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Overview
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The ClpXP control module enables the ATP-dependent, targeted degradation of ssrA-tagged proteins.
It is based on the complex formed by the AAA+ ATPase ClpX and the tetradecameric peptidase ClpP.
The Module can be implemented using purified protein, in situ expressed proteins from DNA templates, or combinations thereof.
Cartoon of the general mechanism of protein degradation by ClpXP, ATP-dependent protease. Adapted from R. Wedam, et al.
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Usage
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DNA Parts
pT7-ClpX - Nucleus v0.5.0 Distribution Plate upcoming.pT7-ClpP - Nucleus v0.5.0 Distribution Plate upcoming.pT7-deGFP ssrA - Nucleus v0.5.0 Distribution Plate upcoming.Protein components
Cell components
Reaction Construction
Reaction Table 1. The control module can be implemented from purified proteins alone.
| Component | Sample 1 | Sample 2 | Sample 3 | Control |
|---|---|---|---|---|
| Purified deGFP-ssrA (41.2 uM) | 0.5 | 0.5 | 0.5 | 0.5 |
| Purified ClpP (79.9 uM) | 0.5 | 0.5 | 0 | 0 |
| Purified ClpX (53.7 uM) | 0.5 | 0 | 0.5 | 0 |
| NEB PURExpress Solution A | 4 | 4 | 4 | 4 |
| NEB PURExpress Solution B | 3 | 3 | 3 | 3 |
| RNase Inhibitor | 0.5 | 0.5 | 0.5 | 0.5 |
| Nucleus Free Water | 1 | 1.5 | 1.5 | 2 |
| Total | 10 | 10 | 10 | 10 |
Reaction Table 2. The control module can also be implemented from in situ expressed proteins. Steady state levels can be tuned by varying the concentration of in situ expressed ClpXP proteins.
| Component | Sample 1 | Sample 2 | Sample 3 | Sample 4 |
|---|---|---|---|---|
| pT7-deGFP ssrA (63.5 ng/ul) | 0.5 | 0.5 | 0.5 | 0.5 |
| pT7-ClpP (17.5 ng/ul) | 0.4 | 0.4 | 0.6 | 0.8 |
| pT7-ClpX (17.5 ng/ul) | 0.4 | 0.4 | 0.6 | 0.8 |
| NEB PURExpress Solution A | 4 | 4 | 4 | 4 |
| NEB PURExpress Solution B | 3 | 3 | 3 | 3 |
| RNase Inhibitor | 0.5 | 0.5 | 0.5 | 0.5 |
| Nucleus Free Water | 1.6 | 1.2 | 0.8 | 0.4 |
| Total | 10 | 10 | 10 | 10 |
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Performance Data
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