Part:BBa_K3523005
T7 pro-His-SOD-His-T7 ter
BBa_K3523005 contains BBa_K3523000, encoding the superoxide dismutases (SOD). SOD is a group of enzymes that catalyze the dismutation of superoxide radicals (O2−) to molecular oxygen (O2) and hydrogen peroxide (H2O2), providing cellular defense against reactive oxygen species.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Contribution and Biology
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. This part can be used for topics related to the degradation of ROS in the future.
Engineering Success
Characterization of the biochemical characteristics of SOD:
SOD was expressed in Escherichia coli, bacterial cells were collected and broken, and SOD solution was obtained through isolation and purification, and further confirmed by the SDS-Page method, protein bands of the corresponding size were found (Fig.2).
We used the classic nitroblue tetrazolium (NBT) color development method. Superoxide anion (O2-.) was produced by Xanthine and Xanthine Oxidase (XO) reaction system to reduce NBT to blue formazan, which had strong absorption at 560nm. While SOD can remove superoxide anions, so dirty formation is inhibited. The bluer the reaction solution is, the lower the activity of superoxide dismutase is, and vice versa. The activity level of superoxide dismutase can be calculated by colorimetric analysis. The detection principle is shown in Fig.3, and the detected absorbance is shown in Table.1.
The data is substituted into the formula for calculation:
Inhibition percentage=[(Ablank1-Ablank2) - (Asample-Ablank3)]/(Ablank1-Ablank2) * 100%=69.543%
Enzyme activity of sample=inhibition percentage / (1-inhibition percentage) (units)=2.283 U
Specific activity of SOD= enzyme activity of sample / amount of protein (units/mg)=1936.12 U/mg.
The results showed that SOD protein became dissolved in this E. coli expressing a condition, and the target protein is very pure. And SOD had excellent catalytic properties, which could successfully degrade ROS into H2O2
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