Overview¶
Base Cytosol is a molecular system with a defined set of components, including purified proteins and E. coli ribosomes and tRNAs that together can perform transcription and translation. It builds on the PURE system and is optimized for integration and extension. The Base Cell is formed by encapsulating Base Cytosol in a liposome. Base Cytosol transcribes a DNA template that carries a T7 promoter and translates a transcript encoding an E. coli ribosome binding site (RBS) and a coding sequence (CDS). It also recycles energetic molecules (rNTPs) from creatine phosphate (CP) and processes waste products (PPi) with a simple energy module (PPiase, Creatine Kinase, adenylate kinase and nucleoside diphosphate kinase). Plasmid designs for the PURE proteins are maintained as pET28a expression vectors in the DNA Distribution repository.
Schematic of components in Base Cytosol and their function. Figure by Ganesh and Maerkl, 2024 used under CC-BY-4.0 / cropped from original with numerical annotations removed.
Reference Composition¶
Base Cytosol is assembled from five components — Protein Mix (PMix), Small Molecule Mix (SMix), ribosomes, tRNA, and an optional RNase inhibitor. The tabs below give each component at its stock concentration, plus the Final Reaction composition with every component at its in-reaction concentration.
Reaction Assembly.
| Component | Stock Concentration | Reaction Concentration |
|---|---|---|
| Protein Mix (PMix) | 15 µg/µL (8.33×) | 1.8 µg/µL |
| Small Molecule Mix (SMix) | 3.33× | 1× |
| Ribosomes | 10 µM (5.55×) | 1.8 µM |
| tRNA | 35 µg/µL (10×) | 3.5 µg/µL |
| (optional) RNase Inhibitor | 40 000 U/mL (20×) | 2000 U/mL |
The RNase inhibitor is optional, but recommended.
PMix is added at 1.2 µL per 10 µL reaction, a 0.12× dilution from its stock: the 15 000 ng/µL total of Protein Mix (PMix) becomes 1800 ng/µL, and each protein is at 0.12 times its PMix stock concentration. Protein molecular weights are those of the assembled complex (monomer weight × oligomeric state, as listed for PMix), and molar concentrations are of that complex. See Assemble Base Cytosol for the volumes of every component.
Composition of Base Cytosol at reaction concentration.
| Category | Molecule | Molecular Weight | Concentration (molar) | Concentration (mass) |
|---|---|---|---|---|
| Buffers | HEPES-KOH | 238.3 g/mol | 50 mM | 11.9 µg/µL |
| Potassium Glutamate | 203.23 g/mol | 100 mM | 20.3 µg/µL | |
| Magnesium Acetate | 214.45 g/mol | 7.5 mM | 1.61 µg/µL | |
| Nucleotides | rATP | 2 mM | ||
| rGTP | 2 mM | |||
| rCTP | 1 mM | |||
| rUTP | 1 mM | |||
| Amino Acids | Alanine (Ala) | 89.1 g/mol | 0.3 mM | 26.7 ng/µL |
| Arginine (Arg) | 210.66 g/mol | 0.3 mM | 63.2 ng/µL | |
| Asparagine (Asn) | 132.1 g/mol | 0.3 mM | 39.6 ng/µL | |
| Aspartic Acid (Asp) | 133.1 g/mol | 0.3 mM | 39.9 ng/µL | |
| Cysteine (Cys) | 157.62 g/mol | 0.3 mM | 47.3 ng/µL | |
| Glutamic Acid (Glu) | 147.1 g/mol | 0.3 mM | 44.1 ng/µL | |
| Glutamine (Gln) | 146.2 g/mol | 0.3 mM | 43.9 ng/µL | |
| Glycine (Gly) | 75.1 g/mol | 0.3 mM | 22.5 ng/µL | |
| Histidine (His) | 209.63 g/mol | 0.3 mM | 62.9 ng/µL | |
| Isoleucine (Ile) | 131.2 g/mol | 0.3 mM | 39.4 ng/µL | |
| Leucine (Leu) | 131.2 g/mol | 0.3 mM | 39.4 ng/µL | |
| Lysine (Lys) | 182.65 g/mol | 0.3 mM | 54.8 ng/µL | |
| Methionine (Met) | 149.2 g/mol | 0.3 mM | 44.8 ng/µL | |
| Phenylalanine (Phe) | 165.2 g/mol | 0.3 mM | 49.6 ng/µL | |
| Proline (Pro) | 115.1 g/mol | 0.3 mM | 34.5 ng/µL | |
| Serine (Ser) | 105.1 g/mol | 0.3 mM | 31.5 ng/µL | |
| Threonine (Thr) | 119.1 g/mol | 0.3 mM | 35.7 ng/µL | |
| Tryptophan (Trp) | 204.2 g/mol | 0.3 mM | 61.3 ng/µL | |
| Tyrosine (Tyr) | 181.2 g/mol | 0.3 mM | 54.4 ng/µL | |
| Valine (Val) | 117.1 g/mol | 0.3 mM | 35.1 ng/µL | |
| Stabilizers | Spermidine | 145.25 g/mol | 2 mM | 290 ng/µL |
| TCEP-HCl | 286.65 g/mol | 1 mM | 287 ng/µL | |
| Cofactors | Creatine Phosphate | 327.14 g/mol | 20 mM | 6.54 µg/µL |
| Folinic Acid | 511.5 g/mol | 0.02 mM | 10.2 ng/µL | |
| Ribonucleics | tRNA | 3.5 µg/µL | ||
| Ribosomes | ~2.7 MDa | 1.8 µM | 4.9 µg/µL | |
| tRNA Synthetases | AlaRS | 384.0 kDa | 0.340 µM | 130.3 ng/µL |
| ArgRS | 64.7 kDa | 0.0557 µM | 3.6 ng/µL | |
| AsnRS | 105.2 kDa | 0.389 µM | 40.9 ng/µL | |
| AspRS | 131.8 kDa | 0.114 µM | 15.0 ng/µL | |
| CysRS | 52.2 kDa | 0.0414 µM | 2.2 ng/µL | |
| GlnRS | 63.5 kDa | 0.111 µM | 7.0 ng/µL | |
| GluRS | 53.8 kDa | 0.437 µM | 23.5 ng/µL | |
| GlyRS | 223.2 kDa | 0.0802 µM | 17.9 ng/µL | |
| HisRS | 94.0 kDa | 0.0154 µM | 1.5 ng/µL | |
| IleRS | 104.4 kDa | 0.715 µM | 74.6 ng/µL | |
| LeuRS | 97.2 kDa | 0.0778 µM | 7.5 ng/µL | |
| LysRS | 115.2 kDa | 0.103 µM | 11.9 ng/µL | |
| MetRS | 152.6 kDa | 0.0283 µM | 4.4 ng/µL | |
| PheRS | 248.4 kDa | 0.127 µM | 31.7 ng/µL | |
| ProRS | 127.4 kDa | 0.146 µM | 18.7 ng/µL | |
| SerRS | 96.8 kDa | 0.0372 µM | 3.6 ng/µL | |
| ThrRS | 148.0 kDa | 0.0786 µM | 11.6 ng/µL | |
| TrpRS | 74.8 kDa | 0.156 µM | 11.6 ng/µL | |
| TyrRS | 95.0 kDa | 0.0126 µM | 1.2 ng/µL | |
| ValRS | 108.2 kDa | 0.0311 µM | 3.4 ng/µL | |
| Initiation Factors | IF1 | 8.2 kDa | 2.27 µM | 18.7 ng/µL |
| IF2 | 97.3 kDa | 0.767 µM | 74.6 ng/µL | |
| IF3 | 20.6 kDa | 0.902 µM | 18.7 ng/µL | |
| Elongation Factors | EF-G | 77.6 kDa | 1.2 µM | 93.3 ng/µL |
| EF-Tu | 43.3 kDa | 21.5 µM | 931.7 ng/µL | |
| EF-Ts | 30.4 kDa | 3.07 µM | 93.3 ng/µL | |
| Release Factors | RF1 | 40.5 kDa | 0.46 µM | 18.7 ng/µL |
| RF2 | 41.3 kDa | 0.45 µM | 18.7 ng/µL | |
| RF3 | 59.6 kDa | 0.312 µM | 18.7 ng/µL | |
| RRF | 20.6 kDa | 0.902 µM | 18.7 ng/µL | |
| Metabolism | MTF | 34.2 kDa | 1.09 µM | 37.3 ng/µL |
| CK | 86.6 kDa | 0.0872 µM | 7.5 ng/µL | |
| AK | 23.6 kDa | 0.234 µM | 5.6 ng/µL | |
| NDK | 93.0 kDa | 0.0206 µM | 1.9 ng/µL | |
| PPiase | 118.2 kDa | 0.0162 µM | 1.9 ng/µL | |
| Transcription | T7RNAP | 98.9 kDa | 0.188 µM | 18.7 ng/µL |
Expected Behavior¶
The behavior of Base Cytosol is characterized using the Reporter: deGFP Module.
Processes¶
Make Protein Mix: makes PMix, the 36 purified PURE proteins at set molar ratios.
Make OnePot Protein Mix: makes OnePot Protein Mix, which has the same proteins as PMix but at molar ratios that cannot be controlled. It is a functional equivalent of PMix, with less control.
Make Small Molecule Mix: makes SMix.
Make Amino Acid Mix: makes the equimolar mix of the 20 canonical amino acids that SMix uses.
Make Ribosomes: makes the ribosomes.
Make tRNAs: makes the tRNA.
Assemble Base Cytosol: combines the mixes into the reaction.
Constituent Modules¶
Protein Mix (PMix) — the 36 PURE proteins in Protein Buffer
Small Molecule Mix (SMix) — the buffers, rNTPs, amino acids, stabilizers, and cofactors
Credits¶
Reformulated by Yemo Ku and Jon Calles (b.next) from the PURE system, developed by the Ueda Lab (Shimizu et al., 2001).
Supported by¶
DevNotes that support this specification include:
- Shimizu, Y., Inoue, A., Tomari, Y., Suzuki, T., Yokogawa, T., Nishikawa, K., & Ueda, T. (2001). Cell-free translation reconstituted with purified components. Nature Biotechnology, 19(8), 751–755. 10.1038/90802
- Ganesh, R. B., & Maerkl, S. J. (2024). Towards self-regeneration: exploring the limits of protein synthesis in the PURE cell-free transcription-translation system. 10.1101/2024.04.03.587879