For the severe epidemic in Shanghai in the first half of the year and our school policy, our wet experiments stared very late. Therefore, the content of our experiment was greatly reduced and the experiment was carried out in a hurry. Our experiment was divided into three groups: wild-type (group 1), single mutant (group 2), double mutant (group 3). You can see what we have done below.

The plasmid containing the target gene of double mutant was transformed into E.coli BL21.

Rejuvenation, amplification and preservation of B.subtilis WB800.
Liquid and solid LB medium were prepared.
The plasmid containing the target gene of single mutant and the wild-type were transformed into E.coli BL21.

The plasmids were transformed into E.coli BL21 again.
The double mutant monoclonal strain was selected and cultured.
The double mutant strain was induced to express bromelain by IPTG.

The monoclonal strains of single mutant and wild-type were selected and cultured.
Both single mutant and wild-type strains were induced to express bromelain by IPTG.
The bacterial solution induced by IPTG was collected, and the bacteria were cleaved by ultrasound.
Bromelain was isolated and purified.
The expression of bromelain was confirmed by SDS-PAGE.

Characterized the part of the 2021 team of Tongji_China: BBa_K3823001 SQR (Sulfide:QuinoneOxidoreductase) from Acidithiobacillus spp.
Detected the activity and stability of double mutant bromelain.
Single factor induction experiment of IPTG.
Characterization experiment of soybean meal.
Preparation of competent cells of B.subtilis WB800 and plasmid transformation.