Part:BBa_K533006:Experience
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Applications of BBa_K533006
This is the expression device of mCherry.
When IPTG is added, strong expression of mCherry can be detected. However, mCherry cannot be properly folded above 20 centigrade, as shown by our experimental results.
| Group | 1 | 2 |
|---|---|---|
| Volume | 3ml | |
| IPTG(mM) | 0.1 | |
| 0.5 | ||
| 1.0 | ||
| Temperature | 30oC | 18oC |
| Duration | 12h | 12h |
| Result | Media remained yellowish. E. coli white |
Media turned purple. E. coli cherry pink |
After Pro-rich mCherry expression, significant color change can be observed (bacteria cells turned cherry red as a result of mCherry expression)
From left to right, IPTG concentration: 0, 0.1mM, 0.5mM, 1mM. All induced at 18 centigrade.
We purified the protein by Ni column. After loading the cell lysate onto the column, we first washed the column with 5mM immidazole and then 20mM immidazole. The elute is a bit pink but the majority of protein is still on the column.
The remaining protein is eluted by 200mM immidazole solution.
| 1 | 2 | 3 | 4 | 5 | 6 | Marker | 7 | 8 | 9 | 10 | Marker |
| 1. Sonicated | 2. Supernatant | 3. Pellet |
| 4. Flowthrough | 5. Buffer wash | 6. Resin |
| 7. 5mM Elution | 8. 20mM Elution | 9. Resin 2 |
| 10. 200mM Elution |
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