Difference between revisions of "Part:BBa K3937007"

Latest revision as of 23:00, 21 October 2021

T7+Lac_Operator+BADE

It is the part we used to test the information of BADE, we build it into pET28a to express the enzyme.

By cloning aflatoxin degradation enzyme into plasmid PET-28A (+), lactose operon and T7 promoter can enhance BADE expression in Escherichia coli.

Usage and Biology

IPTG was used to express the aflatoxin degradation enzyme BADE, and the target protein was isolated and purified by affinity purification method.

Experiment method

1/Search for the gene of interest We obtained the gene sequence of an aflatoxin degradation enzyme BADE from the invention patent- CN 107760655 B.With the help of Beijing Tsingke Biotechnology Co.Ltd.，the gene was cloned into a PET-28A (+) plasmid. Also, Comparing with the original gene sequence, we optimized the gene sequence into E. coli bias. In the end, we successfully got the gene which was linked between Nedl and Xhol cleavage sites of restriction enzyme and retained histidine labels at both the amino and carboxyl ends.

2/Activation of seeded emulsion The plasmid synthesized by BBeijing Tsingke Biotechnology Co.Ltd. was stored in DH5α.To activated it, the strains were needed to inoculate into 20 mL LB medium and placed in 37 ℃ constant temperature shaker , and cultured for 24h with 200r/min. The composition of LB medium :yeast powder 5 g, peptone 10 g, sodium chloride 10 g, 1000 mL distilled water.Then adjusted pH to 7.2.Last,121 ℃ autoclave sterilization for 20 min.

3/Preparation of plasmid extract liquid The activated strain liquid was added to 20 mL LB medium with 1% inoculation volume, placed in 37 ℃ constant temperature shaker, and cultured for 16 h with 200r/min.

4/Extract plasmid

Plasmid extraction was carried out by using plasmid extraction Kit (D3350 Bacterial DNA Kit).

5/Construction of expression host

Escherichia coli BL21 (DE3) prepared by calcium chloride method was removed from -80 ℃ and thawed on ice. Add 1 uL target plasmid, mix well, and leave on ice for 30 min. The centrifugal tube was placed in a water bath at 42 ℃ and heated for 60 s. Ice bath the centrifuge tube for 2 min. Add 800 uL LB liquid medium without antibiotics and resuscitate for 45 min at 120 r/min at 37 ℃. Centrifuged at 5000 RPM for 1 min, 800 uL supernatant was discarded, and then coated evenly on LB plate containing kanamycin after re-suspension(final concentration of kanamycin wasd 50 ug/mL). The plate was placed upside down in an incubator at 37 ℃ and cultured for 12-16 h. Colony growth was observed. The LB plate was prepared by adding 2 % agar powder into LB liquid medium.

8/creening of positive clones The colony obtained on the plate was regarded as the positive vector transferred to the target plasmid.

6/Culture of fermented seed liquid Single colonies were selected from plates and inoculated in 20 mL LB liquid medium with a final concentration of 50 ug/mL kanamycin, and placed in 37 ℃ constant temperature shaker,cultured for 16 h with 200r/min

7/Expanded culture The fermentation seed liquid was inoculated in 400 mL LB liquid medium with a final concentration of 50 ug/mL of kanamycin at a constant temperature of 37 ℃ for 200 r/min for 3 h. The OD value of the bacteria solution was 0.638.

8/. Induction The OD value of bacterial solution was 0.638. IPTG with final concentration of 0.1 mM was added to induce BADE protein expression. It was placed in a constant temperature shaker at 30 ℃ for 200 r/min and cultured for 16h.

9/. Purification of objective protein ①Thallus collection: 10000 RPM, 2 min centrifugation. ②Preparation of buffer Table 1 Cracking buffer formulation Composition Concentration PB,pH=7.4 50mM NaCl 500mM Imidizole 30mM phenylmethylsulfonyl fluoride(PMSF) 1mM Lysosome 500mg/L Note: The equilibrium buffer contains only the first three parts of the cracking buffer; Imidazole solution with final concentration of 300 mM was prepared with equilibrium buffer, namely eluting buffer. ③Thallusfragmentation (1) Centrifugation (10000 × g, 4 ℃, 2-3 min) to collect thallus. (2)Re-suspended the thallus obtained by centrifugation with thallus lysis buffer in the proportion of 1 g wet thallus adding 10 mL lysis buffer ( ensure thallus was completely dispersed). (3) Ice bath for 30 min to ensure lysozyme was fully applied to cell wall. (4) Ultrasonic crushing (power 50%). The ultrasound was performed for 3 seconds with an interval of 5 seconds for a total of 15 min. (5) The crushing solution was centrifuged at 10000 × g and 4 ℃ for 15 min. The precipitation was discarded and the supernatant of the bacterial crushing solution was collected. (6) The supernatant was filtered by 0.22 um ultrafiltration membrane and filtrate was collected for affinity chromatography. ④Affinity chromatography (all buffer 4 ℃ or ice bath) (1)Used a balanced buffer to balance 3-5 column volumes (2) Loaded the filtrated crushing liquid supernatant onto a well-balanced chromatography column.

 (3) Used balance buffer des miscellaneous protein (~ 20 column volume)
 (4) Used the elution buffer des target protein(2ml) 
 (5) Cleaned the column with 500 mM imidazole eluting buffer (3-5 volume) 
 (6) Cleaned the chromatographic column with pure water (8-10 volume) 
 (7) Preserved the chromatographic column with 20% ethanol (3-5 volume) 
 (8) Sealed the chromatographic column and stored at 4-8 ℃.

⑤Desalting (desalting buffer: 50 mM PB pH 7.4) (1)Cleaning: clean the desalting column with 3-5 column volumes in pure water

 (2) Balancing: desalting buffer balanced chromatography column (3-5 column volumes) 
 (3) Sample loading: desalting column on 1.0 mL protein solution after affinity chromatography 
 (4) Elution: 2mL desalting buffer elution target protein .
 (5) Balance:Desalting buffer balanced chromatography column( 3-5 column volume)
 (6) Desalting the next sample (repeat steps 2-4) 
 (7) Cleaning the desalting column with pure water (3-5 column volume) 
 (8) Preserving the desalting column with 20% ethanol (3-5 column volume) 
 (9) Sealed the desalting column and stored at 4-8 ℃.

10/SDS-PAGE electrophoresis ①SDS-PAGE electrophoresis during the purification of protein expression Note:Preparation method of electrophoretic loading sample (1)Before adding IPTG,retained 1ml bacterial sample solution.Centrifuge at 10000rpm for 1min. The supernatant was discarded and resuspended by adding 100 mL deionized water. Then 20 mL 6× protein loading buffer was added. After boiling, the supernatant was centrifuged at 10,000 RPM for 1min and could be used for electrophoresis. (2)After adding IPTG for 16 h, the sample was retained ,next step was the same as the first step. (3) After 16 h induction, the collected thalli were added into lysis buffer and centrifuged at 10000 RPM for 1min after ultrasonic crushing. Respectively, to retain the supernatant and precipitatestep.Before electrophoresis, 50 mL supernatant was taken and 10 mL 6× protein loading buffer was added for electrophoresis spot sample. (4)The precipitation collected in step 3 was suspended with 200 mL deionized water, and then 40 mL 6× protein loading buffer was added. After boiling, centrifugation at 10000 RPM for 1min was used for electrophoresis sampling. (5)After the supernatant obtained in step 3 was added to the nickel column, the last outflow was retained for sample retention. Before electrophoresis, 50 mL was taken and 10 mL of 6× protein loading buffer was added for sample spot electrophoresis. (6)When the elution buffer was added for the last elution, the last effluent was retained for sample retention before electrophoresis, 50 mL was added to 10 mL of 6 protein loading buffer for electrophoresis point sample. (7)After adding 50 mL of target protein into 10 mL 6×protein loading buffer, it can be used for electrophoresis spot sample.

Result

                               Figure 1:Electrophoretic results

M: Protein marker; 1: Thallus crushing solution before IPTG induction; 2: Thallus crushing solution after IPTG induction; 3: Supernatant after ultrasonic fragmentation of thallus; 4: Precipitation after ultrasonic crushing of thallus; 5: The outflow of supernatant through the column; 6: The last eluent; 7: Target protein.

According to the electrophoretic diagram, the expression of the target protein was improved after induction with IPTG, and the protein was successfully purified. ②SDS-PAGE electrophoresis results of purified protein are shown in figure 2.

           Figure 2:electrophoresis results

M:Protein marker; 1:8μg target protein; 2:6μg target protein

According to the electrophoretic diagram, the molecular weight of the purified protein was between 35-48 kD, which was close to the theoretical value, indicating that the protein was successfully purified.

11/Protein concentration determination (1)Prepare standard protein according to BCA protein concentration determination kit (sw101-02). (2)Add 2 uL of the sample to be tested into the 96-well plate and supplement with PBS to 20 µ L. (3) add 200 uL of BCA working solution to the microwell plate and mix well at 37 ℃ for 30 minutes. (4)The absorbance value at 562 nm was measured and the reading was recorded. (5) The absorbance value of the sample without BSA was used as blank control. (6) The standard curve was drawn with BSA content as abscissa and A562 as ordinate.

Figure 3:BCA standard curve

Results 3:The concentration of purified protein was 3630 ug/mL by BCA method. It was also known that 21.78 mg of target protein was purified in 400mL medium.

12/Used ELISA kit to determine the specific enzyme activity (1)Add 900 uL purified protein +100 uL AFB1 (100 μg/L) +3 mL LB medium into test tube, and react at 37 ℃ for 72 h under light protection. (2)Used 900 uL 20 mM PB buffer +100 uL AFB1 (100 μg/L) +3 mL LB medium as control group. (3)The content of residual aflatoxin was determined by ELISA kit instruction.Aflatoxin ELISA kit was purchased from Jianglai Bio.

Figure 4:Standard curve of relation between aflatoxin content and OD value

Result 4:Aflatoxin content was 0.62 ug/L by ELISA kit after 72 h, and 1.99 ug/L in blank group. About 66.8% of aflatoxin was degraded within 72 h. The specific activity was calculated to be 0.2918 ng·h-1·mg-1 (unit is the amount of aflatoxin that can be degraded by 1 mg protein per hour)

References:

[1]贾如. 枯草芽孢杆菌黄曲霉毒素降解酶的分离纯化、基因克隆及表达[D].中国农业大学,2016.

[2]计成,马秋刚,赵丽红,贾如. 由枯草芽孢杆菌分泌的黄曲霉毒素降解酶及其应用[P]. 北京：CN107760655A,2018-03-06.

Sequence and Features