Team:NPU-CHINA/Proof Of Concept
PROOF OF
CONCEPT
In order to simultaneously realize the expression of the degradation enzyme and the initiation of autolysis, we designed the following two protocols
1.1.1Conjured the aflatoxin degradation enzyme BADE to the P43 promoter. Because P43, as an integrated promoter in Bacillus subtilis, does not need to be added as inducer compared with T7 and lactose operon, so it will be safer when adding feed.
The lytic protein (BsrG, lytC) and the lytic protein promoter (PyqfD, PmmgA) were conjugated to each other by pin-pair combination.
Finally, the degradation enzyme part and the lyase part were simultaneously cloned into the PHT01 plasmid, as shown in the figure. It can simultaneously express aflatoxin degradation enzyme, and express lysate protein after a certain period of time, start autolysate to release aflatoxin degradation enzyme, and finally complete the degradation of aflatoxin in the feed.
1.1.2According to the investigation, the pAX01 plasmid can be integrated into the genome of Bacillus subtilis, so four combinations of 1 lysin and lysin promoter can be cloned into the pAX01 plasmid respectively, and then integrated into the genome after transfer into Bacillus subtilis. The BADE gene of aflatoxin degradation enzyme was cloned into pHT01 by connecting with the P43 promoter. Finally, the constructed plasmid was transferred into Bacillus subtilis integrating with lytic protein, as shown in the figure. The expression and autolysis of degradation enzymes were realized.
CONTACT NPU CHINA
NPU-CHINA is the iGEM team of Northwestern Polytechnical University
Address: 127 West Youyi Road Beilin District, Xi'an,
Shaanxi 710072, China
Contact: npuigem@outlook.com
bilibili: NPU-iGEM
