Difference between revisions of "Part:BBa K3523005"
(→Contribution and Biology) |
(→Contribution and Biology) |
||
| Line 9: | Line 9: | ||
[[File:T--Xiamen city--BBa K3523005 Fig 0.png|500px|thumb|center|Figure 1]] | [[File:T--Xiamen city--BBa K3523005 Fig 0.png|500px|thumb|center|Figure 1]] | ||
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. | 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: | 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 corresponding size were found (Fig.2). | 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 corresponding size were found (Fig.2). | ||
[[File:T--Xiamen_city-BBa_K3523006_figure 1.jpg|500px|thumb|center|Figure 2 SDS-Page assay the expression of SOD protein]] | [[File:T--Xiamen_city-BBa_K3523006_figure 1.jpg|500px|thumb|center|Figure 2 SDS-Page assay the expression of SOD protein]] | ||
| + | M: Protein Ladder; FT: Flow-through sample; W: Washing sample; 50: Elution sample with 50mM imidazole; 100: Elution sample with 100mM imidazole; 250: Elution sample with 250mM imidazole; 500: Elution sample with 500mM imidazole. | ||
<!-- --> | <!-- --> | ||
Revision as of 16:32, 26 October 2020
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.
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 corresponding size were found (Fig.2).
M: Protein Ladder; FT: Flow-through sample; W: Washing sample; 50: Elution sample with 50mM imidazole; 100: Elution sample with 100mM imidazole; 250: Elution sample with 250mM imidazole; 500: Elution sample with 500mM imidazole.
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]