Part:BBa_K3408007
Pnar-B0034-phy(ycD)-B0015
Used Pnar(BBa_K3408000), RBS(BBa_B0034), phy(ycD)(BBa_E0040) and terminator(BBa_B0015) to express Phy(ycD) under the control of Pnar. When our Bacillus subtilis was in an anerobic environment, Pnar was activated by FNR so that Phy(ycD) could be expressed. But when our Bacillus subtilis was in an aerobic environment, Pnar could be suppressed and we couldn’t detect more Phy(ycD).
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 fragment to construct the recombinant expression vector pWB980-DB-Pnar-phy(ycD).
Plasmid profile
Fig.1. The expression vector of device Pnar-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 10μg/mL kanamycin, and incubate them overnight at 37°C. Send transformants to biotechnology company for sequencing.
1.3.Phytase 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 10μg/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
②Preparation of phosphorus standard curve
③Determination of phytase activity
1.5.Verification of the effect of phytase to dissolve phosphorus and solid lead
①Experimental reagents: sodium phytase solution 1.5mM, phytase solution, 230mg/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, 15ml of 230mg/L PbCl2 was added to react for 1h, and then the lead content in the reaction system was determined by dithizone colorimetry.
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
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