Our goal of this project is to construct an engineered bacteria which will scavenge superoxide compounds (ROS) in gut quickly and efficiently. To achieve it, we selected a classical enzymes -- superoxide dismutase (SOD), which are capable of effectively degrading ROS, for overexpression and purification in Escherichia coli BL21 (DE3). By monitoring ROS consumption, the ability of the engineered strain to degrade ROS was verified.
We use T7 promoter to start SOD protein transcription, and T7 terminator to end transcription. At the same time, insert a His protein tag into SOD protein for purification of SOD protein on the nickel column.
We replace the SOD coding sequence with the CAT coding sequence for producing BBa_K3523006. This part can be used for topics related to the degradation of ROS in the future.
We use an existing Part: BBa_J23116 to start classical enzymes -- superoxide dismutase (SOD) and catalase (CAT) protein transcription without a terminator. The new composite Part: BBa_K3523007 can be used to transformative into Escherichia coli Nissle 1917 to construct an engineered bacteria which will scavenge superoxide compounds (ROS) in the gut quickly and efficiently.
Furthermore, this composite part can be used for projects related to the degradation of ROS in the future.
In order to eliminate ROS of the intestinal tract more efficiently and rapidly, we further designed the fusion expression of superoxide dismutase (SOD) and catalase(CAT) with membrane proteins (Ag43)9 of E. coli, so that the enzymes responsible for superoxide degradation were displayed on the surface of the cell membrane. This strategy can promote the contact of enzymes and substrates and accelerate the process of ROS degradation by engineered bacteria theoretically. We have constructed plasmids for this purpose and will further test this hypothesis.
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