Cytosol Documentation

Getting Started

Cytosols are the insides of synthetic cells. Cytosols are aqueous mixtures of proteins, RNAs, small organic molecules, and salts that together recapitulate a desired biological function (e.g., transcription and translation).

We currently 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, and is commercially available.

PURE is broadly composed of two (2) classes of components:

1. Protein Mix: thirty-six (36) proteins that reconstitute transcription, translation, amino acylation, and energy recycling
2. Energy Mix: thirty-one (31) small organic molecules and salts, plus forty-three (43) unique tRNAs, that support transcription and translation

Guides

PURE Guide

Introduction to PURE and how to make it.

Protocols

PURE Protocols

Making PURE

1. Prepare Buffers and Columns

Preparing and setting up buffers and materials, as well as the energy mix used in PURE.

2. Prepare Bacterial Lysates for Protein Purification

Growing, inducing, and preparing lysate from strains that over-express proteins used in PURE.

3. Purify Proteins

Purifying proteins from prepared lysates

4. Run PURE reactions

Assembling and running PURE reactions

Measuring PURE

Protocols for QA/QC and measuring PURE performance.

Spec Sheet

Our latest cytosol release is Nucleus PURE v0.1.2, with specifications as below. We achieved a 2x and 4x yield improvement with v0.1.2 relative to v0.1.1 and v0.1.0 respectively, and are currently at ~70% the performance of NEB PURE.

Composition (v0.1.2)

#	Enzyme	Final Conc. [ng/uL]
1	Ala RS	538
2	Arg RS	15
3	Asn RS	169
4	Asp RS	62
5	Cys RS	9
6	Gln RS	29
7	Glu RS	97
8	Gly RS	74
9	His RS	6
10	Ile RS	308
11	Leu RS	31
12	Lys RS	49
13	Met RS	18
14	Phe RS	131
15	Pro RS	77
16	Ser RS	15
17	Thr RS	48
18	Trp RS	48
19	Tyr RS	5
20	Val RS	14
21	IF1	77
22	IF2	308
23	IF3	77
24	EF-G	385
25	EF-Tu	3846
26	EF-Ts	385
27	RF1	77
28	RF2	77
29	RF3	77
30	RRF	77
31	MTF	154
32	CK	31
33	MK	23
34	NDK	8
35	PPiase	8
36	T7RNAP	77
	Total	7430

Performance

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