KR20160005605A - Red Fluorescence Protein Variant and Biosensor for detecting phenol-based compounds using thereof - Google Patents
Red Fluorescence Protein Variant and Biosensor for detecting phenol-based compounds using thereof Download PDFInfo
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Abstract
Description
The present invention relates to a novel red fluorescent protein variant and a use thereof. More particularly, the present invention relates to a novel red fluorescent protein variant and a use thereof. More particularly, the present invention relates to a novel red fluorescent protein variant and a use thereof, Red fluorescent protein variants.
Fluorescent proteins have been used as a variety of technological tools in many fields in studying living organisms. Examples include protein-protein interactions, protein and cell tracing, and the first green fluorescent protein found was responsible for this function. However, the green fluorescent protein is in the ultraviolet region of the wavelengths necessary for this, and thus, there is a considerable risk to irradiate living cells. Recently, red fluorescence proteins, which can be directly detected by naked eyes, have been spotlighted in the wavelength range of visible light.
Among the red fluorescent proteins, DsRed found in Discosoma sp. Is excited at 558 nm and emitted at 583 nm, so that fluorescence can be easily observed with the naked eye, and quantum yield and light stability are high. However, DsRed has a disadvantage in that it takes a long time to form an active (quaternary) structure than other fluorescent proteins because four monomers gather to form a single protein. To overcome these drawbacks, many conventional variants have been reported. Among them, DsRed2 is most widely used as an optimized mutant. However, such DsRed2 also has drawbacks such as long maturation time for protein stabilization, delayed fluorescence, and interference upon expression. Therefore, there is a continuing need in the industry for new DsRed variants that can overcome these shortcomings through mutagenesis.
Phenol, on the other hand, is one of the world's well-known environmental pollutants. Phenol is toxic at low concentrations and is highly carcinogenic, so rapid detection is very important when it is contaminated. At present, the cost of equipment is high due to the mass and chromatographic detection, and it is difficult for the chemicals used in this case to cause secondary pollution and to be widely used. However, biosensors are cheap enough, have high sensitivity, and are portable enough to replace them.
Until now, enzymes, antibodies and microorganisms have been used as biological components of biosensors. Among them, biosensors using microorganisms are known as the most suitable targets due to their low cost, proper pH, suitable temperature, and high lifetime. A biosensor using such a microorganism typically has a plasmid composed of a regulatory protein gene that reacts with phenol, a promoter, and a reporter gene regulated by the promoter.
In this regard, Korean Patent No. 10-0464068 discloses a biosensor for phenol detection, which is transformed with a phenol-based protease control gene capR, a promoter gene Pr of the gene, and a plasmid containing a firefly luciferase gene as a reporter molecule Lt; / RTI > This is effective for analyzing the sample at a low cost in a short time without a preprocessing step. However, the industry is still demanding the development of new microbial biosensors with excellent phenol detection performance.
The present invention has been made in view of the above technical background, and an object of the present invention is to provide a novel red fluorescent protein and a gene thereof that exhibit a strong fluorescence by making a stable quaternary structure in a short time.
It is another object of the present invention to provide a biosensor for detecting a phenol compound containing the novel red fluorescent protein gene as a reporter gene.
In order to achieve the above object, the present invention provides a red fluorescent protein comprising an amino acid Lys-Ile-Lys inserted at the N-terminus of a wild-type DsRed2 gene.
The amino acid Lys-Ile-Lys is preferably added after the start codon (Met) at the N-terminus of the DsRed2 gene.
The present inventors have attempted to produce variants capable of overcoming the disadvantages of DsRed2 through directed-mutagenesis, that is, mutants that produce a stable quaternary structure in a shorter period of time and exhibit strong fluorescence. For this purpose, various mutant libraries were constructed based on the sequence of DsRed2, and the most bright fluorescence was observed for a short time in the mutants. Three amino acids Lys-Ile-Lys were inserted into the N-terminal of the amino acid sequence Respectively.
On the other hand, the control of the transcription step was confirmed by real-time PCR on the mutants, but no significant difference was observed. As a result of examining the regulation of the translation step through protein electrophoresis, it was confirmed that the level of protein was changed every
The thus obtained red fluorescent protein mutant of the present invention can be preferably represented by the amino acid sequence of SEQ ID NO:
The present invention also provides a red fluorescent protein gene encoding the amino acid sequence of SEQ ID NO: 1, wherein the gene is preferably represented by the nucleotide sequence of SEQ ID NO: 2.
The red fluorescent protein mutant of the present invention is useful as a reporter molecule of a whole cell biosensor which is easy to detect a phenol compound which is attracting attention as an environmental pollutant in the world due to the above characteristics.
That is, the present invention provides a biosensor for detecting a phenolic compound containing a gene encoding the red fluorescent protein variant as a reporter gene.
In a specific example, the biosensor of the present invention showed a large difference in the phenol concentration from 0.01 mM to 1 mM as compared with the control group DsRed2 at 12 hours, while the other phenol compounds, 2-methylphenol and 2-chlorophenol, . The results showed that there were significant differences at various concentrations. Even in the case of catechol 3-methylphenol, lysocynol, 2,5-dimethylphenol, 4-ethylphenol, 2-nitrophenol, 3,4-dimethylphenol, 4-methylphenol and 4-chlorophenol It was confirmed that there was no significant difference but an effect (see FIGS. 8 and 9). Therefore, it can be seen that the biosensor using the red fluorescent protein mutant of the present invention as a reporter gene is superior to DsRed2 in all the phenol compounds and thus is useful as a sensor for detecting phenolic compounds.
Preferably, the biosensor of the present invention comprises a regulatory gene capR, its promoter and operator reacting with phenol, and an expression vector containing a gene encoding the red fluorescent protein of the present invention as a reporter gene, Or a transformant introduced into a cell.
At this time, the regulatory gene capR, its promoter and operator that react with the phenol may be those known in the art. For example, those derived from a microorganism having a phenol decomposing activity can be used, and Pseudomonas putida , more preferably Pseudomonas putida KCTC 1452, can be used.
Further, a description of such capR, its promoter and operator can be found in Korean Patent No. 10-0464068. More specifically, the regulatory gene capR may have the nucleotide sequence of SEQ ID NO: 3, and the promoter and operator may have the nucleotide sequence of SEQ ID NO: 4. However, the present invention is not limited thereto.
As described above, the biosensor of the present invention includes a transformant into which a recombinant expression vector containing the red fluorescent protein mutant gene of the present invention has been introduced into the host cell as the regulatory gene capR, its promoter and operator, and a reporter gene.
As the vector for preparing the recombinant expression vector, known vectors or plasmids commonly used in the art can be used without limitation. For example, pET-21a (+) vector can be used.
Specifically, the recombinant expression vector can be prepared by replacing the T7 promoter with a regulatory gene capR, a promoter and an operator that react with phenol, in a pET vector into which DNA encoding the red fluorescent protein variant of the present invention is inserted. Such substitutions may be made through cleavage and ligation with appropriate DNA restriction enzymes. For reference, pCapRP-mRFP of Fig. 7B shows a schematic diagram of such a recombinant expression vector (plasmid). In FIG. 7B, it can be seen that the mutant (mRFP) of the present invention is inserted into the capR regulatory gene, the promoter and the sub-region of the operator.
The host cell may specifically be a prokaryotic microorganism or a eukaryotic microorganism. More specifically, the prokaryotic microorganism may be E. coli , and the eukaryotic microorganism may be yeast, but is not limited thereto. Examples of the E. coli include E. coli DH5 alpha E. coli BL21 (DE3) and the like.
The phenolic compound is not limited as long as it is a phenol-based compound, and specifically it may be phenol, alkyl or halogen-substituted phenol, and more specifically, catechol, 2-methylphenol, 3-methylphenol, Resorcinol, 2,5-dimethylphenol, 4-ethylphenol, 2-nitrophenol, 3,4-dimethylphenol, 4-methylphenol, 4- Phenol, and the like.
Meanwhile, the biosensor of the present invention may further include means for measuring the activity of the intracellular reporter protein when the transformant (cell) is exposed to the phenolic compound. For example, such means may be a device or instrument for measuring the fluorescence of the reporter protein, preferably a transportable fluorometer.
The biosensor for detecting a phenolic compound of the present invention can be produced by a known method. Amplifying an insertion gene comprising a regulatory gene capR, its promoter and an operator, which specifically react with phenol; Cutting the amplified insert genes with an appropriate restriction enzyme; A step of cleaving an expression vector comprising the red fluorescent protein mutant gene of the present invention with a suitable restriction enzyme; And cloning the insert gene and the expression vector to transform the competent cell, such as Escherichia coli, into a competent cell. At this time, restriction enzymes that cut the amplified inserted genes, or restriction enzymes that cut the expression vector containing the red fluorescent protein mutant gene of the present invention, can be used as the same or compatible end, For example, XbaI and BamHI, or XbaI and BglII can be used. However, the present invention is not limited thereto.
The red fluorescent protein variant according to the present invention complements the disadvantages of DsRed2, and it is useful as a reporter gene of a biosensor for detecting a phenolic compound, by forming a stable quaternary structure in a shorter time and exhibiting strong fluorescence.
Figure 1 is a schematic diagram of a recombinant plasmid for producing a mutant library. Here, pET-DsRed2 is a control group and pET-mRFP is a mutant.
2A is a photograph comparing fluorescence between wild type DsRed2, the red fluorescent protein mutant mRFP of the present invention, and Mock in a solid LB medium. Figs. 2B and 2C are graphs and photographs showing the fluorescence patterns of wild-type DsRed2, mutant mRFP, and morphs varying in time after incubation in liquid LB medium.
FIG. 3 shows the results of comparing the expression between the wild type DsRed2 at the transcription level, the mutant mRFP of the present invention, and the Mock through the reverse transcription PCR (3A) and the real time PCR (3B).
FIG. 4 shows the results of comparing the expression between the wild-type DsRed2 at the translational level, the red fluorescent protein mutant mRFP of the present invention, and the mock by SDS-PAGE.
FIG. 5 is a schematic diagram of a recombinant expression vector pCapRP-luciferase prepared to confirm the regulatory gene capR reacting with phenol, the promoter thereof, and the operation of an operator using a luciferase reporter gene.
FIG. 6 shows the result of measuring the activity of relative luciferase after treating phenol by concentration using Escherichia coli transformed with the recombinant expression vector pCapRP-luciferase.
FIG. 7A is a schematic diagram of a recombinant expression vector pCapRP-DsRed2 containing a wild-type DsRed2 as a regulatory gene capR reacting with phenol, a promoter thereof, an operator and a reporter gene, FIG. 7B shows a regulatory gene capR reactive with phenol, its promoter, And a recombinant expression vector pCapRP-mRFP comprising the red fluorescent protein mutant mRFP of the present invention as a reporter gene.
FIG. 8 shows a pellet photograph (FIG. 8A) and a relative fluorescence activity measurement result (FIG. 8B) after treating phenol by concentration using E. coli transformed with the recombinant expression vectors pCapRP-DsRed2 and pCapRP-mRFP, respectively.
FIG. 9 shows the result of measuring the relative fluorescence activity after treating various phenols by concentration using E. coli transformed with the recombinant expression vectors pCapRP-DsRed2 and pCapRP-mRFP, respectively. Wherein (A) is catechol, (B) is 2-methylphenol, (C) is 3-methylphenol, (D) is 2-chlorophenol, Dimethylphenol, (J) is 4-methylphenol, (K) is 5-dimethylphenol, (G) is 4-ethylphenol, 4-chlorophenol.
Hereinafter, the present invention will be described in more detail with reference to specific examples. However, it should be understood that the present invention is not limited by the following embodiments, and that various modifications and changes may be made without departing from the spirit and scope of the present invention.
Here, unless otherwise defined in the technical terms and scientific terms used, those having ordinary skill in the art to which the present invention belongs have a general meaning.
1. Host and culture conditions
The strains used for mutant production are shown in Table 1 below. E. coli DH5? E. coli BL21 (DE3) was used as a transformant and expression host in the present invention. Pseudomonas putida was purchased from KCTC and cultured at 25 ° C in nutrient broth. The transformed E. coli was cultured in 37 Luria Bertani medium supplemented with 50 ug / ml ampicillin.
2. Plasmid
The plasmids used in the present invention are shown in Table 2 below. pDsRed2-N1 was used to obtain DsRed2. PET-21a (+), an expression vector, was used to construct a mutant library and to express red fluorescent protein variants, and was also used in the production of biosensors.
3. DNA manipulation and transformation of E. coli
Plasmid extraction was performed using the GeneAll Exprep Plasmid kit (GeneAll, Korea) and DNA was cut with appropriate restriction enzymes. The DNA confirmed by agarose gel electrophoresis was extracted using GenoAid PCR / Gel Combo kit (Genotech, Korea). Transformation of E. coli was performed by the Hanahan method. Competent cells and plasmid DNA were incubated in ice for 30 minutes, then heat shocked for 42 to 90 seconds, incubated in ice for 60 seconds, and incubated in LB medium for 60 minutes. The cells were then plated on LB solid medium containing ampicillin and colonization was confirmed one day later.
Example 1: Preparation of mutant library, selection of mutant of the present invention and characterization
1. Directed-mutagenesis and mutagenesis libraries
DsRed2 was amplified using DsRed2-F and DsRed2-R primers and cloned into pET-21a (+) vector. The vector prepared at this time was named pET-DsRed2 and used as a control (see FIG. 1A).
For the preparation of the mutant library, the F1 to F6 and DsRed2-R primers shown in Table 3 below were amplified and then cloned into the pET-21a (+) vector. NdeI and XhoI were used as restriction enzymes. The PCR conditions were denaturation at 94 for 40 seconds, annealing at 58 for 40 seconds, extension at 72 for 40 seconds, and 30 cycles. 1B is a schematic diagram of a recombinant plasmid having a RFP (Red Fluorescence Protein) mutant library of the present invention. The amplified gene was ligated for 12 hours after appropriate restriction enzyme treatment.
The recombinant DNA was transformed into E. coli BL21 (DE3), plated on LB solid medium containing ampicillin, and the variants exhibiting the fastest fluorescence in comparison with the control group, DsRed2, Mock, were selected. The results are shown in Fig. 2A. Sequence analysis of this mutant revealed that three amino acids KIK (lysine-isoleucine-lysine) were inserted at the N-terminus of the protein and was named mRFP as the red fluorescent protein variant of the present invention.
2. Fluorescence intensity measurement
After the selected clones were cultured, fluorescence was measured in crude extract. After incubation, the crude enzyme solution was centrifuged to obtain the same amount of pellet, and lysate buffer (20 mM Tris-HCl (pH 7.4), 1 Mm EDTA, 200 mM NaCl, 1% Triton X-100 and 1 mM PMSF) And then the cells were pulverized using an ultrasonic mill. The crude enzyme solution was quantitated to the same amount (1 mg), placed in a 96-well black plate, and the fluorescence was measured with a fluorometer (Tecan, Sweden). The results are shown in Figs. 2B and 2C. As shown in the figure, it can be seen that the mutant mRFP of the present invention shows remarkably strong fluorescence continuously for up to 48 hours compared to the wild type DsRed2 as the control group.
3. E. coli RNA Extraction and PCR Analysis
In order to comparatively analyze the expression between the wild-type DsRed2 at the transcription level and the mRFP and mock of the present invention through transcriptional PCR and real-time PCR, the following experiment was conducted and the results are shown in FIG.
(1) Real-time PCR
The total RNA of E. coli was extracted using an RNeasy mini kit (Qiagen, Germany). Reverse transcriptase was performed using Superscript II Reverse Transcriptase (Invitrogen, USA). For real-time PCR, cDNA was synthesized by reverse transcription using 3 μg of RNA as template. The PCR was performed using a Corbett RG-6000 apparatus (Corbett Life Science, Australia) with denaturation at 95 for 10 seconds, annealing at 58 for 10 seconds, elongation at 72 for 15 seconds, and 30 cycles. The Ct value of the glyceraldehyde-3-phosphate dehydrogenase gene (gapA) was used as a normailizer to measure the relative expression of DsRed2. The sequence of the GapA primer is as follows.
forward primer: 5-tta ccg ctg aac gtg atc cg-3 (SEQ ID NO: 13)
reverse primer: 5-ttg gaa acg atg tcc tgg cc-3 (SEQ ID NO: 14)
(2) reverse transcription PCR
For PCR, cDNA was obtained by reverse transcription with 3 ug of RNA as a template and PCR was performed using AccuPower HotStart PCR Premix (Bioneer, Korea). Supercycler Gradient Cycler (Kyratec, Australia) was used for the reaction. The reaction conditions were denaturation at 94 for 40 seconds, annealing at 58 for 20 seconds, elongation at 72 for 40 seconds, and 30 cycles. The DsRed2 primer is as follows.
forward primer: 5-cgg ctc caa ggt gta cgt g-3 (SEQ ID NO: 15)
reverse primer: 5-ggt ctt ctt ctg cat cac gg-3 (SEQ ID NO: 16)
4. SDS-PAGE analysis
SDS-PAGE was performed to determine if differences in fluorescence were due to differences in protein expression. Escherichia coli transformants carrying pET-DsRed2 and pET-mRFP were cultured at 37 OD 600 until 2, and cells were inoculated 3% in a fresh medium. After 12 hours, the same amount of cells were harvested and lysed. The same amount (300 ug) was loaded on 12% acrylamide gel and electrophoresed. After staining with Brilliant Blue, the result was confirmed by decolorization. The results are shown in Fig.
As shown in FIG. 3 and FIG. 4, the control of the transcription step was confirmed through real-time PCR, but no significant difference was observed. Protein electrophoresis showed the level of translation at each time point Respectively. From these results, it was suggested that the insertion of amino acid affects the stability of protein, resulting in a difference in fluorescence.
Example 2: Production of a biosensor for detecting a phenolic compound of the present invention
1. Preparation of Recombinant Expression Vector pCapRP-luciferase
The recombinant expression vector pCapRP-luciferase was prepared by using luciferase as a reporter gene in order to confirm whether capR, a promoter known to react with phenol, and an operator works normally in pET21-a (+). Luciferase was amplified and cloned into the pGL3-basic vector by PCR using the following primers. The PCR conditions were: denaturation at 94 for 60 seconds, annealing at 58 for 40 seconds, and elongation at 72 for 100 seconds. The restriction enzymes used here are NdeI and XhoI. A schematic diagram of the expression vector prepared is shown in Fig.
luciferase-F primer: 5-agt cat atg gaa gac gcc aaa aac ata aa gaa-3 (SEQ ID NO: 17)
luciferase-R primer: 5-tga ctc gag tta cac ggc gat ctt tcc gcc-3 (SEQ ID NO: 18)
2. Measurement of luciferase activity
To measure luciferase activity, Escherichia coli transformants carrying pCapRP-luciferase were cultured at 37 for 24 hours, and the cells were inoculated 3% in a fresh medium. The cells were harvested 2 hours after the treatment, and 400 μl of lysis buffer (1.25 mg / ml lysozyme, 2.5 mg / ml BSA, 1 × CCLR) The supernatant was obtained after separation. For the measurement of luciferase activity, 50 μl of firefly luciferin solution was added to 50 μl of the supernatant, and then measured with a luminometer (Berthold, Germany).
The results are shown in Fig. As shown in FIG. 6, the E. coli transformant having pCapRP-luciferase showed luciferase activity from 0.01 mM to phenol treated at each concentration, and showed the maximum activity of luciferase at 1 mM. This result indicates that the regulatory gene capR, the promoter and the operator, are functioning normally in the present expression system.
3. Production of recombinant expression vectors pCapRP-DsRed2, pCapRP-mRFP and the biosensor of the present invention
To extract genomic DNA of Pseudomonas putida KCTC 1452, GeneAll Ex Gene Cell SV kit (GeneAll, Korea) was used. The regulatory gene kapR, the promoter and the operator, reacting with phenol were obtained by PCR after genomic DNA extraction. The primers were as follows: 94 to 60 seconds, 58 to 40 seconds, 72 to 120 seconds. The amplified gene was recovered by electrophoresis on 1% agarose gel and then through GenoAid PCR / Gel Combo kit (Genotech, Korea).
CapRP-F primer: 5-tga ctc aga taa cga gtg agc tga tcg aaa-3 (SEQ ID NO: 19)
CapRP-R primer: 5-cgg gga tcc cta gcc ttc gat gcc gat ttt3 (SEQ ID NO: 20)
The recovered DNA was digested with restriction enzymes XbaI and BamHI, and then the pET vector (pET-DsRed2, pET-mRFP) inserted with DsRed2 and mRFP was digested with XbaI and BglII restriction enzymes, Vectors pCapRP-DsRed2 and pCapRP-mRFP were prepared. At this time, BamHI and BglII can be ligated to the compatible end.
FIG. 7A is a schematic schematic diagram of a recombinant expression vector pCapRP-DsRed2 comprising the wild-type DsRed2, and FIG. 7B shows a schematic diagram of a recombinant expression vector pCapRP-mRFP comprising the mutant mRFP of the present invention. As shown in FIGS. 7A and 7B, the recombinant expression vector has the capR, the promoter and the operator inserted therein in place of the T7 promoter of the pET vector, and DsRed2 and mRFP as reporter genes, respectively.
E. coli BL21 was transformed with each of the recombinant expression vectors described above to obtain colonies, thereby obtaining a biosensor for detecting the phenolic compounds of the present invention.
Example 3: Characteristic measurement of the biosensor of the present invention
1. Treatment of phenol / phenol compounds and measurement of fluorescence intensity
Phenol and phenolic compounds were purchased from Sigma (USA), dissolved in ethanol and treated at appropriate concentrations. E. coli BL21 harboring the respective plasmids obtained in Example 2 was cultured at 37 for one day, and cells were inoculated 3% in 10 ml of the new medium. Phenol and phenol compounds were treated at 1 mM at 1 μM for 12 hours, and the same amount of cells were harvested. Cells were harvested in 96-well black plates (corning, USA) And fluorescence was measured using a fluorescence analyzer (Tecan, Sweden).
The results are shown in Fig. 8 and Fig. Figure 8 is the result for phenol, Figure 9 is the result for various phenolic compounds (Figure 9A is catechol, 9B is 2-methylphenol, 9C is 3-methylphenol, 9D is 2- 9H is 4-methylphenol, 9G is 4-ethylphenol, 9H is 2-nitro phenol, 9I is 3,4-dimethylphenol, 9J is 4-methylphenol, 9K is 4-chlorophenol phenol). The biosensor using the mutant of the present invention showed a large effect from 0.01 mM to 1 mM in the 12 hour period compared to the control group DsRed2 in the phenol. The other phenolic compounds, 2-methylphenol and 2-chlorophenol, showed significant differences at various concentrations ranging from 0.001 mM to 1 mM. Even in the case of catechol 3-methylphenol, lysocynol, 2,5-dimethylphenol, 4-ethylphenol, 2-nitrophenol, 3,4-dimethylphenol, 4-methylphenol and 4-chlorophenol It was confirmed that there was no significant difference but it was effective. Therefore, it was confirmed from these experimental results that the biosensor using the red fluorescent protein mutant of the present invention as a reporter gene is superior to DsRed2 in all the phenol compounds, and thus it is useful as a sensor for detecting phenolic compounds.
<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Red Fluorescence Protein Variant and Biosensor for detecting phenol-based compounds using them <130> KL-P-00952 <160> 20 <170> Kopatentin 2.0 <210> 1 <211> 228 <212> PRT <213> mRFP <400> 1 Met Lys Ile Lys Ala Ser Ser Glu Asn Val Ile Thr Glu Phe Met Arg 1 5 10 15 Phe Lys Val Arg Met Glu Gly Thr Val Asn Gly His Glu Phe Glu Ile 20 25 30 Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly His Asn Thr Val Lys 35 40 45 Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp Ile Leu 50 55 60 Ser Pro Gln Phe Gln Tyr Gly Ser Lys Val Tyr Val Lys His Pro Ala 65 70 75 80 Asp Ile Pro Asp Tyr Lys Lys Leu Ser Phe Pro Glu Gly Phe Lys Trp 85 90 95 Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val Ala Thr Val Thr Gln 100 105 110 Asp Ser Ser Leu Gln Asp Gly Cys Phe Ile Tyr Lys Val Lys Phe Ile 115 120 125 Gly Val Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys Thr Met 130 135 140 Gly Trp Glu Ala Ser Thr Glu Arg Leu Tyr Pro Arg Asp Gly Val Leu 145 150 155 160 Lys Gly Glu Thr His Lys Ala Leu Lys Leu Lys Asp Gly Gly His Tyr 165 170 175 Leu Val Glu Phe Lys Ser Ile Tyr Met Ala Lys Lys Pro Val Gln Leu 180 185 190 Pro Gly Tyr Tyr Tyr Val Asp Ala Lys Leu Asp Ile Thr Ser His Asn 195 200 205 Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Thr Glu Gly Arg His 210 215 220 His Leu Phe Leu 225 <210> 2 <211> 687 <212> DNA <213> mRFP <400> 2 atgaaaatta aggcctcctc cgagaacgtc atcaccgagt tcatgcgctt caaggtgcgc 60 atggagggca ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc 120 tacgagggcc acaacaccgt gaagctgaag gtgaccaagg gcggccccct gcccttcgcc 180 tgggacatcc tgtcccccca gttccagtac ggctccaagg tgtacgtgaa gcaccccgcc 240 gacatccccg actacaagaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg 300 aacttcgagg acggcggcgt ggcgaccgtg acccaggact cctccctgca ggacggctgc 360 ttcatctaca aggtgaagtt catcggcgtg aacttcccct ccgacggccc cgtgatgcag 420 aagaagacca tgggctggga ggcctccacc gagcgcctgt acccccgcga cggcgtgctg 480 aagggcgaga cccacaaggc cctgaagctg aaggacggcg gccactacct ggtggagttc 540 aagtccatct acatggccaa gaagcccgtg cagctgcccg gctactacta cgtggacgcc 600 aagctggaca tcacctccca caacgaggac tacaccatcg tggagcagta cgagcgcacc 660 gagggccgcc accacctgtt cctgtag 687 <210> 3 <211> 1692 <212> DNA <213> capR <400> 3 atgccgatca agtacaagcc tgaaatccag cactccgatt tcaaggacct gaccaacctg 60 atccacttcc agagcacgga aggcaagatc tggcttggcg aacaacgcat gctgttgctg 120 caggtttcag caatggccag ctttcgccgg gaaatggtca ataccctggg catcgaacgc 180 gccaagggct tcttcctgcg ccagggttac cagtccggcc tgaaggatgc cgaactggcc 240 aggaagctta gaccgaatgc cagcgagtac gacatgttcc tcgccggccc gcagctgcat 300 tcgctcaagg gtctggtcaa ggtccgcccc accgaggtcg atatcgacaa ggaatgcggg 360 cgcttctatg ccgagatgga gtggatcgac tccttcgagg tggaaatctg ccagaccgac 420 ctggggcaga tgcaagaccc ggtgtgctgg actctgctcg gctacgcctg cgcctattcc 480 tcggcgttca tgggccggga aatcatcttc aaggaagtaa gctgccgcgg ctgcggcggc 540 gacagtgcc gggtcattgg caagccggcc gaagagtggg acgacgttgc cagcttcaaa 600 cagtatttca agaacgaccc catcatcgag gaactctacg agttgcaatc gcaactggtg 660 tcgctgcgta ccaacctcga caaacaggaa ggccagtact acggcatcgg tcagaccccg 720 gcctaccaga ccgtgcgcaa tatgatggac aaggccgcac agggcaaagt ctcggtgctg 780 ctgcttggcg agaccggggt cggcaaggag gtcatcgcgc gtagcgtgca cctgcgcagc 840 aaacgcgccg ccgagccctt tgtcgcggtg aactgtgcgg cgatcccgcc ggacctgatc 900 gagtccgaat tgttcggcgt ggaaaaaggc gccttcaccg gcgccaccca gtcacgcatg 960 ggccgcttcg agcgggccga caagggcacc atcttccttg acgaggtgat cgaactcagc 1020 ccgcgcgctc aggccagtct gctgcgcgtg ctgcaagaag gcgagctgga gcgagttggc 1080 gacaaccgca cgcgcaagat cgacgtaagg gttatcgcag ccacccacga ggacctggcc 1140 gaagcggtca aggccgggcg ttttcgcgcc gacctgtact accggctgaa cgttttcccg 1200 gtggcgatcc cggcgttgcg cgaacgccgc gaggacattc cactgctggt tgagcacttc 1260 cttcagcgct tccaccagga gtacggcaag agaaccctcg gcctttcaga caaagccctg 1320 gaggcctgcc tgcattacag ttggccgggc aatatccgtg agctggagaa cgtcatcgag 1380 cgcggcatca tcctcaccga tccgaacgaa agcatcagcg tgcaggcgct gttcccacgg 1440 gcgccggaag agccgcagac cgccagcgag cgggtgtcgt cggacggcgt gctgattcag 1500 ccaggcaatg gccagggcag ttggatcagc cagttgttga gcagcggcct gagcctcgac 1560 gagatcgagg aaagcctgat gcgcgaagcc atgcaacagg ccaaccaaaa cgtctccggt 1620 gccgcgcgct tgctcggcct aagccgaccg gcactggcct atcggctgaa gaaaatcggc 1680 atcgaaggct ag 1692 <210> 4 <211> 455 <212> DNA <213> Promoter and Operator of capR <400> 4 taacgagtga gctgatcgaa agtcggtgtg ggggtattgg tcacggccat ctccaggttg 60 gcggattgcg caggacaaag tgcaacagct gtgccaaggt ctgaaaaccg acctcaaggt 120 attgtttttc aatgtgtttc taatttttag aatgatcgga gcgagcgaaa ttaagccgcg 180 cttgcgcagg ctttttaagc atttgatcaa ttgcccaagg ccgcttgagc aaatgctcat 240 ggcgcagctg aaggctgatc tctagcacta aagtcactgc cgtcgattga tcatttggtt 300 gacttttgcc agatactgag gtcggctatg gggagctggc gcaggtgaaa aaactgccga 360 ttttccccat gaccccatct ggaatcgccg cctgccttgc gctatagcgg cgaccctgat 420 ttccccatct aaaaataaat aggggcctcg cttac 455 <210> 5 <211> 28 <212> DNA <213> DsRed2-F primer <400> 5 agtcatatgg cctcctccga gaacgtca 28 <210> 6 <211> 29 <212> DNA <213> DsRed2-R primer <400> 6 tgactcgagc tacaggaaca ggtggtggc 29 <210> 7 <211> 37 <212> DNA <213> F1 primer <400> 7 agtcatatgn nnattaaggc ctcctccgag aacgtca 37 <210> 8 <211> 37 <212> DNA <213> F2 primer <400> 8 agtcatatga ttaagnnngc ctcctccgag aacgtca 37 <210> 9 <211> 34 <212> DNA <213> F3 primer <400> 9 agtcatatgn nnattgcctc ctccgagaac gtca 34 <210> 10 <211> 34 <212> DNA <213> F4 primer <400> 10 agtcatatga ttnnngcctc ctccgagaac gtca 34 <210> 11 <211> 34 <212> DNA <213> F5 primer <400> 11 agtcatatgn nnaaggcctc ctccgagaac gtca 34 <210> 12 <211> 34 <212> DNA <213> F6 primer <400> 12 agtcatatga agnnngcctc ctccgagaac gtca 34 <210> 13 <211> 20 <212> DNA <213> GapA Forward primer <400> 13 ttaccgctga acgtgatccg 20 <210> 14 <211> 20 <212> DNA <213> GapA reverse primer <400> 14 ttggaaacga tgtcctggcc 20 <210> 15 <211> 19 <212> DNA ≪ 213 > DsRed2 Forward primer for PCR <400> 15 cggctccaag gtgtacgtg 19 <210> 16 <211> 20 <212> DNA ≪ 213 > DsRed2 Reverse primer for PCR <400> 16 ggtcttcttc tgcatcacgg 20 <210> 17 <211> 32 <212> DNA <213> Luciferase-F primer <400> 17 agtcatatgg aagacgccaa aaacataaag aa 32 <210> 18 <211> 30 <212> DNA <213> Luciferase-R primer <400> 18 tgactcgagt tacacggcga tctttccgcc 30 <210> 19 <211> 30 <212> DNA <213> CapRP-F primer <400> 19 tgactcagat aacgagtgag ctgatcgaaa 30 <210> 20 <211> 29 <212> DNA <213> CapRP-R primer <400> 20 cggggatccc tagccttcga tgccgattt 29
Claims (11)
Wherein the red fluorescent protein comprises the amino acid sequence of SEQ ID NO: 1.
A red fluorescent protein gene comprising the nucleotide sequence set forth in SEQ ID NO: 2.
A phenol compound detection method comprising a transformant into which an expression vector comprising a regulatory gene capR, which reacts with phenol, a promoter and an operator thereof, and an expression vector containing a gene encoding a red fluorescent protein according to claim 1, For example.
Wherein the regulatory gene capR, the promoter and operator thereof, which react with the phenol are derived from Pseudomonas putida KCTC 1452.
Wherein the regulatory gene capR that reacts with the phenol has the nucleotide sequence of SEQ ID NO: 3, and the promoter and the operator have the nucleotide sequence of SEQ ID NO: 4.
Wherein the host cell is a prokaryotic microorganism or a eukaryotic microorganism.
Wherein the prokaryotic microorganism is E. coli and the eukaryotic microorganism is yeast.
The phenolic compound may be selected from the group consisting of phenol, catechol, 2-methylphenol, 3-methylphenol, 2-chlorophenol, resorcinol, 2,5-dimethylphenol, Phenol, 2-nitrophenol, 3,4-dimethylphenol, 4-methylphenol, and 4-chlorophenol.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116334119A (en) * | 2023-03-13 | 2023-06-27 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | Plasmid for itaconic acid detection and application thereof |
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| KR100464068B1 (en) | 2002-05-16 | 2005-01-03 | 학교법인 동서학원 | biosensor for detecting phenolic compounds and the manufacturing method thereof |
| KR101101986B1 (en) | 2009-04-30 | 2012-01-02 | 전남대학교산학협력단 | Fast maturating red fluorescent protein, FmRed, as a novel reporter and molecular probe |
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2014
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100464068B1 (en) | 2002-05-16 | 2005-01-03 | 학교법인 동서학원 | biosensor for detecting phenolic compounds and the manufacturing method thereof |
| KR101101986B1 (en) | 2009-04-30 | 2012-01-02 | 전남대학교산학협력단 | Fast maturating red fluorescent protein, FmRed, as a novel reporter and molecular probe |
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| 2. 정권영, 표적지향 돌연변이법을 이용한 적색형광단백질(RFP) 변이체의 선발과 특성조사, 2012 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116334119A (en) * | 2023-03-13 | 2023-06-27 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | Plasmid for itaconic acid detection and application thereof |
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