Cytosols are the “insides” of synthetic cells, aqueous mixtures of proteins, RNAs, small organic molecules, and salts that recapitulate user desired biological functions. We recommend the PURE system as our cytosol of choice. PURE is a defined set of molecules that recapitulate transcription and translation. PURE is commercially available in small quantities (~1 mL) from several providers, or can be prepared by following 
PURE Protocols.
PURE GuideIntroduction to PURE and how to make it.
Our latest cytosol release is Nucleus PURE v0.3.0. Our Protein Mix and Energy Mix are working at ~100% the performance of NEB PURExpress. We are working on our Ribosome purification protocol; stay tuned for v0.4.
For a comprehensive list of protocols used to make PURE, check out PURE Protocols.
Making PURE
Make Protein Mix - Prepare the consumables you will need. Make buffers and cell culture media. Assemble gravity columns, pack them with affinity resin, equilibrate, and store.
Prepare Columns, 
Make Protein Purification Buffers and Media - Grow out E.coli expression strains expressing the thirty-six (36) protein of PURE, induce protein expression, and collect bacterial pellets for downstream purification. Grow and Induce Expression Strains
- Lyse your bacteria using a sonicator, allowing you to access the proteins inside. The resulting mixture of cell debris and target proteins is called “lysate”. Remove debris by centrifugation and filtering to make “clarified lysate”.
Lyse Bacteria by Sonication - Purify your target proteins from clarified lysate by flowing them through your affinity columns. PURE proteins are expressed with a polyhistidine affinity tag, which selectively binds Ni2+ affinity resin. Flow your clarified lysate through your columns, wash out debris, and elute your target proteins with a high salt (Imidazole) buffer.
Purify Proteins by Ni2+ Gravity Column - Wash and Concentrate your proteins simultaneously using a centrifugal filter. Load your sample, remove the high salt buffer by centrifugation. Then, wash with fresh buffer, centrifuge, and repeat. Finally, centrifuge to remove buffer from your sample until you reach your target concentration.
Exchange Buffers and Concentrate by Spin Filtration - Measure the concentration and purity of your proteins by protein gel electrophoresis and Pierce660 Assay, respectively.
Protein Gel 
Pierce660 Assay. - Assemble Protein Mix by combining each purified protein at its specified concentration. Aliquot and store at -80C. Assemble Protein Mix
Make Energy Mix - Make tRNAs by growing out an E. coli culture, extracting small nucleic acids with acid phenol, and purifying tRNAs from small DNA by alcohol precipitation. Measure the purity and concentration of your tRNAs by Urea gel elecrophoresis and UV-Vis spectroscopy (Nanodrop), respectively.
Make tRNAs
- Make Amino Acid Mix by weighing, resuspending, and combining twenty (20) amino acids. Make Amino Acid Mix
- Assemble Energy Mix. Prepare the remaining eleven (11) components of Energy Mix. Then, combine with tRNA and Amino Acid Mix. Aliquot and store at -80C. Make Energy Mix . Make Energy Mix
Assemble PURE Reactions
Original PURE paper
OnePot PURE
