Cytosol Documentation

Getting Started

Cytosols are the insides of synthetic cells, aqueous mixtures of “components” (e.g., proteins, RNAs, small organic molecules, and salts) that recapitulate user desired biological functions (e.g., transcription and translation).

We use the PURE system as our cytosol of choice. PURE is a defined set of 108 molecules (proteins, RNAs, small organic molecules, and salts) 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.

Guides

PURE Guide

Introduction to PURE and how to make it.

Spec Sheet

Our latest cytosol release is Nucleus PURE v0.2.0. We improved yield ~ 4x over v0.1.1, and are currently at ~70% the performance of NEB PURExpress.

Protocols

For a comprehensive list of protocols used to make PURE, check out PURE Protocols.

Making PURE

1. Make Protein Mix
1. Prepare Columns and Make Protein Purification Buffers and Media - get ready to purify proteins by making all the buffers and media you will need, as well as prepare the columns that you will use to purify the proteins.
2. Grow and Induce Expression Strains - Using thirty-six (36) E. coli expression strains, grow bacterial cultures, induce protein expression, and collect bacterial pellets for downstream purification.
3. Lyse Bacteria by Sonication - Take your pellets and break open the bacteria using a sonicator so that you can access the proteins inside. The resulting mixture of cell debris and target proteins is called “lysate”.
4. Purify Proteins by Ni-His Gravity Column - Purify your target proteins from lysate by running them over an affinity column. PURE proteins have a purification tag (6xHis) that makes them selectively bind the column, allowing the cell debris to wash away. Elute PURE proteins off of the column using a high salt (Imidazole) buffer.
5. Exchange Buffers and Concentrate by Spin Filtration - Take your purified proteins out of elution buffer and concentrate them down to easier to handle concentrations by using centrifugal filters.
6. Check the purity of your protein preps by Protein Gel. Check the concentration of your protein stocks by Pierce660 Assay
7. Assemble Protein Mix - Assemble proteins into Protein Mix.
2. Make Energy Mix
1. Make Amino Acid Mix - One buffer stock for each of twenty (20) amino acids.
2. Make twelve (12) other component buffers (see Make Energy Mix)
3. Combine Amino Acid Mix and component buffers into Energy Mix (see Make Energy Mix)
3. Assemble PURE Reactions - Assemble and run PURE.

Developer Notes

Developer Notes

Name	Tags
PURE protein expression in pOpen vs pET28a	Cytosol

Further Reading

Original PURE paper

Cell-free translation reconstituted with purified components

Nature Biotechnology - Cell-free translation reconstituted with purified components

doi.org

OnePot PURE

A Simple, Robust, and Low-Cost Method To Produce the PURE Cell-Free System

We demonstrate a simple, robust, and low-cost method for producing the PURE cell-free transcription–translation system. Our OnePot PURE system achieved a protein synthesis yield of 156 μg/mL at a cost of 0.09 USD/μL, leading to a 14-fold improvement in cost normalized protein synthesis yield over existing PURE systems. The one-pot method makes the PURE system easy to generate and allows it to be readily optimized and modified.

doi.org

OnePot PURE Cell-Free System | Text Page

Scientific Article | The defined PURE (protein synthesis using recombinant elements) transcription-translation system provides an appealing chassis for...

dx.doi.org