Make Energy Mix

Getting Started

You want to make PURE. You’ll need Energy Mix, the combination thirty-one (31) small molecules (e.g., rNTPs, amino acids, buffering salts, etc.) and tRNAs (from E. coli) needed to fuel transcription and translation.

Here, we describe the components of Energy Mix, how to make it, and how to store it properly.

Reagent	Concentration in Energy Mix (mM)	Concentration in Final Reaction (mM)
HEPES-KOH (pH 7.6)	125	50
Potassium glutamate	250	100
Magnesium acetate	29.5	11.8
rATP	5	2
rGTP	5	2
rCTP	2.5	1
rUTP	2.5	1
Amino Acids (each)	0.75	0.325
Creatine phosphate	50	20
Folinic acid	0.05	0.02
Spermidine	5	2
TCEP	2.5	1
tRNA (ug/uL)	8.75	3.5

Prerequisite Protocols

Make Amino Acid Mix (3.25 mM):

Make Amino Acid Mix

Make tRNAs (40 ug/uL):

Make tRNAs

Nucleus_v0.3.0_EMix-worksheet.xlsx11.4KB

Materials and Equipment

Creatine phosphate	Sodium creatine phosphate dibasic tetrahydrate, ≥98.0%(NT)	Sigma-Aldrich	27920-1G	$102	-25C to -15C	[link]
Name	Product	Manufacturer	Part #	Price	Storage Conditions	Link
Potassium Hydroxide	Potassium hydroxide - ACS reagent, ≥85%, pellets	Sigma-Aldrich	221473-1KG	$99.90	4C to 30C	[link]
Hydrochloric acid (1 M)	Hydrochloric acid 1 N, Reagent Grade, Packaging=Amber Bottle, Size=500 mL	VWR	E447-500L	$72.46	4C to 30C	[link]
HEPES	HEPES, Crystalline Powder, ≥99.5% (titration), Poly bottle	Sigma-Aldrich	H3375-500G	$431	4C to 30C	[link]
Potassium Glutamate	L-Glutamic acid potassium monohydrate	Sigma-Aldrich	G1501-100G	$74.80	4C to 30C	[link]
Magnesium acetate	Magnesium acetate tetrahydrate	Sigma-Aldrich	M0631-100G	$34.80	4C to 30C	[link]
rATP	Adenosine 5′-triphosphate disodium salt hydrate, BioXtra, ≥99% (HPLC), from microbial	Sigma-Aldrich	A7699-1G	$125.00	-25C to -15C	[link]
rGTP	Guanosine 5′-triphosphate sodium salt hydrate, ≥ 95% (HPLC), powder	Sigma-Aldrich	G8877-100MG	$155	-25C to -15C	[link]
rCTP	Cytidine 5′-triphosphate disodium salt, ≥95%	Sigma-Aldrich	C1506-100MG	$102.00	-25C to -15C	[link]
rUTP	Uridine 5′-triphosphate trisodium salt hydrate, Type IV, ≥93.0% (HPLC)	Sigma-Aldrich	U6750-100MG	$58.20	-25C to -15C	[link]
TCEP-HCl (500 mM; presuspended)	Thermo Scientific, Bond-Breaker® Tris[2-Carboxyethyl]phosphine Neutral Solution (TCEP), Application=Water-Soluble Reducing Agent, Molecular Weight=286.65, Size=5 mL	Thermo Scientific	77720	$175.65	4C to 30C	[link]
Folinic acid	Folinic acid calcium salt hydrate	Sigma-Aldrich	F7878-100MG	$108	4C to 30C	[link]
BME	2-Mercaptoethanol	Sigma-Aldrich	M6250-100ML	$65.50	4C to 30C	[link]
Spermidine	Spermidine, ≥99% (GC)	Sigma-Aldrich	S2626-5G	$176	-25C to -15C	[link]
tRNA	Nucleus Total tRNA (50 ug / uL; see Make tRNAs)	Sigma-Aldrich	R1753-100UN	$69.00	-25C to -15C	[link]
Amino Acids	Amino Acid Mix (3.25 mM; see Make Amino Acid Mix)				-80C to -20C

Protocol

Folinic Acid Preparation

For more information please see our developer note:

36-Pot PURE

Prepare Folinic Acid Stock (2.56 mg / mL, 5 mM).

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Notes: This step converts Folinic acid to 5,10-methenyl-THF

Prepare 50 mM BME by adding 17.5 uL pure BME (14.3 M) to 50 mL H2O.

Weigh 25 mg Folinic acid.

Resuspend in 2 mL BME (50 mM).

Add 220 uL HCl (1M).

Incubate at 25C / 3 hr. The solution should turn yellow.

Dilute by adding 7.55 mL deionized water and mix by inversion.

Aliquot and freeze at -20C.

One-Pot PURE

Prepare Folinic Acid Stock (5 mM)

Weigh 12.5 mg folinic acid

Dissolve to a final volume of 4.89 mL

Aliquot and freeze at -20C.

Make the following stock solutions

Folinic acid (5 mM)	511.50	See above		-25C to -15C	no	no
Reagent	MW (g/mol)	Amount (g)	Final Volume (mL)	Storage	Needs pH adjustment?	Needs Sterilization?
Potassium Hydroxide (1 M)	56.11	14.0	250	4C to 30C	no	no
HEPES-KOH (pH 7.6, 1 M)	238.3	59.5	250	4C to 30C; dark	yes	no
Potassium glutamate (2.5 M)	203.23	21.8	50	4C to 30C	no	no
Magnesium acetate (1 M)	214.45	10.8	50	4C to 30C	no	no
Creatine phosphate (500 mM)	327.14	1	9.43	-25C to -15C	no	no
Spermidine (200 mM)	145.25	3.64	50	-25C to -15C	no	no
Amino Acid Mix			see Make Amino Acid Mix	-85C to -15C	yes	yes

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Tips: adjust pH then volume

Thaw tRNAs on ice immediately before use.

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Notes: tRNAs are fragile!

Combine Energy Mix components.

Reagent	Stock Concentration (mM)	Concentration in Energy Mix (mM)	Volume to Add (uL)
HEPES-KOH (pH 7.6)	1000	125	62.5
Potassium glutamate	2500	250	50.0
Magnesium acetate	1000	29.5	14.8
rATP	100	5	25.0
rGTP	100	5	25.0
rCTP	100	2.5	12.5
rUTP	100	2.5	12.5
Creatine phosphate	500	50	50.0
TCEP	500	2.5	2.5
Folinic acid	5	0.05	5.0
Spermidine	500	5	5.0
Amino Acid Mix	3.25	0.75	115.4
tRNA (ug/uL)	40	8.75	87.5
Water	n/a	n/a	32.3
Total			500

Aliquot Energy Mix into 1.5 mL microfuge tubes (between 50 uL and 100 uL per aliquot) and store at -80C.

Resources and References

The OnePot PURE methods paper (JOVE) has great interactive tables!

Use Supplementary Table 4 for making stock solutions

OnePot-JOVE-Table-S4-stock-solutions.xlsx11.5KB

Use Supplementary Table 5 for assembling Energy Mix

OnePot-JOVE-Table-S5-energy-mix-worksheet.xlsx10.9KB

Papers

Original PURE paper

Cell-free translation reconstituted with purified components

Nature Biotechnology - Cell-free translation reconstituted with purified components

doi.org

Cell-free translation reconstituted with purified components

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

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

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...