1 -fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or more compared with the reference value is considered as an “increased” degree of queuosine modification. On the other hand, a decrease in the degree of queuosine modification below the reference value of at least 0.9-fold, 0.8-fold, 0.7-fold, 0.6-fold, 0.5-fold, 0.2-fold, 0.1 -fold, 0.05-fold, 0.025-fold, 0.02-fold, 0.01 -fold, 0.005-fold or less compared with the reference value is considered as a “decreased” degree of queuosine modification. "Reference value", as used herein in the context of the invention, refers to a laboratory value used as a reference for values / data obtained by laboratory examination of representative samples, such as a sample from a standard environment or a sample from a subject or subjects. The reference value or reference level can be an absolute value; a relative value; a value that has an upper and / or lower limit; a range of values; an average value; a median value, a mean value, or a value as compared to a particular control or baseline value. A reference value can be based on an individual sample value, such as for example, a value obtained from a sample from the subject or from the environment being tested, but at an earlier point in time or from a noncancerous tissue. The reference value can be based on a large number of samples or based on a pool of samples including or excluding the sample to be tested. In the context of the invention, detecting queuosine and / or a queuosine precursor in a sample necessarily results in a positive or negative outcome (i.e., a yes or no result), wherein a positive result can be determined when the queuosine concentration is above a certain threshold value. In the context of the invention, measuring queuosine and / or a queuosine precursor in a sample results in a quantifiable result, typically expressed as a concentration value.

[0032] As used herein, “sample” refers to a limited quantity of a larger amount of a certain object, which maintains the characteristics and is representative of said object, that is taken for analysis, testing or investigation. “Biological sample” means biological material isolated from a subject (i.e., from an animal or from a human subject) or from a biological environment, such as cellular extracts from bacteria, eukaryotes or archaea; cell culture supernatants; water samples (taken from rivers, lakes, ponds, salt flats, etc.), soils (soil, sediments, etc.), food products, biofilms and / or communities of microorganisms associated with biotic or abiotic surfaces. The biological sample may contain any biological material suitable for detecting and or for measuring queuosine and / or a queuosine precursor. The sample can be isolated from any suitable biological tissue or fluid such as, for example, blood, plasma, serum, sputum, bronchoalveolar lavage, urine or cerebrospinal fluid (CSF). In the case of solid tissue samples, it would be necessary to pre-treat the sample to liquefy it, resuspend the sample in an appropriate buffer and treat the sample under a mechanical or sonication procedure.

[0033] In another aspect, the invention refers to the use of a genetic construct according to the invention, or of a host cell according to the invention, for identifying whether a gene a protein or a chemical compound is an inhibitor or an activator of the biosynthesis of Q-tRNA. The term "inhibitor" as used herein, refers to a compound which is capable of reducing, in a detectable manner, the biosynthesis of Q-tRNA. The biosynthesis of Q-tRNA is considered reduced when detectable Q-tRNA levels decrease with respect to the reference value by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, or by at least 100% (i.e., absent). The term "activator" as used herein, refers to a compound which is capable of increasing, in a detectable manner, the biosynthesis of Q-tRNA. The biosynthesis of Q-tRNA is considered increased when detectable Q-tRNA levels increase with respect to the reference value by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50%, by at least 55%, by at least 60%, by at least 65%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 100%, or more. The reference value refers to the level of detectable Q-tRNA levels in a control sample or in a control experiment.

[0034] In another aspect, the invention refers to a method for measuring the concentration of queuosine and / or a queuosine precursor in a sample, preferably in a biological sample, wherein the method comprises: a) incubating calibration standard dilutions of queuosine and / or queuosine precursor in the presence of a host cell according to the invention in a cell culture medium; b) incubating a sample dilution in the presence of a host cell according to the invention in a cell culture medium; c) quantifying the signal associated with the reporter protein in the host cell for the calibration standard dilutions and calculating a calibration curve; d) quantifying the signal associated with the reporter protein in the host cell for the sample dilution and determining the concentration of queuosine and / or of the queuosine precursor in the sample in relation to the calibration curve of step (c).

[0035] The host cells according to the invention may be grown in any suitable cell culture medium, which is appropriate for the correct growth of the host cell (that is, media that contains the essential elements for the host organism), and which should be readily apparent to a person skilled in the art. Suitable cell culture mediums in the context of the instant invention are, without limitation, M63 medium, Luria-Bertani (LB), Tryptone Broth, TY broth, Terrific broth, M9 medium, Msgg medium (for Bacillus subtilis and others) or MM medium (for Streptomyces sp. and others), and the like. Ideally, minimal media are preferable, which do not contain any component that may contain queuosine or any of its precursors.

[0036] In another aspect, the invention refers to a method for identifying whether a chemical compound is an inhibitor or an activator of the biosynthesis of Q-tRNA, wherein the method comprises: a) incubating calibration standard dilutions of queuosine and / or queuosine precursor in the presence of a host cell according to the invention in a cell culture medium, wherein the cell culture medium is a blank control medium; b) incubating calibration standard dilutions of queuosine and / or queuosine precursor in the presence of a host cell according to the invention in a cell culture medium, wherein the cell culture medium comprises a library of chemical compounds; c) quantifying the signal associated with the reporter protein in the host cell for the calibration standard dilutions in blank control medium and calculating a first calibration curve; d) quantifying the signal associated with the reporter protein in the host cell for the library of chemical compounds and calculating a second calibration curve; e) comparing the first calibration curve obtained in step (c) with the second calibration curve obtained in step (d), wherein significant variation between the first and second calibration curves is indicative that the chemical compound is an inhibitor or an activator of the biosynthesis of Q-tRNA. In another aspect, the invention refers to a method for identifying whether a gene or a gene product is an inhibitor or an activator of the biosynthesis of Q-tRNA, wherein the method comprises: a) incubating calibration standard dilutions of queuosine and / or queuosine precursor in the presence of a host cell according to the invention in a cell culture medium; b) incubating calibration standard dilutions of queuosine and / or queuosine precursor in the presence of a host cell according to the invention in a cell culture medium, wherein the host cell has been transfected with a metagenomic library; c) quantifying the signal associated with the reporter protein in the host cell for the calibration standard dilutions in blank control medium and calculating a first calibration curve; d) quantifying the signal associated with the reporter protein in the host cell for the library of chemical compounds and calculating a second calibration curve; e) comparing the first calibration curve obtained in step (c) with the second calibration curve obtained in step (d), wherein significant variation between the first and second calibration curves is indicative that the chemical compound is an inhibitor or an activator of the biosynthesis of Q-tRNA.

[0037] In the context of the instant invention, the expression “significant variation” refers to any statistically significant variation (i.e., values are significantly increased, significantly decreased, or significantly not altered after an appropriate statistical analysis is carried out). In a particular embodiment, any statistically significant variation will depend on the standard deviation (S.D.) of the intersect with the "y"-axis and the slope of the calibration curves: if said parameters of the calibration curves of the test sample are statistically different than those of the control sample after repeating several iterations of the experiment, it would be considered that there is a significant difference, and that the chemical compound, gene or gene product could be an inhibitor or activator of the biosynthesis of Q-tRNA. In particular embodiments, 3, 5, 10, 15, 20, 30, 40, 50, 100, or more iterations are needed, in order to determine statistical significance. As the skilled person would readily understand, three (3) iterations can be typically sufficient to obtain statistically significant data. However, if the differences between conditions are small, a greater number of iterations (5 - 10, or even more) may be necessary. The values of the "y"-axis intersect and the slope of the calibration curves are used to calculate enzymatic parameters Vmax(app) and KM(aPP), that is, apparent maximum velocity and apparent Michaelis constant for the complete reaction of biosynthesis of Q-tRNA from preQi precursor, assuming that all enzymes involved in this process stablish an ordered sequential reaction following Michaelis-Menten kinetics. Thus, in particular embodiments of the invention, the experiment is repeated at least 3 times and the values of the mean and associated standard deviation (SD) for the y-axis intersect and slope parameters are calculated. Next, the unpaired t test (or ANOVA if more than two conditions are compared, or any other appropriate statistical analysis) is applied to study whether the parameters are significantly different between conditions (p-value < 0.05). Other non-parametric methods (without assuming normality) such as the Mann-Whitney method, or methods that do not assume equality of variance, such as the unpaired t test with Welch's correction, can also be used.

[0038] Accordingly, the uses and methods of the invention can be used to determine whether a chemical compound, gene or gene product is an inhibitor of activator of the biosynthesis of Q-tRNA, wherein:

[0039] • A competitive inhibitor increases KM(aPP), while Vmax(aPP) is not altered.

[0040] • A non-competitive inhibitor decreases Vmax(apP), while KM(apP) is not altered.

[0041] • A mixed inhibitor decreases Vmax(apP) and increases or decreases KM(3PP).

[0042] • An activator increases Vmax(apP) and / or decreases KM(3PP).

[0043] In another aspect, the invention refers to a kit or assay device comprising reagents adequate for detecting and / or measuring queuosine and / or a queuosine precursor in a sample, wherein the kit or assay device comprises a genetic construct according to the invention, or of a host cell according to the invention, and wherein said reagents comprise at least 10% of the reagents present in the kit. In the context of the present invention, "kit" or "assay device" is understood as a product or device containing the different reagents necessary for carrying out the methods of the invention packed in a manner that allows for their transport and storage. Materials suitable for packing the components of the kit include glass, plastic, paper, and the like. The kits of the invention can contain instructions for the appropriate use of the components within the kit. Said instructions can be in the form of printed material or in the form of an electronic support capable of storing instructions such that they can be read by a subject, such as electronic storage media, internet repositories and the like. In a preferred embodiment, the reagents adequate for detecting and / or measuring queuosine and / or a queuosine precursor in a sample comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% of the total amount of reagents forming the kit. Finally, the invention relates to the following additional aspects:

[0044] 1. A genetic construct, wherein the genetic construct comprises: the promoter of the histidine operon (Pws); the histidine leader gene (hisL)', the His operon regulatory elements between hisL and the following gene of the His operon (hisG , a gene encoding for a reporter protein replacing hisG, and wherein all NAU codons in the gene encoding for the reporter protein have been modified to NAC codons; a transcriptional terminator.

[0045] 2. The genetic construct according to aspect 1, wherein the reporter protein is EGFP(NAC).

[0046] 3. The genetic construct according to aspect 2, wherein the construct has a sequence with an 80% homology to SEQ ID NO:1.

[0047] 4. A host cell, wherein the host cell can only use exogenous queuosine or an exogenous queuosine precursor selected from the group consisting of preQo, preQi, and q, and wherein the host cell comprises a genetic construct according to any one of aspects 1 to 3.

[0048] 5. The host cell according to aspect 4, wherein the host cell is a AqueF mutant.

[0049] 6. The host cell according to aspect 5, wherein the host cell is a AqueF mutant strain of Escherichia coli or a AqueF mutant strain of Bacillus subtilis.

[0050] 7. Use of a genetic construct according to any one of aspects 1 to 3, or of a host cell according to any one of aspects 4 to 6, for measuring the degree of queuosine modification of tRNA in a cell, for detecting queuosine and / or a queuosine precursor in a sample, preferably in a biological sample, or for measuring queuosine and / or a queuosine precursor in a sample, preferably in a biological sample.

[0051] 8. Use of a genetic construct according to any one of aspects 1 to 3, or of a host cell according to any one of aspects 4 to 6, for identifying whether a gene, a protein or a chemical compound is an inhibitor or an activator of the biosynthesis of Q-tRNA. 9. A method for measuring the concentration of queuosine and / or a queuosine precursor in a sample, preferably in a biological sample, wherein the method comprises: a) incubating calibration standard dilutions of queuosine and / or queuosine precursor in the presence of a host cell according to any one of aspects 4 to 6 in a cell culture medium; b) incubating a sample dilution in the presence of a host cell according to any one of aspects 4 to 6 in a cell culture medium; c) quantifying the signal associated with the reporter protein in the host cell for the calibration standard dilutions and calculating a calibration curve; d) quantifying the signal associated with the reporter protein in the host cell for the sample dilution and determining the concentration of queuosine and / or of the queuosine precursor in the sample in relation to the calibration curve of step (c).

[0052] 10. A method for identifying whether a chemical compound is an inhibitor or an activator of the biosynthesis of Q-tRNA, wherein the method comprises: a) incubating calibration standard dilutions of queuosine and / or queuosine precursor in the presence of a host cell according to any one of aspects 4 to 6 in a cell culture medium, wherein the cell culture medium is a blank control medium; b) incubating calibration standard dilutions of queuosine and / or queuosine precursor in the presence of a host cell according to any one of aspects 4 to 6 in a cell culture medium, wherein the cell culture medium comprises a library of chemical compounds; c) quantifying the signal associated with the reporter protein in the host cell for the calibration standard dilutions in blank control medium and calculating a first calibration curve; d) quantifying the signal associated with the reporter protein in the host cell for the library of chemical compounds and calculating a second calibration curve; e) comparing the first calibration curve obtained in step (c) with the second calibration curve obtained in step (d), wherein significant variation between the first and second calibration curves is indicative that the chemical compound is an inhibitor or an activator of the biosynthesis of Q-tRNA.

[0053] 11 . A method for identifying whether a gene or a gene product is an inhibitor or an activator of the biosynthesis of Q-tRNA, wherein the method comprises: a) incubating calibration standard dilutions of queuosine and / or queuosine precursor in the presence of a host cell according to any one of aspects 4 to 6 in a cell culture medium; b) incubating calibration standard dilutions of queuosine and / or queuosine precursor in the presence of a host cell according to any one of aspects 4 to 6 in a cell culture medium, wherein the host cell has been transformed or transfected with a metagenomic library; c) quantifying the signal associated with the reporter protein in the host cell for the calibration standard dilutions in blank control medium and calculating a first calibration curve; d) quantifying the signal associated with the reporter protein in the host cell for the library of chemical compounds and calculating a second calibration curve; e) comparing the first calibration curve obtained in step (c) with the second calibration curve obtained in step (d), wherein significant variation between the first and second calibration curves is indicative that the chemical compound is an inhibitor or an activator of the biosynthesis of Q-tRNA.

[0054] 12. A kit or assay device comprising reagents adequate for detecting and / or measuring queuosine and / or a queuosine precursor in a sample, wherein the kit comprises a genetic construct according to any one of aspects 1 to 3, or of a host cell according to any one of aspects 4 to 6, and wherein said reagents comprise at least 10% of the reagents present in the kit.

[0055] All the terms and embodiments described herein are equally applicable to all aspects of the invention. It should be noted that, as used in the specification and in the appended claims, the singular forms "a", "an", and "the" include their plural referents unless the context clearly indicates otherwise. Similarly, the term "comprises" or "comprising" as used herein also describes "consists of" or "consisting of" in accordance with generally accepted patent practice.

[0056] EXAMPLES

[0057] The following invention is hereby described by way of the following examples, which are to be construed as merely illustrative and not limitative of the scope of the invention.

[0058] Example 1 : Queuosine reporter gene construction While carrying out research on the biological function of queuosine in bacteria, the inventors observed an activation of the histidine (His) operon in a bacterial strain of Escherichia coli uncapable of producing queuosine de novo. They concluded that the regulatory region of the histidine operon (Figure 1A) is sensitive to queuosine levels within the cell. In view of these results, the inventors decided to fuse the histidine operon of Escherichia coli (Figure 1 B) to a reporter gene, where the construct takes advantage of the regulation mechanism of the histidine operon. This includes a promoter (P S) and a leader gene, hisL. In particular, the inventors designed a DNA construction in which P^ / s, together with hisL, were followed by a reporter gene encoding for the Enhanced Green Fluorescence Protein (EGFP), such that the levels of EGFP fluorescence could be correlated with the levels of Q-tRNA(His). The reporter gene replaces the second gene of the histidine operon, hisG. The gene encoding for EGFP was modified to replace all the NAU codons with NAC codons [EGFP(NAC)], so that EGFP translation was not dependent on Q-tRNA levels. Therefore, the invention consists of a construction that comprises the histidine operon regulatory sequence (P / )fe+ hisL) followed by a gene encoding for a reporter protein (EGFP) in which all NAU codons are replaced by NAC codons (Figure 2A, Figure 2B, and SEQ ID NO: 2).

[0059] The inventors also introduced this synthetic construct in a cell for measuring Q-tRNA levels. Escherichia coli DH10B was used for these experiments. The host bacterium was modified such that Q can only be produced from a certain precursor. Seven enzymes are involved in the biosynthesis of Q in bacteria: QueD, QueE and QueC form the precursor preQo, which is then converted into the precursor preQi by QueF. Then, Tgt catalyzes the preQi replacing of the guanine located at position 34 in the tRNAs of His, Asp, Asn and Tyr (NAC / NAU codons) to form preQi-tRNA. Finally, QueA and QueG transform preQi-tRNA into Q-tRNA. In addition, E. coli can import preQo and preQi using YhhQ transporter. Other bacteria and eukaryotes can also import queuine, the Q nucleobase, to form Q (Figure 3). The objective of the invention was also to develop a reporter strain for preQi. For this reason, it was necessary to ensure that all of the Q-tRNA was formed from the exogenous preQi of a certain sample, in order to measure preQi concentration is said sample. For that purpose, queF gene was deleted, preventing the biosynthesis de novo of preQi. Thus, the reporter strain is E. coli DH10B AqueF Pms-hisL-AhisG egfp(NAC).

[0060] Example 2: preQi quantification with E. coli DH10B AqueF hisL- hisG::egfp(NAC).

[0061] The inventors devised a three-day protocol in order to quantify concentration of queuosine precursor preQi using the experimental tools described in Example 1 above.

[0062] On the first day of this procedure, 5 ml of LB medium were inoculated with the reporter strain E. coli DH10B AqueF PhiS-hisL-AhisG egfp(NAC) and incubated at 37 °C and 200 rpm until exponential phase (~8h). Then, 2 ml of the cell culture were centrifuged at 13,000 rpm for 2 min. The supernatant was decanted and discarded, and the cell pellet was resuspended in 1 ml of M63 medium supplemented with Ampicillin (Amp). Finally, the cell pellet was diluted 1 / 100 in 5 ml of M63-Amp medium and incubated at 37 °C 200 rpm overnight.

[0063] The following day, samples and preQi standards were prepared as follows:

[0064] • preQi standard preparation: To prepare preQi standards, a 25-pl aliquot of 10 mM preQi is diluted to 25 pM with M63 medium. Then, a serial dilution was performed to prepare the following preQi standard concentration (pM) in M63 medium: 10, 5, 2.5, 1.5, 1.25, 1 , 0.75, 0.5. 0.25, 0. preQi standards 0 to 1.5, and 25 pM were used for test, being finally diluted 1 / 100; that is, 0-15 nM and 250 nM.

[0065] • Sample preparation: samples from which the concentration of preQi wanted to be measured were prepared using M63 medium for dilution. The dilution factor of the sample should be within the optimal measure range (5-15 nM). The dilution factor may be optimised using the “Dilution factor test”, described in the following paragraph. Samples may need to be pre-treated, as per the “Pre-treatments” section described in the following paragraph.

[0066] • Dilution factor test: In order to determine the order of magnitude of preQi concentration in the sample of interest, a previous test is needed to set the optimal dilution factor for the sample: (1) Label three 2-mL tubes per each dilution of the sample of interest; (2) Prepare M63 with 2-fold Amp and reporter cells at an OD600 of 0.04. Add 0.3 mL in each tube; (3) Prepare 0.9 mL of 2-fold serial dilutions of the sample, as many dilutions as necessary; (4) Add 0.3 mL of each sample dilution to their corresponding three tubes, notice that sample dilution will be multiplied by two in this step; (5) Prepare standards as described above; (6) Perform the normal protocol with M63 only as a blank experiment; (7) Add 6 pL of standards 25 pM, 1 pM, or 0 pM to the three tubes of each dilution sample; (8) Incubate tubes inside the racks at 37 °C 200 rpm overnight.

[0067] • Pre-treatments: In some case, it will be necessary to pre-treat the sample, for instance, because the sample is not sterile, or because reporter strain could not grow in the sample of interest unless some component is removed. There are some strategies that can be used: (1) If the sample is not sterile, it may be filtered. Do not use nitrocellulose filters if the sample has DMSO (for example, for adding a known concentration of preQi from standards to check something), because DMSO reacts with the material of some filters, altering the measurement. (2) Treatment with heat: incubation at 100 °C during 30 min does not affect preQi concentration. If, for some reason, DMSO has been added to the sample before heating, do not heat the sample in a 15-mL of 50-mL tube, heat it in 1.5-, 2-mL tubes. Some plastics can react with DMSO and measurement is affected. (3) Components of a complex sample can be separated by size using a Microcon centrifugal filter, or similar. (4) It is possible to perform standard protein precipitation with trichloroacetic acid (TCA) or hydrochloric acid (HCI), preQi is not affected. Adjust pH and salinity before inoculating the reporter strain in the sample. (5) When measuring preQi in biological samples, such as bacterial cultures, serum, urine, etc., it is better to pre-treat the sample with some of the pre-treatments described above for a better growth of the reporter strain. Usually, using 3 KDa Microcon is a very good choice.

[0068] On this same day, the cell suspension and cell cultures are prepared as follows:

[0069] • Cell suspension: 5-ml culture inoculated overnight was centrifuged at 6,000 rpm 5 min. The supernatant was decanted and discarded, and the cell pellet was resuspended in 1 ml of M63 medium supplemented with Amp, and its optical density at 600 nm (OD600) was measured. OD600 should be around 6-7. The amount of cell suspension needed to reach a final OD600 of 0.02 when added to 16-ml diluted samples was calculated.

[0070] • Cell cultures: 16 ml of a diluted sample (as per sample preparation, above) were mixed with 16 pl of Amp and the volume of cell suspension such that the OD600 was 0.02. Then, 1.8 ml of said mix were added to one of the tubes of each triplicate together with 18 pl of each preQi standard, and the final volume was split into the three tubes of each triplicate (0.6 ml per tube). The tubes were incubated at 37 °C 200 rpm overnight.

[0071] On the final day, a 96-well plate was filled with 200 pl of the content of each tube. Duplicates for each tube were done. OD600 and fluorescence intensity (excitation: A=470 nm, emission: A=530 nm) were measured. Optimal gain should be set, if possible. If all fluorescence values are similar and high, probably the sample contains too much preQi (>30 nM) and further dilutions are needed.

[0072] From these data, the concentration of preQi was calculated as follows: (1) The OD600 noise was subtracted from OD600 measurements. (2) Fluorescence intensity was divided by corrected OD600. (3) Each technical duplicate was averaged. (4) The technical averages were divided by the average of the three tubes of 250 nM preQi standard (Signal to noise; S / N). At 250 nM, all tRNAs will be Q-modified, so fluorescence must be minimal (noise). In some cases, a higher concentration of preQi might be required to reach minimal S / N; for example, with the presence of an inhibitor. (5) S / N values were transformed with natural logarithm (Ln). (6) The relationship between the preQi standards concentrations and triplicates of In (S / N) was modelled by linear regression, fitting a linear equation to said data (see Figure 4). Values of S / N lower than 0.45-0.5 were not used and outliers were removed. (7) The equation of the line, the slope, and the interception with y axis for control and sample were calculated. With software like GraphPad Prism, the standard deviation (S.D.) of y-axis interception value may be calculated.

[0073] In order to calculate the concentration of queuosine precursor preQi, the sample tendency line (linear fit to the scatter plot with circular markers, Figure 4) is extrapolated to calculate the preQi concentration necessary to reach the y-intercept of the control tendency line (linear fit to the scatter plot with squared markers, Figure 4). For that, sample y-intercept is subtracted from control y-intercept. Then, it is divided by the slope of the sample tendency line. The absolute value of the result is preQi concentration. [preQi] value is finally multiplied by dilution factor of the sample. Control data refers to M63 medium samples.

[0074] Table 1 : Equations for calculating [preQ1] concentration.

[0075] As control experiments, Figure 5 shows the preQi concentration obtained for a sample containing M63 medium alone, M63 medium plus 10 nM of preQi, culture supernatant of an E. coli DH10B queF mutant strain that does not produce preQi (E. coli DH10B AqueF), and the same supernatant plus 10 nM of preQi. As expected, preQi was only detected on the samples to which an exogenous preQi has been added.

[0076] To prove the suitability of the genetic construct of the invention, preQi concentration was measured in supernatants of different E. coli strains (ST131 , DH10B), in Staphylococcus aureus and, in sera from foetal bovine, mouse and human, human urine, and in filtered LB (see Figure 6). Thus, the EGFP(NAC) fluorescence levels of the reporter strain can be correlated to the levels of preQi in a sample where preQi concentration needs to be measured. Using the AqueF mutant from Escherichia coli, preQi is imported by the YhhQ transporter, increasing Q-tRNA(His) levels, which reduces the translation of the Phis-hisL-AhisG egfp(NAC) construction, and therefore, the fluorescence levels. The higher the preQi concentration levels in the sample, the lower the fluorescence signal (see Figure 7).

[0077] Example 3: Use of queuosine reporter gene construction to search for novel genes, proteins or chemical compounds that inhibit the biosynthesis of Q-tRNA

[0078] The above reporter gene construction can be used to search for novel genes, proteins or compounds that inhibit the biosynthesis of Q-tRNA. For that purpose, two different strategies can be designed: i) the strain E. coli DH10B AqueF Phis-hisL-AhisG egfp(NAC) can be incubated with a library of compounds to observe whether the quantitative relationship between the extracellular preQi concentration and fluorescence changes in the presence of a specific compound, when compared with the results of incubating the reporter strain in M63 medium (control), and ii) the strain E. coli DH10B AqueF PhiS-hisL-AhisG egfp(NAC) can be transformed with metagenomic libraries to study whether the expression of certain genes from environmental organisms could alter the relationship between the extracellular preQi concentration and fluorescence.

[0079] For the first strategy, the effect of indole in the biosynthesis of Q has been tested. The main reason is that indole has been characterised has a quorum sensing molecule that seems to inhibit biofilm formation and virulence in certain bacteria, while Q and / or its precursors appear to work as quorum sensing molecules that produce the opposite effect, that is, they increase biofilm formation and virulence in bacteria. Thus, the strain E. coli DH10B AqueF Phis-hisL-AhisG egfp(NAC) was incubated with and without different concentrations of indole, and the linear regressions between the extracellular preQi concentration and fluorescence were modelled (see Figure 8). For each indole concentration, slope and “y”- axis intercept are calculated. These parameters would be used to calculate the parameters Vmax(app) and KM(app), that is, apparent maximum velocity and apparent Michaelis constant for the complete reaction of biosynthesis of Q-tRNA from preQi precursor, assuming that all enzymes involved in this process stablish an ordered sequential reaction following Michaelis-Menten kinetics.

[0080] • Apparent maximum velocity of the global reaction is reached when the added preQi substrate is present in excess. The more apparent maximum velocity, the bigger the difference of fluorescence measurement in the presence of 0 nM preQi and excess preQi. Therefore, Vmax(app) would be proportional to "y"-axis intersect (see Table 2).

[0081] • Apparent Michaelis constant of the global reaction is defined as the concentration of substrate needed to reach the half of the maximum velocity (see Table 2).

[0082] Table 2: Equations for calculating Vmax(aPP) and KM<3PP).

[0083] An indole dose-dependent decrease in Vmax(aPP) and KM<3PP) was observed (see Figure 9). Therefore, this result supports that indole may be inhibiting the modification of tRNA with Q, probably as a mixed inhibitor. This approach could similarly be scaled up to search for Q-tRNA inhibitors in big libraries of compounds.

[0084] For the second strategy, the search of genes from environmental organisms that inhibit the Q-modification of tRNA was addressed. For that purpose, the strain E. coli DH10B AqueF Phis-hisL-AhisG egfp(NAC) was transformed with a metagenomic library constructed using environmental DNA isolated from a rhizosphere of an Antarctic plant, harboured into the plasmid pBluescript (pSKII+). Transformation with the plasmid number 84 was observed to produce a decrease in Vmax(aPP), but an increase in KM<3PP) (see Figure 10 and Figure 11). Furthermore, the environmental DNA fragment harboured in plasmid 84 contained two genes: 84-orf1 and 84-orf2. It was separately tested which gene produced the observed effect, and data indicated that gene 84-orf1 was individually responsible for the phenotype (see Figure 10 and Figure 11). Therefore, these results suggest that 84-orf1 encodes for a gene product that may act as a mixed inhibitor of the Q-modification of tRNAs. In a similar fashion, metagenomic libraries harboured in different plasmids, fosmids or cosmids could be transformed into a strain with the construction of the invention, and it could be used to perform high-throughput screenings (by coupling with microfluidics, for example) to search for genes that inhibit the Q-modification of tRNAs.

Claims

CLAIMS1 . A genetic construct, wherein the genetic construct comprises: an operon; a histidine leader gene (hisL)', histidine operon regulatory elements between hisL and the following gene of the histidine operon (hisG)', a gene encoding for a reporter protein replacing hisG, wherein the gene encoding for the reporter protein lacks NAU codons; and a transcriptional terminator, wherein the genetic construct comprises a sequence with at least an 80% homology to SEQ ID NO: 1.

2. The genetic construct according to claim 1 , wherein the genetic construct comprises: the promoter of the histidine operon (Pws); the histidine leader gene (hisL)', the histidine operon regulatory elements between hisL and the following gene of the histidine operon hisG ', a gene encoding for a reporter protein replacing hisG, wherein the gene encoding for the reporter protein lacks NAU codons; and a transcriptional terminator, wherein the genetic construct comprises a sequence with at least an 80% homology to SEQ ID NO: 2.

3. The genetic construct according to any one of claims 1 or 2, wherein the reporter protein is encoded by a modified version of the egfp gene, wherein all NAU codons have been replaced by NAC codons (EGFP(NAC)).

4. The genetic construct according to claim 3, wherein the construct has a sequence with an 80% homology to SEQ ID NO: 3.

5. A host cell, wherein the host cell can only use exogenous queuosine or an exogenous queuosine precursor selected from the group consisting of preQo, preQi, and q, and wherein the host cell comprises a genetic construct according to any one of claims 1 to 4.

6. The host cell according to claim 5, wherein the host cell is a AqueF mutant.

7. The host cell according to claim 6, wherein the host cell is a AqueF mutant strain of Escherichia coli or a AqueF mutant strain of Bacillus subtilis.

8. Use of a genetic construct according to any one of claims 1 to 4, or of a host cell according to any one of claims 5 to 7, for measuring the degree of queuosine modification of tRNA in a cell, for detecting queuosine and / or a queuosine precursor in a sample, preferably in a biological sample, or for measuring queuosine and / or a queuosine precursor in a sample, preferably in a biological sample.

9. Use of a genetic construct according to any one of claims 1 to 4, or of a host cell according to any one of claims 5 to 7, for identifying whether a gene, a protein or a chemical compound is an inhibitor or an activator of the biosynthesis of Q-tRNA.

10. A method for measuring the concentration of queuosine and / or a queuosine precursor in a sample, preferably in a biological sample, wherein the method comprises: a) incubating calibration standard dilutions of queuosine and / or queuosine precursor in the presence of a host cell according to any one of claims 5 to 7 in a cell culture medium; b) incubating a sample dilution in the presence of a host cell according to any one of claims 5 to 7 in a cell culture medium; c) quantifying the signal associated with the reporter protein in the host cell for the calibration standard dilutions and calculating a calibration curve; d) quantifying the signal associated with the reporter protein in the host cell for the sample dilution and determining the concentration of queuosine and / or of the queuosine precursor in the sample in relation to the calibration curve of step (c).

11. A method for identifying whether a chemical compound is an inhibitor or an activator of the biosynthesis of Q-tRNA, wherein the method comprises: a) incubating calibration standard dilutions of queuosine and / or queuosine precursor in the presence of a host cell according to any one of claims 5 to 7 in a cell culture medium, wherein the cell culture medium is a blank control medium; b) incubating calibration standard dilutions of queuosine and / or queuosine precursor in the presence of a host cell according to any one of claims 5 to 7 in a cell culture medium, wherein the cell culture medium comprises a library of chemicalcompounds; c) quantifying the signal associated with the reporter protein in the host cell for the calibration standard dilutions in blank control medium and calculating a first calibration curve; d) quantifying the signal associated with the reporter protein in the host cell for the library of chemical compounds and calculating a second calibration curve; e) comparing the first calibration curve obtained in step (c) with the second calibration curve obtained in step (d), wherein significant variation between the first and second calibration curves is indicative that the chemical compound is an inhibitor or an activator of the biosynthesis of Q-tRNA.

12. A method for identifying whether a gene or a gene product is an inhibitor or an activator of the biosynthesis of Q-tRNA, wherein the method comprises: a) incubating calibration standard dilutions of queuosine and / or queuosine precursor in the presence of a host cell according to any one of claims 5 to 7 in a cell culture medium; b) incubating calibration standard dilutions of queuosine and / or queuosine precursor in the presence of a host cell according to any one of claims 5 to 7 in a cell culture medium, wherein the host cell has been transformed or transfected with a metagenomic library; c) quantifying the signal associated with the reporter protein in the host cell for the calibration standard dilutions in blank control medium and calculating a first calibration curve; d) quantifying the signal associated with the reporter protein in the host cell for the library of chemical compounds and calculating a second calibration curve; e) comparing the first calibration curve obtained in step (c) with the second calibration curve obtained in step (d), wherein significant variation between the first and second calibration curves is indicative that the chemical compound is an inhibitor or an activator of the biosynthesis of Q-tRNA.

13. A kit or assay device comprising reagents adequate for detecting and / or measuring queuosine and / or a queuosine precursor in a sample, wherein the kit comprises a genetic construct according to any one of claims 1 to 4, or of a host cell according to any one of claims 5 to 7, and wherein said reagents comprise at least 10% of the reagents present in the kit.

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