CN113234749B

CN113234749B - Method for detecting leucine level in cells in real time

Info

Publication number: CN113234749B
Application number: CN202110546393.6A
Authority: CN
Inventors: 滕昕辰; 王丽蓉; 王桂琴
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2021-05-19
Filing date: 2021-05-19
Publication date: 2023-08-25
Anticipated expiration: 2041-05-19
Also published as: CN113234749A

Abstract

The invention relates to a method for detecting leucine level in cells in real time, which clones a promoter sequence of LEU4 gene to YY by means of a molecular cloning technology ₁ Among the plasmids, a new plasmid pr-LEU4-GFP was obtained. It is known that in s.cerevisiae cells, leucine synthesis is feedback-inhibited by the LEU4 gene, and that when the intracellular leucine level is too high, the expression of the LEU4 gene is inhibited, and vice versa. The pr-LEU4-GFP plasmid is successfully transferred into saccharomyces cerevisiae, and when the leucine level in the cell of the saccharomyces cerevisiae is reduced, the expression of the LEU4 gene is promoted, so that the expression of the pr-LEU4-GFP plasmid is started, the expression quantity of GFP in the cell is increased, and otherwise, the expression quantity of GFP is reduced. According to the method, the expression quantity of GFP is detected by a Western blot technology, the change of the leucine level in the yeast cells can be reflected in real time, the sample preparation is simple, the detection cost is low, the environment is friendly, and the change trend of the leucine level in the cells can be reflected conveniently and rapidly for experiments which do not need accurate quantification.

Description

Method for detecting leucine level in cells in real time

Technical Field

The invention belongs to the technical field of detection, and particularly relates to a method for detecting the level of leucine in cells in real time.

Background

Amino acids are an important class of compounds in animals, plants and microorganisms, which are the basic units that make up proteins, the latter being the primary contributor to vital activity. Scientific studies have shown that amino acid metabolic abnormalities are associated with a variety of diseases, such as albinism due to tyrosine metabolic abnormalities, phenylketonuria due to phenylalanine metabolic abnormalities, and the like. Therefore, the qualitative and quantitative analysis of free amino acids in organisms is of great importance for understanding the pathological mechanism of diseases.

Traditional amino acid analysis is planar chromatography, which has been replaced with instrumental analysis in recent years. The existing analysis methods mainly comprise an amino acid automatic analyzer method, a gas chromatography method, a reversed-phase high-performance liquid chromatography method and a high-performance anion chromatography-integral pulse amperometry method. Among them, the most used is the automatic amino acid analyzer method, and the fastest growing is the high performance liquid chromatography, but since both methods require pre-column or post-column derivatization, the results have a certain error, so the selection of efficient derivatization reagents is the future development direction. The high-efficiency anion chromatography-integral pulse amperometric method can directly analyze amino acid, has the advantages of high resolution, good reproducibility, accurate qualitative and quantitative, lower cost and the like, is applied to a certain degree in recent years, and has bright development prospect.

However, the existing method for analyzing amino acid is mainly instrument analysis, and the detection cost is too high due to the fact that the used instrument has a high manufacturing cost generally. And most amino acids do not contain a chromogenic group, pre-or post-column derivatization is typically required to effect detection of the signal. The mobile phase methanol, acetonitrile and other organic solvents used in high performance liquid chromatography can pollute the environment. And the instrument analysis generally requires that the concentration of the sample reach the detection limit, the required concentration of the sample is relatively high (40 OD), and the requirement on the quality of the sample is relatively high.

Disclosure of Invention

In order to solve the technical problems, the invention provides a method for detecting the leucine level in cells in real time. The invention reflects the change of the content of leucine in cells by detecting the expression quantity of GFP in the cells. The detection of the fusion protein GFP can be realized by Western blot or fluorescence technology.

The first object of the present invention is to provide a biosensor for detecting intracellular leucine levels in real time, which comprises a promoter of the expression gene LEU4 of alpha-isopropyl propionate synthase II, and a fluorescent protein gene expressed by the promoter.

Further, the nucleotide sequence of the promoter is shown as SEQ ID NO. 1.

Further, the fluorescent protein gene is a green fluorescent protein gene.

Further, the green fluorescent protein gene is an enhanced green fluorescent protein gene yEGFP.

Further, the promoter and the fluorescent protein gene are located on the vector Ycplac 33.

The second object of the present invention is to provide a recombinant plasmid for detecting leucine level in cells in real time, wherein the recombinant plasmid comprises a promoter of an expression gene LEU4 of alpha-isopropyl propionate synthase II, and a fluorescent protein gene expressed by the promoter.

Further, the recombinant plasmid takes the Ycpac 33 as a vector.

A third object of the present invention is to provide a method for detecting the intracellular leucine level in real time, which comprises transferring the biosensor into a leucine-producing cell, and detecting the change of the intracellular leucine level by detecting the expression level of intracellular fluorescent protein.

Further, the cells are Saccharomyces cerevisiae cells.

Further, the expression level of the fluorescent protein is detected by western blotting.

By means of the scheme, the invention has at least the following advantages:

the invention clones the promoter sequence of LEU4 (alpha-isopropyl propionate synthase II) gene to YY by means of molecular cloning technology ₁ Among the plasmids, a new plasmid pr-LEU4-GFP was obtained. It is known that in s.cerevisiae cells, leucine synthesis is feedback-inhibited by the LEU4 gene, and that when the intracellular leucine level is too high, the expression of the LEU4 gene is inhibited, and vice versa. The pr-LEU4-GFP plasmid is successfully transferred into saccharomyces cerevisiae, and when the leucine level in the cell of the saccharomyces cerevisiae is reduced, the expression of the LEU4 gene is promoted, so that the expression of the pr-LEU4-GFP plasmid is started, the expression quantity of GFP in the cell is increased, and otherwise, the expression quantity of GFP is reduced. According to the method, the expression quantity of GFP is detected by a Western blot technology, the change of the leucine level in the yeast cells can be reflected in real time, the sample preparation is simple, the detection cost is low, the environment is friendly, and the change trend of the leucine level in the cells can be reflected conveniently and rapidly for experiments which do not need accurate quantification.

The foregoing description is only an overview of the present invention, and is intended to provide a better understanding of the present invention, as it is embodied in the following description, with reference to the preferred embodiments of the present invention.

Drawings

FIG. 1 is a YY1 plasmid map;

FIG. 2 is a diagram showing the specificity verification of pr-LEU4-GFP plasmid;

FIG. 3 is a time sensitivity verification of pr-LEU4-GFP plasmid;

FIG. 4 is a graph showing the sensitivity verification of pr-LEU4-GFP plasmid to leucine concentration variation;

FIG. 5 shows the intracellular leucine concentration as determined by high performance liquid chromatography.

Detailed Description

Example 1: construction of plasmids inducing leucine levels in Saccharomyces cerevisiae cells

The invention firstly uses PCR technology to amplify the promoter sequence of LEU4 gene, uses agarose gel to separate PCR product, then uses gel recovery kit to recover and purify the obtained PCR product, and uses Nanodrop 200D software to measure the product concentration. Purified LEU4 gene promoter sequence and YY ₁ The plasmid was double digested to produce a cohesive end, run on agarose gel, after which the purified digested product was recovered and its concentration was determined. Then the LEU4 gene promoter sequence and YY after enzyme digestion ₁ By T ₄ DNA ligase to clone the promoter sequence of the LEU4 gene into YY ₁ On the plasmid, a new plasmid pr-LEU4-GFP was constructed.

Transferring the newly constructed plasmid pr-LEU4-GFP into escherichia coli, coating the transformed escherichia coli on an LB plate containing ampicillin for screening, and culturing in an incubator at 37 ℃ for 12-14 hours in an inversion mode after uniform coating. 3-4 single clones are selected on an LB plate in the next day and cultured in an LB liquid medium overnight, plasmids are extracted by a plasmid small pump kit, the concentration is measured, enzyme digestion verification is carried out, and after the enzyme digestion verification is successful, the extracted plasmids are delivered to Jin Weizhi company for sequencing verification.

Finally transferring plasmids which are successfully sequenced and verified into experimental saccharomyces cerevisiae, coating the transformed bacteria on a culture plate of CSH-Ura, inversely culturing the bacteria in a constant temperature incubator at 30 ℃ for 14-16 hours, selecting monoclonal on the next day, culturing the bacteria in a liquid culture medium of CSH-Ura for overnight, and finally mixing the overnight bacteria with 45% glycerol in a volume ratio of 1:2, mixing uniformly, and storing in a refrigerator at-80 ℃.

The specific operation is as follows:

(1) PCR amplification of the promoter sequence of the LEU4 Gene

A.PCR System

PCR procedure

i. Pre-denaturation: 95 ℃ for 3min

Denaturation: 95 ℃,15s

Annealing: 54 ℃,15s

Extension: 72℃for 1min (repeating steps ii-iv 30 times)

Final extension: 72 ℃ for 5min

Holding: 4 DEG C

C. Glue recovery (operating according to glue recovery kit)

(2) Restriction enzyme target fragment and plasmid vector YY ₁

YY ₁ The plasmid is a plasmid map obtained by inserting a PCR amplified product of yEGFP (enhanced green fluorescent protein gene) into Ycpac 33 through HindIII and BamHI double cleavage sites by using the Ycpac 33 plasmid (a commercial yeast expression plasmid) as a skeleton, as shown in FIG. 1.

A. Enzyme cutting system

a. Cleavage of the fragment of interest

Component (A)	Volume (mu L)
		Target fragment	200ng
10 Xgreen fast-cutting enzyme buffer	3
		HindⅢ	1
BamHⅠ	1
		Sterilizing double distilled water	Constant volume to 30 mu L

Enzyme digestion for 30min at 37 DEG C

b. Cleavage of plasmid vector

Component (A)	Volume (mu L)
		Plasmid vector	1000ng
10 Xgreen fast-cutting enzyme buffer	2
		HindⅢ	1
BamHⅠ	1
		Sterilizing double distilled water	Constant volume to 20 mu L

Enzyme digestion for 30min at 37 DEG C

B. Glue recovery (operating according to glue recovery kit)

(3) Ligation of cleavage products

A. Connection system

PCR procedure: 16 ℃, connect overnight

(4) Conversion of ligation products

A. E.coli Top 10 was thawed on ice, and 50. Mu.L of E.coli Top 10 cells were added with all the ligation products, gently swirled and mixed. Standing the mixture on ice for 20min;

B. heating the mixture in a water bath at a temperature of 42 ℃ for 45S, and immediately standing on ice for 2min;

C. then, 500. Mu.L of LB liquid medium without ampicillin was added to the above mixture, and the mixture was cultured in a shaker at 37℃and 200rpm for 1 hour;

d.5000rpm centrifugation for 1min of the above mixture, discarding the supernatant, resuspending the cells with 100. Mu.L of sterilized double distilled water, plating the cells on LB plates containing ampicillin, culturing overnight in a constant temperature incubator at 37℃and selecting the monoclonal on the next day;

(5) Clone validation

A. 3-4 single clones are selected from each transformation plate, amplified and cultured overnight in LB liquid medium containing ampicillin, plasmids of each clone are extracted according to the specification of a plasmid small extract kit on the next day, and the concentration of the plasmids is measured;

B. enzyme digestion verification

PCR system

Component (A)	Volume (mu L)
		Plasmid(s)	500ng
10 Xgreen fast-cutting enzyme buffer	1
		Bam HⅠ	0.5
HindⅢ	0.5
		Sterilizing double distilled water	Constant volume to 10 mu L

C. Sequencing verification (commission Jin Weizhi company)

D. Transfection of Yeast

a. The frozen BY4741 strain was streaked onto CSH solid plates and incubated at 30℃for 2 days in a thermostated incubator. Then picking the monoclonal on the plate, and culturing in 2mL CSH liquid medium for 14-16 h at 30 ℃ and 280 rpm.

b. Determination of overnight bacterial OD ₆₀₀ The concentration of the bacterial liquid is diluted to OD ₆₀₀ ＝0.25。

c. Culturing for 4h to make OD ₆₀₀ About=0.8 (i.e. logarithmic growth phase), 1mL of yeast solution was taken, centrifuged at 13000rpm for 1min, and the supernatant was discarded.

d. The yeast pellet was resuspended in 1mL of 0.1M lithium acetate solution and centrifuged at 13000rpm for 1min, and the supernatant discarded. The mixture was resuspended in 100. Mu.L of 0.1M lithium acetate solution and gently swirled to mix, which was competent cells.

e. Denatured salmon sperm DNA, each transfection was boiled at 100℃for 5min with 5. Mu.L, and immediately placed on ice.

f. In a super clean bench, 0.5. Mu.g of the above plasmid, 5. Mu.L of denatured salmon sperm DNA, 350. Mu.L of PLATE MIX were added to 100. Mu.L of competent yeast in this order, and the mixture was intermittently shaken until complete mixing was achieved.

g. Placing the mixture into a shaking table at 30 ℃ and 280rpm for culturing for 30min, and then heating in a water bath at 42 ℃ for 10min. The supernatant was discarded after centrifugation at 13000rpm for 1min, and the cells were resuspended in 100. Mu.L of sterilized double distilled water, plated onto C-U selective plates and incubated upside down at 30℃for 2d.

h. 3 to 4 monoclonals are selected on a conversion plate and are amplified and cultured for 14 to 16 hours at a speed of 280rpm in 2mL of C-U liquid culture medium, and the monoclonals are frozen in a refrigerator at a temperature of minus 80 ℃ for later use.

Example 2: verification of the specificity of the plasmid

Yeast sampling

1. Shaking: BY4741 strain transferred with pr-LEU4-GFP plasmid is inoculated in a 12mL culture tube, 4mL CSH-Ura liquid culture medium is added into the culture tube, the tube is shaken for 5 times, and the temperature is 30 ℃ and the speed is 280rpm for overnight culture for 14-16 hours.

2. The next day, the OD of the yeast liquid was measured separately ₆₀₀ Values, and recorded. Each tube was centrifuged at 13000rpm for 1min with 6OD of the bacterial liquid and the supernatant was discarded and further cultured with 6mL of CSH-Ura-20% Leu medium for 1h.

After 3.1h, the OD of the yeast liquid was measured separately ₆₀₀ Values, and recorded. 3 parts of 2OD bacterial solutions are taken from each tube, centrifuged at 13000rpm for 1min, the supernatant is discarded, 1 part of the bacterial solutions are stored in a refrigerator at-20 ℃ as a 0h sample, the rest 2 parts of the 2OD bacterial solutions are mixed together, and 4mL of CSH-Ura-20% Leu, CSH-Ura-10% Met, CSH-Ura-10% His, CSH-Ura-Arg and CSH-Ura-Gln are used for further culture for 6h respectively.

4. OD of each tube of bacterial liquid is measured in 3h and 6h respectively ₆₀₀ And recorded. 2OD bacterial liquid is taken from each tube, and centrifuged at 13000rpm for 1min,the supernatant was discarded. Samples were taken as 3h and 6h respectively.

(II) sample preparation

1. Preparing cell lysate: 1/20 of dithiothreitol and 1/500 of protease inhibitor are added to a certain amount of SDS cell lysate.

2. Lysis of Yeast cells: 200. Mu.L of the above cell lysate and 100. Mu.L of the acid-washed glass beads were added to each yeast cell, and the mixture was shaken at high speed on a vortex mixer for 45s, then immediately placed on ice for 30s, and repeated 4 times. The protein was then denatured by heating at 100℃for 5min.

(III) Western blot

1. Electrophoresis: sequentially adding 15 mu L of cell lysate into the sample loading holes of SDS-polyacrylamide gel, and respectively adding 2 mu L of PageRuler into the holes at the two sides of the gel ^TM Constant pressure 100V, electrophoresis 120min.

2. Transferring: after electrophoresis, the proteins in SDS-polyacrylamide gel were transferred to PVDF (about 5min before methanol activation), and the reaction was carried out at a constant pressure of 100V for 90min.

3. Closing: the PVDF membrane was blocked with 5% skim milk on a shaking machine (80 rpm) for 45min.

4. Applying an antigen: the films were cut between 34 and 43Kda, with the upper film applied Pgk, the lower film applied GFP, and the refrigerator at 4 ℃ overnight.

5. Washing the film: the primary antibody was recovered and washed with 1 XPBST on a shaking machine (100 rpm) for 10min, and this step was repeated 4 to 5 times.

6. And (3) secondary antibody: incubation was performed with 3mL of murine antibody for 1h at room temperature, respectively.

7. Washing the film: this step was repeated 4 to 5 times by washing with 1 XPBST on a shaking machine (100 rpm) for 5 minutes.

8. And developing and imaging.

FIG. 2 demonstrates the specificity of the pr-LEU4-GFP plasmid. The BY4741 strain transformed with the plasmid is used as a control, CSH-Ura-20% Leu is used as a control, experiments are respectively carried out under the conditions of CSH-Ura-10% Leu (37), CSH-Ura-10% Met (7.4), CSH-Ura-10% His (7.4) and CSH-Ura-Arg, and the experimental results show that GFP expression level is obviously increased under the condition of CSH-Ura-10% Leu, and no obvious sign of increase is caused under the condition of reducing or deleting other amino acids, which indicates that the pr-LEU4-GFP plasmid can actually reflect the level of leucine in yeast cells specifically.

Example 3: time sensitivity of pr-LEU4-GFP plasmid

Yeast sampling

1. Shaking: BY4741 strain transferred with pr-LEU4-GFP plasmid is inoculated in a 12mL culture tube, 7mL CSH-Ura liquid culture medium is added into the culture tube, 2 tubes are shaken, and the culture is carried out at 30 ℃ and 280rpm for 14-16 hours.

2. The next day, the OD of the yeast liquid was measured separately ₆₀₀ Values, and recorded. Each tube was centrifuged at 13000rpm for 1min at 14OD and the supernatant was discarded and cultured with 7mL of CSH-Ura-20% Leu medium for 1h.

After 3.1h, the OD of the yeast liquid was measured separately ₆₀₀ Values, and recorded. 7 parts of 2OD bacterial solutions are taken from each tube, centrifuged at 13000rpm for 1min, the supernatant is discarded, 1 part of the bacterial solutions are stored in a refrigerator at-20 ℃ as a 0h sample, the rest 6 parts of the 2OD bacterial solutions are mixed together, and 8mL of CSH-Ura and CSH-Ura-10% Leu are respectively used for continuous culture for 6h.

4. OD of each tube of bacterial liquid was measured at 0.25, 0.5, 1, 2, 3, and 6 hours, respectively ₆₀₀ And recorded. 2OD of bacterial liquid is taken from each tube, centrifuged at 13000rpm for 1min, and the supernatant is discarded. Samples were taken at 0.25, 0.5, 1, 2, 3, and 6h, respectively.

(II) sample preparation and Western blot were as in example 2.

FIG. 3 time sensitivity of pr-LEU4-GFP plasmid was tested using BY4741 strain design experiments transformed with pr-LEU4-GFP plasmid. The CSH-Ura-100% Leu is used as a control, the CSH-Ura-10% Leu is used as an experimental group, the GFP expression levels in the cells of 0, 0.25, 0.5, 1, 2, 3 and 6 hours are respectively detected, the GFP expression level in the experimental group is obviously higher than that in the control group in 0.25 hours, the response time of the plasmid is relatively short, the GFP expression level in the cells at any time point can be detected by using the method in theory, only 2OD cells are needed at a certain time point, the sampling is convenient, and the sample preparation time is short. While High Performance Liquid Chromatography (HPLC) requires 40OD of cell sample, sampling is more time-consuming and difficult to monitor in real time. (Note: 1 OD. Apprxeq. 3X 10. Times.7 yeast cells).

Example 4: sensitivity of pr-LEU4-GFP plasmid to leucine concentration variation

Yeast sampling

1. Shaking: BY4741 strain transferred with pr-LEU4-GFP plasmid is inoculated in a 12mL culture tube, 7mL CSH-Ura liquid culture medium is added into the culture tube, and the culture is carried out at 30 ℃ and 280rpm for 14-16 h.

2. The next day, the OD of the yeast liquid was measured separately ₆₀₀ Values, and recorded. 4 tubes of the bacterial liquid were centrifuged at 6 OD/13000 rpm for 1min, the supernatant was discarded, and each tube was further cultured with 6mL of CSH-Ura-20% Leu medium for 1h.

After 3.1h, the OD of the yeast liquid was measured separately ₆₀₀ Values, and recorded. 3 parts of 2OD bacterial solutions are respectively taken from each tube, centrifuged at 13000rpm for 1min, the supernatant is discarded, 1 part of the bacterial solutions are stored in a refrigerator at the temperature of minus 20 ℃ as a 0h sample, the rest 6 parts of the 2OD bacterial solutions are mixed together, and 8mL of CSH-Ura, CSH-Ura-30% Leu, CSH-Ura-20% Leu and CSH-Ura-10% Leu are respectively used for continuous culture for 6h.

4. OD of each tube of bacterial liquid was measured at 3 rd and 6h, respectively ₆₀₀ And recorded. 2OD of bacterial liquid is taken from each tube, centrifuged at 13000rpm for 1min, and the supernatant is discarded. Samples 3 and 6h were obtained.

(II) sample preparation and Western blot were as in example 2.

FIG. 4 shows the sensitivity of pr-LEU4-GFP plasmid to leucine concentration changes using BY4741 strain design experiments transformed with pr-LEU4-GFP plasmid. Taking CSH-Ura as a control group, taking CSH-Ura-30% Leu, CSH-Ura-20% Leu and CSH-Ura-10% Leu as experimental groups, examining the expression amount of GFP in cells of 0, 3 and 6h, it can be seen that when the leucine concentration is reduced to 20% of the control group, the expression amount of GFP is obviously increased, and the lower the leucine concentration is, the higher the expression amount of GFP is.

FIG. 5 shows the concentration of intracellular leucine measured by High Performance Liquid Chromatography (HPLC) by the company Pa Mi Nuoke, suzhou. The concentration of intracellular leucine was determined 3h in CSH-20% Leu, CSH-10% Leu, BY BY4741 strain experiments. It can be seen that the difference between the two groups is not significant. However, if we used this method, the lower the leucine concentration, the higher the GFP expression level, and the more likely it was to reflect a slight difference in the change in leucine concentration.

Regarding the detection limit of this method, it is known from the existing experimental results that the lower the leucine concentration is, the stronger the GFP signal is, and the theoretical detection limit can be made infinitely low. In general, to measure the concentration of a substance by using high performance liquid chromatography, it is necessary that the concentration of the substance in a sample reaches a detection limit, and the substance cannot be detected if the concentration is not reached, but our method is more sensitive to low concentration reaction and is a supplement to the conventional method.

The above is only a preferred embodiment of the present invention, and it should be noted that it should be understood by those skilled in the art that several improvements and modifications can be made without departing from the technical principle of the present invention, and these improvements and modifications should also be considered as the protection scope of the present invention.

Sequence listing

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Claims

1. The biosensor for detecting the intracellular leucine level in real time is characterized by comprising a promoter of an expression gene LEU4 of alpha-isopropyl propionate synthase II and a fluorescent protein gene expressed by the promoter, wherein the nucleotide sequence of the promoter is shown in SEQ ID NO. 1.

2. The biosensor of claim 1, wherein the fluorescent protein gene is a green fluorescent protein gene.

3. The biosensor of claim 2, wherein the green fluorescent protein gene is an enhanced green fluorescent protein gene ygfp.

4. The biosensor of claim 1, wherein the promoter and fluorescent protein gene are located on the vector Ycplac 33.

5. The recombinant plasmid for detecting the intracellular leucine level in real time is characterized by comprising a promoter of an expression gene LEU4 of alpha-isopropyl propionate synthase II and a fluorescent protein gene which is expressed by the promoter, wherein the nucleotide sequence of the promoter is shown as SEQ ID NO. 1.

6. The recombinant plasmid according to claim 5, wherein the recombinant plasmid uses Ycplac33 as a vector.

7. A method for detecting the level of leucine in a cell in real time, which comprises transferring the biosensor according to any one of claims 1 to 3 into a cell producing leucine, and detecting the change in the level of leucine in the cell by detecting the expression level of fluorescent protein in the cell.

8. The method of claim 7, wherein the cells are s.cerevisiae cells.

9. The method of claim 7, wherein the expression level of the fluorescent protein is detected by western blotting.