Table 3.
Established and improved concentrations of reaction components in the coupled Pichia pastoris CFPS system.
| Component | Established concentration | Improved concentration |
|---|---|---|
| HEPES KOH pH 7.4 | 25 mM | 60 mM |
| K-glutamate | 120 mM | 0 mM |
| Mg-glutamate | 6 mM | 6 mM |
| DTT | 2 mM | 2 mM |
| Amino acids mixture | 0.6 mM each 19 amino acids, 0.5 mM Leucine (RTS amino acids mix) | 0.9 mM each 19 amino acids, 0.75 mM Leucine (RTS amino acids mix) |
| ATP | 1.5 mM | 1 mM |
| NTP mixture (CTP/GTP/UTP) | 1 mM | 1 mM |
| Creatine phosphate | 25 mM | 45 mM |
| Creatine phosphokinase | 0.54 mgml−1 | 0.27 mgml−1 |
| RNase Inhibitor | 200 U/ml | 200 U/ml |
| T7 RNA polymerase | 100 U | 100 U |
| DNA Template | 40 nM | 40 nM |