Make Energy Mix

Getting Started

You’ve got a mixture of proteins and ribosomes that you think should be able to perform transcription and translation. But you’ll need more than proteins and riboproteins to run those reactions. Energy mix is the solution! It’s made of the thirty-one (31) small molecules (e.g., rNTPs, amino acids, buffering salts, etc.) and tRNAs (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	125	50
Potassium glutamate	250	100
Magnesium acetate	29.5	11.8
rNTPs (each)	5	2
tRNA [mg/mL]	8.75	3.5
Amino Acids (each)	0.75	0.325
Creatine phosphate	50	20
TCEP	2.5	1
Folinic acid	0.05	0.02
Spermidine	5	2

Prerequisite Protocols

Make amino acid mix (3.25 mM):

Make Amino Acid Mix

Materials and Equipment

Name	Product	Manufacturer	Part #	Price	Link	Storage Conditions
Potassium Hydroxide	Potassium hydroxide - ACS reagent, ≥85%, pellets	Sigma-Aldrich	221473-1KG	$99.90	4C to 30C	[link]
HEPES	HEPES, Crystalline Powder, ≥99.5% (titration), Poly bottle	Sigma-Aldrich	H3375-500G	$431	[link]	4C to 30C
Potassium Glutamate	L-Glutamic acid potassium monohydrate	Sigma-Aldrich	G1501-100G	$74.80	[link]	4C to 30C
Magnesium acetate	Magnesium acetate tetrahydrate	Sigma-Aldrich	M0631-100G	$34.80	[link]	4C to 30C
rATP	Adenosine 5′-triphosphate disodium salt hydrate, BioXtra, ≥99% (HPLC), from microbial	Sigma-Aldrich	A7699-1G	$125.00	[link]	-25C to -15C
rGTP	Guanosine 5′-triphosphate sodium salt hydrate, ≥ 95% (HPLC), powder	Sigma-Aldrich	G8877-100MG	$155	[link]	-25C to -15C
rCTP	Cytidine 5′-triphosphate disodium salt, ≥95%	Sigma-Aldrich	C1506-100MG	$102.00	[link]	-25C to -15C
rUTP	Uridine 5′-triphosphate trisodium salt hydrate, Type IV, ≥93.0% (HPLC)	Sigma-Aldrich	U6750-100MG	$58.20	[link]	-25C to -15C
Creatine phosphate	Sodium creatine phosphate dibasic tetrahydrate, ≥98.0%(NT)	Sigma-Aldrich	27920-1G	$102	[link]	-25C to -15C
TCEP-HCl (0.5M; 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	[link]	4C to 30C
Spermidine	Spermidine, ≥99% (GC)	Sigma-Aldrich	S2626-5G	$176	[link]	-25C to -15C
tRNAs	Ribonucleic acid, transfer from Escherichia coli (Type XX, Strain W, lyophilized)	Sigma-Aldrich	R1753-100UN	$69.00	[link]	-25C to -15C
Amino Acids	Amino Acid Mix (3.25 mM; see Make Amino Acid Mix)					-80C to -20C

Protocol

Make the following stock solutions

MW [g/mol]	Amount [g]	Final Volume [mL]	Storage	Reagent	Needs pH adjustment?	Needs Sterilization?
56.11	14.0	250	4C to 30C	Potassium Hydroxide (1M)	no	no
238.3	59.5	250	4C to 30C; dark	HEPES-KOH (pH=7.6; 1M)	yes	no
203.23	46.3	100	4C to 30C	Potassium glutamate (2.5M)	no	no
214.45	21.5	100	4C to 30C	Magnesium acetate (1M)	no	no
327.14	1	9.43	-25C to -15C	Creatine phosphate (500 mM)	no	no
511.50	0.119	50	1C to 4C	Folinic acid (5 mM)	no	no
145.25	3.64	50	-25C to -15C	Spermidine (200 mM)	no	no
		see Make Amino Acid Mix	-85C to -15C	Amino acid mix	yes	yes

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Tips: adjust pH then volume

Assemble components of Energy Mix (except tRNAs)

Component	Stock Concentration [mM]	Concentration in Energy Mix [mM]	Volume to Add [uL]
HEPES	1000	125	62.5
Potassium glutamate	2500	250	50.0
Magnesium acetate	1000	29.5	14.8
rNTPs (each)	25	5	100.0
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 solution	3.25	0.75	115.4
Water	n/a	n/a	94.9
Total			500

Open a fresh vial of tRNAs (100 U); pipette 476 uL of Energy Mix (except tRNAs) into the vial. Mix by pipetting briefly until tRNAs are resuspended.

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Notes: 100 A260 U ~ 5 mg tRNA

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Notes: tRNAs are fragile!

Aliquot Energy Mix into 1.5 mL microfuge tubes (between 50 uL and 100 uL per aliquot)

Store Energy Mix aliquots frozen (-75C and -85C)

Resources and References

Other Protocols

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

Use Supplementary Table 4 for making stock solutions
Use Supplementary Table 5 for making final Energy Solution

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...