CN114277045B - Construction and application of amide compound biosensor - Google Patents
Construction and application of amide compound biosensor Download PDFInfo
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Abstract
The invention discloses construction and application of an amide compound biosensor, and belongs to the technical field of bioengineering. The invention provides application of an amino acid motif of an AmiC-AmiR mutant in detecting amide compounds. According to the invention, the response of the sensor to amide substances is realized by optimizing RBS and substituting the 83 rd position of AmiC into histidine, and the molar-level content can be detected, and the response range is between 0 and 10 mM. Thereby having good application prospect.
Description
Technical Field
The invention relates to construction and application of an amide compound biosensor, in particular to an amino acid sequence of an amide compound biosensor (AmiC-AmiR), RBS used for expressing the sensor, a plasmid containing a gene for encoding the amino acid sequence, a strain containing the plasmid after transformation and application of the strain in improving the amide compound sensor, and belongs to the technical field of bioengineering.
Background
Amides are a class of nitrogen-containing carboxylic acid derivatives, and structurally, amides can be regarded as compounds in which the hydroxyl group of the carboxyl group in the carboxylic acid molecule is replaced by an amino group or a hydrocarbylamino group (-NHR or-NR 2); it can also be considered as a compound in which hydrogen on nitrogen atoms in ammonia or amine molecules is substituted with acyl groups. The method is mainly used as an industrial solvent, is used for producing vitamins and hormones in the medical industry, and is also used for manufacturing the insecticidal amidine. Acrylamide (Acrylamide) is a very important compound. In industry, the polymer has good flocculation, resistance reduction, thickening and other characteristics, and can be used as tunnel excavation, petroleum exploitation, soil improvement, industrial corrosion prevention and the like; in the aspect of human health, the organic micromolecule with electrophilic groups has extremely strong water solubility, is easy to exist in overcooked food, is absorbed by human bodies and exists in various tissues in the bodies, and causes damage to the bodies.
A biosensor (bio) is a signal that can quickly respond to a target substance in an environment and convert the concentration of the target substance into a signal that is easy to recognize, such as an electrical signal, a color, etc., and people can determine the presence or absence and the content of the target substance according to the received signal.
Acrylamide is a colorless and odorless compound which is difficult to detect after being dissolved in a solvent, and the most common detection means at present is high performance liquid chromatography, but the method has high cost and consumes time. In addition, a colorimetric method can be used for detecting the acrylamide, but the method is easy to be interfered by other substances, has low sensitivity and serious error. Meanwhile, other amide compounds have the same problems as acrylamide, and have high detection difficulty and high detection concentration requirement. Therefore, a means for efficiently and accurately detecting amide compounds, especially acrylamide, from a complex environment is still lacking.
Disclosure of Invention
Aiming at the prior art difficulties and problems, the invention provides an amino acid motif of a transcription regulator AmiC-AmiR from pseudomonas aeruginosa (Pseudomonas aeruginosa) and application thereof in detecting acrylamide.
The invention provides a biosensor, which consists of transcription regulating factors AmiC and AmiR, wherein the AmiC is obtained by carrying out Y83H mutation on the basis of a wild type sequence, the amino acid sequence of the AmiC is shown as SEQ ID NO.1, or the amino acid sequence of the AmiC is shown as the 83 rd position of the sequence shown as SEQ ID NO.1, and the amino acid sequence of the AmiC is the wild type sequence before substitution.
In one embodiment, the amino acid sequence of AmiR is shown in SEQ ID No. 2.
In one embodiment, the RBS sequences of AmiC and AmiR are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.
The invention provides a biological detection system which is used for transferring a biosensor into a host cell to construct recombinant bacteria.
In one embodiment, plasmid pVEO is used as an expression vector comprising AmiC, amiR, and an anti-terminator associated with AmiR.
In one embodiment, the AmiC is obtained by Y83H mutation based on its wild type sequence, the amino acid sequence of which is shown in SEQ ID No.1, or the wild type sequence before substitution of the 83 rd position of the sequence shown in SEQ ID No. 1.
In one embodiment, the amino acid sequence of AmiR is shown in SEQ ID No. 2.
In one embodiment, the RBS sequences of AmiC and AmiR are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.
In one embodiment, the sequence of the AmiR-bound anti-terminator is shown in SEQ ID No. 5.
In one embodiment, E.coli is used as the host cell.
The invention provides a method for detecting the content of amide substances, which utilizes the biological detection system to detect, in particular to a method for culturing recombinant bacteria in a target system and detecting fluorescence value after culturing.
In one embodiment, the amide-based substance content in the target system is 0 to 4mM.
In one embodiment, the recombinant bacteria are cultured at 30-40 ℃ and 150-200 rpm for 7-8 hours to obtain seed liquid, and the seed liquid is added into a target system according to the proportion of 1% -5% for 20-30 hours.
The invention provides application of the biosensor or the biological detection system in detecting amide compounds, nitrile compounds or acid compounds.
In one embodiment, the amide compound comprises acetamide, propionamide, butyramide, valeramide, acrylamide; the nitrile compound includes acrylonitrile; the acid compound comprises acrylic acid.
The invention has the beneficial effects that: the invention provides an amino acid motif of an AmiC-AmiR mutant, and the gene sequence of the RBS can be optimized for normal functioning in a host. Meanwhile, the application of the mutant (Y83H) formed by the mutation of the 83 rd position on the AmiC structural unit of the AmiC-AmiR and the directed evolution of the enzyme and the detection of acrylamide are further described. The mutant Y83H of AmiC-AmiR is compared with the response capability of the wild type to acrylamide, and the response capability of the mutant Y83H is changed from unresponsive to responsive, so that the response capability of the mutant Y83H reaches a micromolar level through the optimization of RBS, and the response range is between 0 and 10 mM. The amino acid residues are described as having an important role in the modification of the responsive ligand of AmiC-AmiR and have an important significance for further insight and understanding of the modification and responsive ligand of AmiC-AmiR.
Drawings
FIG. 1 results of the optimized library construction portion of the RBS of wild-type AmiC-AmiR.
FIG. 2 shows the specificity of wild type and mutant for different substances.
FIG. 3 is a graph showing the response verification of Y83H for different concentrations of acrylamide.
Detailed Description
TABLE 1 primer sequences
(Note: F means upstream primer, R means downstream primer)
TABLE 2PCR amplification System
Example 1: construction of wild-type AmiC-AmiR
The amino acid motif of the transcription regulator amiC-amiR from Pseudomonas aeruginosa (Pseudomonas aeruginosa) was downloaded from NCBI, gene synthesis was performed at Jin Weizhi and codon optimization was performed, and then the synthesized gene was integrated with the laboratory-existing plasmid pVEO (pVEO was constructed by using Pveg promoter instead of T7 promoter on pET24a and inserting GFP gene after promoter) by assembling with the Gibson Assembly method (Gibson Assembly), and then the anti-terminator sequence (nucleotide sequence as shown in SEQ ID NO. 5) bound to amiR was integrated onto the constructed plasmid by the whole plasmid PCR method.
The method comprises the following specific steps:
the plasmid skeleton was amplified by using a primer (AmiC-R_insert-S-R, amiC-R_insert-S-F) and a plasmid pVEO as a template, and the target gene was amplified by using a primer (AmiC-R_insert-R, amiC-R_insert-F) and a synthesized AmiC-AmiR gene as a template (AmiC-AmiR gene sequence is shown as SEQ ID NO. 6), wherein the sequence of the primer is shown as Table 1, the amplification system is shown as Table 2, the PCR amplification reaction conditions are 95℃for 3min,98℃for 15S,55℃for 30S,72℃for 30 cycles, and the extension time is set according to the length of the target gene. Digesting the PCR product with DpnI digestive enzyme for 2-3h, transforming E.coli DH5 alpha, and sequencing and verifying the gene sequence of the positive transformant by Jin Weizhi (Suzhou) limited company; the plasmid with correct sequence was used as template and subjected to whole plasmid PCR using primers (Ter-F, ter-R), the sequences of which are shown in Table 1, the amplification system in Table 2, and the conditions shown above.
Example 2: optimization of RBS of wild-type AmiC-AmiR
The RBS of amiC and amiR was screened for library construction according to the wild-type plasmid pVEO-amiC-amiR-WT constructed as described above, respectively, using degenerate primers for library construction, using primers (RBSNNN-S-F, RBSNNN-S-R) to amplify a backbone fragment using pVEO-amiC-amiR-WT as a template, using primers (RBSNNN-F, RBSNNN-R) to amplify a fragment gene and introducing a mutation. The primer sequences are shown in table 1, the plasmids for library construction are transformed into escherichia coli JM109, acetamide with the final concentration of 10mM is added for culture, fluorescence is detected by an enzyme-labeled instrument, partial results are shown in figure 1, individuals incapable of expressing fluorescence and having over-high background fluorescence are removed, functional individuals capable of normally functioning transcription regulating factors are selected, the RBS sequences of AmiC obtained through sequencing are shown as SEQ ID NO.3, and the RBS sequences of AmiR are shown as SEQ ID NO. 4.
Example 3: construction of mutant AmiC-AmiR-Y83H
The mutant plasmid pVEO-AmiC-AmiR-Y83H (the 83 rd position of AmiC is mutated into histidine) was constructed by the whole plasmid PCR method according to the plasmid pVEO-AmiC-AmiR-WT after optimizing RBS. Firstly, the optimized wild plasmid pVEO-AmiC-AmiR-WT is used as a template, primers are designed to amplify target DNA fragments by PCR, the sequences of the primers are shown in a table 1 (Y83H-F and Y83H-R), the amplification system is shown in a table 2, the PCR amplification reaction conditions are 95 ℃ pre-denaturation for 3min,98 ℃ denaturation for 15s,55 ℃ annealing for 30s and 72 ℃ extension for 1min for 30s, and the total cycles are 30. The PCR product was digested with DpnI digestive enzyme for 2-3h, E.coli DH 5. Alpha. Was transformed, and the gene sequence of the positive transformant was verified by sequencing by Jin Weizhi (Suzhou).
Example 4: verification of the response capability of mutant AmiC-AmiR-Y83H to amide Compounds
(1) Response ability of mutant AmiC-AmiR-Y83H to amide Compounds
Plasmids pVEO-AmiC-AmiR-Y83H and pVEO-AmiC-AmiR-WT were transformed into E.coli JM109, and positive single colonies were picked up to 5mL of LB medium (tryptone 10.0g/L, yeast extract 5.0g/L, naCl 10.0g/L, kanamycin final concentration 50. Mu.g/mL) and cultured at 37℃for 7-8 hours at 200rpm, respectively. The seed solution was transferred to another 5mL of LB added with different amides at a final concentration of 10mM at 1% (v/v), and cultured for 24 hours, after which the fluorescence value was measured using an enzyme-labeled instrument.
The fluorescence detection method comprises the following steps: detection of OD Using an enzyme-labeled Instrument, respectively 600 Absorbance and fluorescence at excitation wavelength 495, absorbance wavelength 525。
The results are shown in FIG. 1: when the amino acid residue is mutated, the response of AmiC-AmiR to acrylamide is converted from nothing to nothing, and the final induction multiplying power can reach more than 6 times; meanwhile, the responses of AmiC-AmiR to acetamide, propionamide and butyramide are also obviously improved compared with wild type, and are respectively improved by 13 times, 12.3 times and 4.4 times, which shows that the amino acid residues possibly bind with the key structural domain of the ligand of AmiC and have important roles to the substrate of AmiC-AmiR response.
(2) Mutant AmiC-AmiR-Y83H response to acrylamide concentrations at different concentrations
Positive transformants containing the plasmid pVEO-AmiC-AmiR-Y83H were picked up and cultured in LB medium at 37℃and 200rpm for 7-8 hours. The seed solution was transferred to 5mL of LB containing different concentrations of ethacrylamide (0.0064 mM, 0.032mM, 0.16mM, 0.8mM, 4mM, and 20mM, respectively) at 1% (v/v), and cultured for 24 hours, and the fluorescence value was measured using an enzyme-labeled instrument after the culture.
As shown in FIG. 2, the mutant has very sensitive response to acrylamide, has better detection capacity to 0.0064mM acrylamide, effectively detects between 0mM and 1mM, and has a certain linear relation.
Comparative example 1
Specific embodiment As in example 3, amiC 83 rd mutation into other amino acids, the mutant plasmid was transformed into E.coli to construct recombinant strains, and the mutant recombinant strains were tested for their ability to respond to acrylamide in the manner of example 4, which showed that these recombinant strains did not respond significantly to acrylamide.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of Jiangnan
<120> BAA211476A
<130> construction of an amide Compound biosensor and use thereof
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Claims (6)
1. A biosensor is characterized by comprising transcription regulating factors AmiC and AmiR, wherein the amino acid sequence of AmiC is shown as SEQ ID NO.1, the amino acid sequence of AmiC is obtained by carrying out Y83H mutation on the basis of a wild type sequence, the amino acid sequence of AmiR is shown as SEQ ID NO.2, and RBS sequences of AmiC and AmiR are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4.
2. A biological detection system, characterized in that Escherichia coli is used as a host cell, and the biosensor of claim 1 is transferred into the host cell to construct recombinant bacteria.
3. The system of claim 2, wherein plasmid pVEO is used as an expression vector comprising AmiC, amiR, and an anti-terminator associated with AmiR.
4. A method for detecting the content of amide substances is characterized in that the biological detection system of claim 2 or 3 is used for detection, specifically, the recombinant bacteria are cultured in a target system, and fluorescence values are detected after the culture; the amide substances are acrylamide, butyramide, propionamide and acetamide.
5. The method according to claim 4, wherein the recombinant bacteria are cultured at 30-40 ℃ and 150-200 rpm for 7-8 hours to obtain seed solution, and the seed solution is added into the target system for 20-30 hours according to the proportion of 1% -5%.
6. Use of the biosensor of claim 1 or the biological detection system of claim 2 or 3 for detecting acrylamide, butyramide, propionamide and acetamide.
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| CN113621600A (en) * | 2021-09-17 | 2021-11-09 | 无锡新晨宇生物工程有限公司 | High-activity nitrile hydratase mutant and application thereof |
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| CN112111468A (en) * | 2020-09-23 | 2020-12-22 | 江南大学 | Gamma-glutamine transpeptidase mutant and application thereof |
| CN113621600A (en) * | 2021-09-17 | 2021-11-09 | 无锡新晨宇生物工程有限公司 | High-activity nitrile hydratase mutant and application thereof |
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