Difference between revisions of "Part:BBa K3408007"

Revision as of 00:56, 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 B.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

①Preliminary identification of engineered bacteria expressing phytase

Pick a single colony of the transformant and inoculate it on a LB solid medium containing calcium phytate, and then observe the hydrolysis circle after culturing in a 37 ℃ incubator for 36 h.

②Preparation of phosphorus standard curve

Configure 50 mmol standard KH₂PO₄ solution (weigh 0.6804 g KH₂PO₄ and dilute it to 100 mL with pH 5.5 acetate buffer), and then dilute it to 0.025, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40 mmol/mL nine concentration gradients with deionized water. Take 1 mL of gradient solution and add 1 mL of 5.0 mmol/L sodium phytate solution, then add 2 mL of stop solution, measure absorbance at 415 nm, do 3 sets of repeated experiments for each concentration gradient, and average within the standard deviation range. Draw a standard curve with the concentration of inorganic phosphorus as the abscissa and the absorbance value as the ordinate.

③Determination of phytase activity

Add 0.8 mL of sodium phytate to 0.2 mL of diluted enzyme solution. After reacting for 15 min at pH 6.5 and temperature of 37 ℃, add 1 mL of 5% TCA (trichloroacetic acid) to stop enzyme activity reaction, and then add 1 mL of ferrous sulfate-ammonium molybdate coloring solution, determine the content of inorganic phosphorus in visible light at 415 nm, and calculate the specific activity. (Enzyme activity unit definition: Under the conditions of 37 °C and pH 6.5, the amount of enzyme that releases 1 μmol of inorganic phosphorus from a 5.0 mmol/L sodium phytate solution per minute is defined as 1 enzyme activity unit (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