Overview¶
A molecular system with a defined set of components including T7 RNA Polymerase, ribosomes, and tRNA capable of transcription and translation. Base Cytosol builds on the PURE system, and is optimized for integration and extension. The Base Cell is formed by encapsulating Base Cytosol in a liposome.
Plasmid designs for the PURE proteins are maintained as pET28a expression vectors in the DNA Distribution repository.
Schematic of components in 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 four components — a protein mix (PMix), a small molecule mix (SMix), ribosomes, and tRNA. The tabs below give the composition of each component at its stock concentration, plus the Final Reaction composition with every component at its in-reaction concentration.
The Protein Mix (PMix) contains all 36 PURE proteins at a total stock concentration of 15 µg/µL. Per-protein stock concentrations are shown below; see the Make PMix and Make OnePot Protein Mix processes for preparation.
Composition of the Protein Mix (PMix) at stock concentration. Molecular weight, stoichiometry, and molarity are computed as described in the note below.
| Category | Protein | Concentration (ng/µL) | Molecular Weight (kDa) | Stoichiometry | Molarity (µM) |
|---|---|---|---|---|---|
| tRNA Synthetases | AlaRS | 1086 | 96.0 | Homotetramer | 2.83 |
| ArgRS | 30 | 64.7 | Monomer | 0.464 | |
| AsnRS | 341 | 52.6 | Homodimer | 3.24 | |
| AspRS | 125 | 65.9 | Homodimer | 0.948 | |
| CysRS | 18 | 52.2 | Monomer | 0.345 | |
| GlnRS | 59 | 63.5 | Monomer | 0.929 | |
| GluRS | 196 | 53.8 | Monomer | 3.64 | |
| GlyRS* | 149 | 223.2 | Heterotetramer (α₂β₂) | 0.668 | |
| HisRS | 12 | 47.0 | Homodimer | 0.128 | |
| IleRS | 622 | 104.4 | Monomer | 5.96 | |
| LeuRS | 63 | 97.2 | Monomer | 0.648 | |
| LysRS | 99 | 57.6 | Homodimer | 0.859 | |
| MetRS | 36 | 76.3 | Homodimer | 0.236 | |
| PheRS* | 264 | 248.4 | Heterotetramer (α₂β₂) | 1.06 | |
| ProRS | 155 | 63.7 | Homodimer | 1.22 | |
| SerRS | 30 | 48.4 | Homodimer | 0.310 | |
| ThrRS | 97 | 74.0 | Homodimer | 0.655 | |
| TrpRS | 97 | 37.4 | Homodimer | 1.30 | |
| TyrRS | 10 | 47.5 | Homodimer | 0.105 | |
| ValRS | 28 | 108.2 | Monomer | 0.259 | |
| Initiation Factors | IF1 | 155 | 8.2 | Monomer | 18.9 |
| IF2 | 622 | 97.3 | Monomer | 6.39 | |
| IF3 | 155 | 20.6 | Monomer | 7.52 | |
| Elongation Factors | EF-G | 777 | 77.6 | Monomer | 10.0 |
| EF-Tu | 7764 | 43.3 | Monomer | 179 | |
| EF-Ts | 777 | 30.4 | Monomer | 25.6 | |
| Release Factors | RF1 | 155 | 40.5 | Monomer | 3.83 |
| RF2 | 155 | 41.3 | Monomer | 3.75 | |
| RF3 | 155 | 59.6 | Monomer | 2.60 | |
| RRF | 155 | 20.6 | Monomer | 7.52 | |
| Metabolism | MTF | 311 | 34.2 | Monomer | 9.09 |
| CK | 63 | 43.3 | Homodimer | 0.727 | |
| AK | 46 | 23.6 | Monomer | 1.95 | |
| NDK | 16 | 15.5 | Homohexamer | 0.172 | |
| PPiase | 16 | 19.7 | Homohexamer | 0.135 | |
| Transcription | T7RNAP | 155 | 98.9 | Monomer | 1.57 |
| Total | 15 000 |
The Small Molecule Mix (SMix) contains the buffers, nucleotides, amino acids, stabilizers, and cofactors. Concentrations below are in the assembled SMix; see the Make SMix process for preparation.
Composition of the Small Molecule Mix (SMix) at stock concentration.
| Category | Molecule | Concentration | Units |
|---|---|---|---|
| Buffers | HEPES-KOH (pH 7.6) | 125 | mM |
| Potassium Glutamate | 250 | mM | |
| Magnesium Acetate | 18.75 | mM | |
| Nucleotides | rATP | 5 | mM |
| rGTP | 5 | mM | |
| rCTP | 2.5 | mM | |
| rUTP | 2.5 | mM | |
| Amino Acids | Each (Ala–Val) | 0.75 | mM |
| Stabilizers | Spermidine | 5 | mM |
| TCEP | 2.5 | mM | |
| Cofactors | Creatine Phosphate | 50 | mM |
| Folinic Acid | 0.05 | mM |
Ribosome stock concentration.
| Component | Stock Concentration | Reaction Concentration |
|---|---|---|
| Ribosomes | 10 µM (5.55×) | 1.8 µM |
tRNA stock concentration.
| Component | Stock Concentration | Reaction Concentration |
|---|---|---|
| tRNA | 35 µg/µL (10×) | 3.5 µg/µL |
Composition of Base Cytosol at reaction concentration.
| Category | Molecule | Concentration | Units |
|---|---|---|---|
| Buffers | HEPES-KOH | 50 | mM |
| Potassium Glutamate | 100 | mM | |
| Magnesium Acetate | 7.5 | mM | |
| Nucleotides | rATP | 2 | mM |
| rGTP | 2 | mM | |
| rCTP | 1 | mM | |
| rUTP | 1 | mM | |
| Amino Acids | Alanine (Ala) | 0.3 | mM |
| Arginine (Arg) | 0.3 | mM | |
| Asparagine (Asn) | 0.3 | mM | |
| Aspartic Acid (Asp) | 0.3 | mM | |
| Cysteine (Cys) | 0.3 | mM | |
| Glutamic Acid (Glu) | 0.3 | mM | |
| Glutamine (Gln) | 0.3 | mM | |
| Glycine (Gly) | 0.3 | mM | |
| Histidine (His) | 0.3 | mM | |
| Isoleucine (Ile) | 0.3 | mM | |
| Leucine (Leu) | 0.3 | mM | |
| Lysine (Lys) | 0.3 | mM | |
| Methionine (Met) | 0.3 | mM | |
| Phenylalanine (Phe) | 0.3 | mM | |
| Proline (Pro) | 0.3 | mM | |
| Serine (Ser) | 0.3 | mM | |
| Threonine (Thr) | 0.3 | mM | |
| Tryptophan (Trp) | 0.3 | mM | |
| Tyrosine (Tyr) | 0.3 | mM | |
| Valine (Val) | 0.3 | mM | |
| Stabilizers | Spermidine | 2 | mM |
| TCEP-HCl | 1 | mM | |
| Cofactors | Creatine Phosphate | 20 | mM |
| Folinic Acid | 0.02 | mM | |
| Ribonucleics | tRNA | 3.5 | µg/µL |
| Ribosomes | 1.8 | µM | |
| tRNA Synthetases | AlaRS | 130.3 | ng/µL |
| ArgRS | 3.6 | ng/µL | |
| AsnRS | 40.9 | ng/µL | |
| AspRS | 15.0 | ng/µL | |
| CysRS | 2.2 | ng/µL | |
| GlnRS | 7.0 | ng/µL | |
| GluRS | 23.5 | ng/µL | |
| GlyRS | 17.9 | ng/µL | |
| HisRS | 1.5 | ng/µL | |
| IleRS | 74.6 | ng/µL | |
| LeuRS | 7.5 | ng/µL | |
| LysRS | 11.9 | ng/µL | |
| MetRS | 4.4 | ng/µL | |
| PheRS | 31.7 | ng/µL | |
| ProRS | 18.7 | ng/µL | |
| SerRS | 3.6 | ng/µL | |
| ThrRS | 11.6 | ng/µL | |
| TrpRS | 11.6 | ng/µL | |
| TyrRS | 1.2 | ng/µL | |
| ValRS | 3.4 | ng/µL | |
| Initiation Factors | IF1 | 18.7 | ng/µL |
| IF2 | 74.6 | ng/µL | |
| IF3 | 18.7 | ng/µL | |
| Elongation Factors | EF-G | 93.3 | ng/µL |
| EF-Tu | 931.7 | ng/µL | |
| EF-Ts | 93.3 | ng/µL | |
| Release Factors | RF1 | 18.7 | ng/µL |
| RF2 | 18.7 | ng/µL | |
| RF3 | 18.7 | ng/µL | |
| RRF | 18.7 | ng/µL | |
| Metabolism | MTF | 37.3 | ng/µL |
| CK | 7.5 | ng/µL | |
| AK | 5.6 | ng/µL | |
| NDK | 1.9 | ng/µL | |
| PPiase | 1.9 | ng/µL | |
| Transcription | T7RNAP | 18.7 | ng/µL |
Expected Behavior¶
The behavior of Base Cytosol is characterized using the deGFP Reporter Module.
Protocols¶
Protocols for assembling Base Cytosol and making its components from scratch can be found on Base Cytosol Processes.
- 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