Part:BBa_K3523006
T7 pro-His-katA-His-T7 ter
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1462
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1334
- 1000COMPATIBLE WITH RFC[1000]
BBa_K3523006 contains Part:BBa_K3523001, encoding sequence of protein catalase. Its function is to decomposes hydrogen peroxide into water and oxygen; serves to protect cells from the toxic effects of hydrogen peroxide.
Contribution
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 -- catalase(CAT), 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 CAT protein transcription, and T7 terminator to end transcription. At the same time, insert a His protein tag into CAT protein for purification of protein on the nickel column. This part can be used for topics related to the degradation of ROS in the future.
Engineering Success
Characterize the biochemical characteristics of CAT. CAT was expressed in E. coli, bacterial cells were collected and broken, and the CAT enzyme solution was isolated and purified, and further confirmed by the SDS-Page Method (Fig1).
Fig. 1 SDS-Page assay the expression of CAT 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.
When hydrogen peroxide is relatively abundant, catalase can catalyze hydrogen peroxide to produce water and oxygen. The residual hydrogen peroxide can oxidize the color substrate under the catalysis of Peroxidase to produce the red product (N-(4-antipyryl) -3-chloro-5-sulfonate-Pbenzoquinonemonoimine), and the maximum absorption wavelength is 520nm.We used the hydrogen peroxide standard to make the standard curve (Fig2), so that the amount of residual hydrogen peroxide in the sample could be measured, and the catalase catalyzed the conversion of hydrogen peroxide to water and oxygen in unit volume of unit time could be calculated, thus the enzyme activity of catalase in the sample could be calculated.
We added different amounts of enzymes to obtain the maximum rate of CAT catalytic substrates, and we found that the catalytic efficiency of enzymes gradually decreased with the increase of enzyme concentration (Fig3).This indicated that CAT could be well expressed in Escherichia coli, and the purified enzyme also had high catalytic efficiency, which could rapidly transform a large amount of hydrogen peroxide into water and oxygen in a short time.
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