Difference between revisions of "Part:BBa K4055525"
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In order to reduce the amount of leakage expression, referring to the structure of BL21 (DE3) +pET28 protein expression system, we hoped to make the T7 promoter and T7RNAP controlled by PhlF at the same time to reduce the leakage activity, so we added PhlO sequence after the T7 promoter. | In order to reduce the amount of leakage expression, referring to the structure of BL21 (DE3) +pET28 protein expression system, we hoped to make the T7 promoter and T7RNAP controlled by PhlF at the same time to reduce the leakage activity, so we added PhlO sequence after the T7 promoter. | ||
</p> | </p> | ||
| + | ===Experience=== | ||
| + | We took 200 microl cultures into 96-well plates and measured 485/510 fluorescence and OD600 using a microplate reader. The expression level of sfGFP was expressed by the ratio of fluorescence value to OD600. The results showed that for PT7-PHLO, PhlF could significantly inhibit its activity, up to 70 times. | ||
Revision as of 14:35, 9 October 2021
pT7-PhlO
An modified T7 phage promoter which can be regulated by the repressor PhlF
In order to reduce the amount of leakage expression, referring to the structure of BL21 (DE3) +pET28 protein expression system, we hoped to make the T7 promoter and T7RNAP controlled by PhlF at the same time to reduce the leakage activity, so we added PhlO sequence after the T7 promoter.
Experience
We took 200 microl cultures into 96-well plates and measured 485/510 fluorescence and OD600 using a microplate reader. The expression level of sfGFP was expressed by the ratio of fluorescence value to OD600. The results showed that for PT7-PHLO, PhlF could significantly inhibit its activity, up to 70 times.
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]