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.
Schematic of components in Cytosol and their function. Figure by Ganesh, R. B. & Maerkl, S. J. used under CC-BY-4.0 / cropped from original with numerical annotations removed.
Plasmid designs are included in the documentation of the DNA Distribution.
Reference Composition¶
Composition of Base Cytosol.
| 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/mL |
| Ribosomes | 1.8 | μM | |
| tRNA Synthetases | AlaRS | 139 | ng/μL |
| ArgRS | 3.87 | ng/μL | |
| AsnRS | 43.7 | ng/μL | |
| AspRS | 16.0 | ng/μL | |
| CysRS | 2.32 | ng/μL | |
| GlnRS | 7.49 | ng/μL | |
| GluRS | 25.1 | ng/μL | |
| GlyRS | 19.1 | ng/μL | |
| HisRS | 1.55 | ng/μL | |
| IleRS | 79.6 | ng/μL | |
| LeuRS | 8.01 | ng/μL | |
| LysRS | 12.7 | ng/μL | |
| MetRS | 4.66 | ng/μL | |
| PheRS | 33.9 | ng/μL | |
| ProRS | 19.9 | ng/μL | |
| SerRS | 3.87 | ng/μL | |
| ThrRS | 12.4 | ng/μL | |
| TrpRS | 12.4 | ng/μL | |
| TyrRS | 1.29 | ng/μL | |
| ValRS | 3.62 | ng/μL | |
| Initiation Factors | IF1 | 19.9 | ng/μL |
| IF2 | 79.6 | ng/μL | |
| IF3 | 19.9 | ng/μL | |
| Elongation Factors | EF-G | 99.5 | ng/μL |
| EF-Tu | 994 | ng/μL | |
| EF-Ts | 99.5 | ng/μL | |
| Release Factors | RF1 | 19.9 | ng/μL |
| RF2 | 19.9 | ng/μL | |
| RF3 | 19.9 | ng/μL | |
| RRF | 19.9 | ng/μL | |
| Metabolism | MTF | 39.8 | ng/μL |
| CK | 8.01 | ng/μL | |
| MK | 5.94 | ng/μL | |
| NDK | 2.07 | ng/μL | |
| PPiase | 2.07 | ng/μL | |
| Transcription | T7RNAP | 19.9 | 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