Difference between revisions of "Part:BBa K3408007"

Revision as of 01:58, 28 October 2020

P_nar-B0034-phy(ycD)-B0015

Used P_nar(BBa_K3408000), RBS(BBa_B0034), phy(ycD)(BBa_E0040) and terminator(BBa_B0015) to express phy(ycD) under the control of P_nar. When our Bacillus subtilis was in an anaerobic environment, P_nar was activated by FNR so that phy(ycD) could be expressed. But when our Bacillus subtilis was in an aerobic environment, P_nar could be suppressed and we couldn’t detect more phytase.

1. Experimental methods

1.1.Construction of the expression vector

The pWB980-DB is digested with enzyme EcoRI and PstI. The target fragment of the promoter, RBS, gene of phytase and terminator of this device are synthesized by the biotechnology company with 6×His tags added. Add EcoRI and PstI restriction sites to both ends of the target fragment respectively. Connect the target fragment to the plasmid vector to construct the recombinant expression vector pWB980-DB-P_nar-phy(ycD).

Plasmid profile

Fig.1. The expression vector of device P_nar-phy(yCD)

1.2.Construction and screening of recombinant engineered bacteria

Using Bacillus subtilis WB800N as the expression host, the secretion expression vector pWB980-DB was transformed by electro-transformation. Inoculate them on LB solid medium coated with calcium phytate and 10 μg/mL kanamycin, and incubate them overnight at 37 °C. Send transformants to biotechnology company for sequencing.

1.3.expression and purification

Set up two groups of experiments:

(1) the control group: recombinant Bacillus subtilis are cultured in an aerobic condition.

(2) the test group: recombinant Bacillus subtilis are cultured in an anaerobic condition.

①Inoculate recombinant Bacillus subtilis in 20 mL of LB liquid medium containing 10 μg/mL kanamycin, and cultivate them overnight at 37 °C with shaking at 180 rpm.

②Inoculate 2% of the overnight cultured bacteria in 100 mL of LB liquid medium containing 10 μg/mL kanamycin, and culture them with shaking at 25 °C for 24 hours. The supernatant was collected by centrifugation to obtain the crude enzyme solution, and the pure enzyme solution was obtained after Ni-NAT affinity chromatography and Superdex-75 gel chromatography. The purified protein is subjected to SDS-PAGE gel electrophoresis, western blot to determine phytase expression. And gel chromatography is used to obtain the elution profile of the enzyme after gel purification.

③Western Blot

1.4.Verification of the effect of phytase on phosphate hydrolysis

①Drawing the standard curve of inorganic phosphorus

The standard solution of 16 mM potassium dihydrogen phosphate is diluted with 100 mM Tris-Hcl solution to 0.0, 3.2, 6.4, 9.6, 12.8 and 16 mM solutions respectively, and react together according to the operating steps above. With inorganic phosphorus content as ordinate (take 0.05 mL of the diluent above and the inorganic phosphorus content is: 0.00, 0.16, 0.32, 0.48, 0.64, 0.8 μmol respectively) and absorbance at the wavelength of 700 nm as abscess coordinate, the standard curve is drawn and the linear regression equation is listed (Y=KX+B). The light absorption value is determined by the molybdenum-blue method.

0.2177 g constant weight potassium dihydrogen phosphate is accurately weighed in 100 ml volumetric flask and fixed with 100 mM Tris-HCl(A) buffer to a concentration of 16 mM.

The standard dilution ratio is shown in the table below.

Standard phosphorus concentration dilution

Table 1. Draw a standard curve of inorganic phosphorus.

②Determination of enzyme activity on expression product

The collected supernatant is used to determine the activity of phytase by the molybdenum-blue method. The specific steps are as follows:

①Use the lysis buffer to suitably dilute the collected supernatant enzyme solution and take 50 μL in a test tube, incubate it in a 37 °C constant temperature water bath for 5 minutes, and add 50 μL enzyme solution to the sample blank control group. Add 950 μL of the sodium phytate substrate solution which is preheated in a 37 °C constant temperature water bath for 5 minutes into the test tubes of sample group, add 1 mL of trichloroacetic acid (TCA) to the blank control group to stop the reaction and start the timer.

②Let phytase react with sodium phytate substrate for 15 minutes at 37 °C, and add 1 mL trichloroacetic acid (TCA) to stop the reaction immediately. At the same time, add 950 μL sodium phytate solution to the control group.

③After the completion of the reaction, add 2 mL of ammonium molybdate ferrous sulfate coloring solution and wait for 10 minutes at room temperature.

④Use a spectrophotometer to measure the absorbance of sample solution A at a wavelength of 700 nm, and adjust the blank control A0 to zero.

$$U = \frac{K \times (A-A_{0})}{S \times m \times 15}\times F$$

Note: U-activity of sample phytase (U/g); K-slope of standard curve; F-the total dilution ratio of the sample solution before reaction; S-sample measured value (S=0.05mL in the table); m-sample mass (g); A0-blank absorbance of working sample; A-absorbance of the sample solution; 15-enzymatic reaction time (min).

Definition of unit of enzyme activity: the amount of enzyme releasing 1 μmol inorganic phosphorus from 5.0 mmol/L sodium phytate solution at 37 ℃ and pH 5.0 per minute is defined as one unit of enzyme activity (U).

1.5.Verification of the effect of phytase to dissolve phosphorus and solid lead

①Experimental reagents: sodium phytate solution 1.5 mM, phytase solution, 230 mg/L PbCl₂ solution

②test group:

Set test groups for determination of phytase activity

③Experimental steps: Set up four groups of experiments, with whether to add sodium phytase solution and whether to add phytase solution as variables. When phytase solution or sodium phytase solution is not added, the same amount of ddH₂O is used instead. In each group of experiments, 15 ml of 230 mg/L PbCl₂ was added to react for 1 h, and then the lead content in the reaction system was determined by dithizone colorimetry.

The specific operation steps are as follows:

First, prepare a lead standard series with lead content of 0, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0 μg, and measure it in the range of 540 nm and pH 8.5- pH 11, and draw a standard curve based on the data.

Secondly, take 10ml of the reaction solution in a 100 ml separatory funnel, add 2 ml 20% ammonium citrate, 1 ml 20% hydroxylamine hydrochloride, 2 d phenol red indicator, adjust the pH from 8.5 to 9.0 with concentrated ammonia and add 1 ml 10% potassium hydride, shake well. Add 10 ml of dithizone chloroform application solution, shake and layer, put the chloroform layer into a clean 10 ml colorimetric tube, measure the spectrophotometry at 540 nm, and find out the corresponding content from the standard curve.

2. Expected results

2.1.Phytase expression and purification

The control group has no production of phytase, and the test group has production of phytase. The molecular weight that can be determined by SDS-PAGE analysis of the expressed enzyme is 45 KD, and the protein can be determined as phytase by Western blot.

Fig.2. Expected results: SDS-PAGE image of predicted expression product.

2.2.Verification of the effect of phytase on phosphorus hydrolysis

①Preliminary screening of strains

Fig.3. Expected results: transparent hydrolysis circle produced by engineered bacteria.

②Phytase activity determination

According to the experiment, the relative activity of phytase can be obtained.

According to literature prediction, the relative activity of phytase is about 40%.

2.3.Verification of the effect of phytase on phosphorus hydrolysis and lead fixation

Only in the group 4, whose reaction system has both phytase and sodium phytate, the lead content is reduced.

Fig.4. Expected results: changes of lead concentration over time.

These results are predicted because of the lack of experiment for the COVID-19.

Sequence and Features