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Overview
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The PPK Energy module consists of a purified protein, the bifunctional polyphosphate kinase (PPK2), and it’s substrate, the 25mer polyphosphate (PolyP). Here, bifunctionality refers to the modules ability to direct the synthesis of ATP and GTP from AMP and GDP, respectively
When used alongside PURE’s standard energy regeneration module based on creatine kinase and creatine phosphate (CP/CK), significant increases in protein expression yields can result.
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Usage
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The PPK energy module is implemented by preparing a custom energy mix and adding in purified PPK2 protein. This module is highly sensitive to amount of Mg++ contained in Cytosol since PolyP acts as a magnesium chelator
DNA Parts
Protein components
Cell components
Reaction Construction
Custom SMix. The construction SMix without creatine phosphate (SMixΔCP) is as follow. Note that normal SMix contains all of the following plus 20 mM CP.
| Component | Stock concentration (mM) | Concentration of components in reaction (mM) | Concentration in Energy solution (mM) | Final volume to add (µL) |
|---|---|---|---|---|
| HEPES | 1000 | 50 | 150 | 30.0 |
| Potassium glutamate | 2500 | 100 | 300 | 24.0 |
| Magnesium acetate | 1000 | 11.8 | 35.4 | 7.1 |
| NTP | 100 | 2 | 6 | 12.0 |
| tRNA (mg/mL) | 40 | 3.5 | 10.5 | 52.5 |
| Creatine phosphate | 1000 | 0 | 0 | 0.0 |
| TCEP | 500 | 1 | 3 | 1.2 |
| Folinic acid | 5 | 0.02 | 0.06 | 2.4 |
| Spermidine | 200 | 2 | 6 | 6.0 |
| Amino acid solution | 3.25 | 0.3 | 0.9 | 55.4 |
| Water | 9.4 | |||
| Energy solution total | Final concentration (fold) | Final volume | ||
| 3 | 200 |
The following reaction table is a self-contained experiment for evaluating the performance of the PPK energy module. Details about the stock solutions and detailed reaction descriptions are available in the toggle list below.
| Component | CP/CK [µL] | PPK/PolyP [µL] | CP/CK + PPK/PolyP [µL] | + DNA Positive Control [µL] | -DNA Negative Control [µL] |
|---|---|---|---|---|---|
| Sol A | 14.00 | 14.00 | |||
| Energy solution-CP | 11.67 | 11.67 | 11.67 | ||
| Sol B | 10.51 | 10.51 | 10.51 | 10.51 | 10.51 |
| plam-GFP DNA | 1.67 | 1.67 | 1.67 | 1.67 | 0.00 |
| Mg-Acetate | 1.40 | 3.15 | 3.15 | 0.00 | 0.00 |
| Creatine phosphate | 0.70 | 0.00 | 0.70 | 0.00 | 0.00 |
| PEG4K 40% | 1.75 | 1.75 | 1.75 | 0.00 | 0.00 |
| PolyP | 0.00 | 2.10 | 2.10 | 0.00 | 0.00 |
| PPK2 | 0.00 | 1.22 | 1.22 | 0.00 | 0.00 |
| Water | 7.30 | 2.93 | 2.23 | 8.82 | 10.49 |
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Performance Data
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Translation kinetics of PURE reactions using different energy modules. The PPK2 module performed approximately 77% lower than the CP/CK module. However, the combination of PPK2 and CP/CK modules resulted in approximately 96% higher final protein yield than the CP/CK module alone.