Step 2: Preparing amino acid, polyphosphates, and maltodextrin-based energy solutions for cell-free reactions

Anibal Arce Medina; Juan Puig Contreras; Fernan Federici

Oct 17, 2018

Version 3

Step 2: Preparing amino acid, polyphosphates, and maltodextrin-based energy solutions for cell-free reactions V.3

DOI

dx.doi.org/10.17504/protocols.io.urzev76

¹Pontificia Universidad Católica de Chile;
²Pontificia Universidad Catolica de Chile

Laboratorio de Tecnologias Libres

Anibal Arce Medina

Pontificia Universidad Catolica de Chile

DOI: dx.doi.org/10.17504/protocols.io.urzev76

Protocol Citation: Anibal Arce Medina, Juan M Puig Contreras, Fernan Federici 2018. Step 2: Preparing amino acid, polyphosphates, and maltodextrin-based energy solutions for cell-free reactions. protocols.io https://dx.doi.org/10.17504/protocols.io.urzev76

License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Protocol status: Working

We use this protocol and it's working

Created: October 17, 2018

Last Modified: October 17, 2018

Protocol Integer ID: 16921

Materials

STEP MATERIALS
Reagentsodium hexametaphosphateMerck MilliporeSigma (Sigma-Aldrich)Catalog #305553 
ReagentDTTMerck MilliporeSigma (Sigma-Aldrich)Catalog #D0632 
ReagentCoAMerck MilliporeSigma (Sigma-Aldrich)Catalog #C4282 
ReagentFolinic AcidMerck MilliporeSigma (Sigma-Aldrich)Catalog #F7878 
ReagentSpermidineMerck MilliporeSigma (Sigma-Aldrich)Catalog #85558 
ReagentNAD (β-Nicotinamide adenine dinucleotide hydrate)Merck MilliporeSigma (Sigma-Aldrich)Catalog #N6522 
ReagentHEPESMerck MilliporeSigma (Sigma-Aldrich)Catalog #H6147 
ReagentATP (Adenosine 5′-triphosphate dipotassium salt hydrate)Merck MilliporeSigma (Sigma-Aldrich)Catalog #A8937 
ReagentGTP (Guanosine 5′-triphosphate sodium salt hydrate)Merck MilliporeSigma (Sigma-Aldrich)Catalog #10106399001 ROCHE 
ReagentUTP (Uridine 5′-triphosphate trisodium salt dihydrate)Merck MilliporeSigma (Sigma-Aldrich)Catalog #94370  
ReagentMaltodextrin (4.0-7.0 dextrose equivalent)Merck MilliporeSigma (Sigma-Aldrich)Catalog #419672 
ReagentL-aminoacidsMerck MilliporeSigma (Sigma-Aldrich)Catalog #LAA21–1KT 
ReagentL-ProlineMerck MilliporeSigma (Sigma-Aldrich)Catalog #81709–25G 
ReagentL-CysteineMerck MilliporeSigma (Sigma-Aldrich)Catalog #30089 
ReagentL-histidineCatalog #53319–25G 
ReagentL-lysineCatalog #L5501–5G 

Protocol materials

Reagentsodium hexametaphosphateMerck MilliporeSigma (Sigma-Aldrich)Catalog #305553 
ReagentL-aminoacidsMerck MilliporeSigma (Sigma-Aldrich)Catalog #LAA21–1KT 
ReagentL-ProlineMerck MilliporeSigma (Sigma-Aldrich)Catalog #81709–25G 
ReagentCoAMerck MilliporeSigma (Sigma-Aldrich)Catalog #C4282 
ReagentSpermidineMerck MilliporeSigma (Sigma-Aldrich)Catalog #85558 
ReagentUTP (Uridine 5′-triphosphate trisodium salt dihydrate)Merck MilliporeSigma (Sigma-Aldrich)Catalog #94370  
ReagentL-CysteineMerck MilliporeSigma (Sigma-Aldrich)Catalog #30089 
ReagentATP (Adenosine 5′-triphosphate dipotassium salt hydrate)Merck MilliporeSigma (Sigma-Aldrich)Catalog #A8937 
ReagentDTTMerck MilliporeSigma (Sigma-Aldrich)Catalog #D0632 
ReagentL-lysineCatalog #L5501–5G 
ReagentFolinic AcidMerck MilliporeSigma (Sigma-Aldrich)Catalog #F7878 
ReagentHEPESMerck MilliporeSigma (Sigma-Aldrich)Catalog #H6147 
ReagentMaltodextrin (4.0-7.0 dextrose equivalent)Merck MilliporeSigma (Sigma-Aldrich)Catalog #419672 
ReagentL-histidineCatalog #53319–25G 
ReagentNAD (β-Nicotinamide adenine dinucleotide hydrate)Merck MilliporeSigma (Sigma-Aldrich)Catalog #N6522 
ReagentGTP (Guanosine 5′-triphosphate sodium salt hydrate)Merck MilliporeSigma (Sigma-Aldrich)Catalog #10106399001 ROCHE 
ReagentL-CysteineMerck MilliporeSigma (Sigma-Aldrich)Catalog #30089 
ReagentL-histidineCatalog #53319–25G 
ReagentL-lysineCatalog #L5501–5G 
ReagentL-aminoacidsMerck MilliporeSigma (Sigma-Aldrich)Catalog #LAA21–1KT 
ReagentL-ProlineMerck MilliporeSigma (Sigma-Aldrich)Catalog #81709–25G 
ReagentSpermidineMerck MilliporeSigma (Sigma-Aldrich)Catalog #85558 
ReagentNAD (β-Nicotinamide adenine dinucleotide hydrate)Merck MilliporeSigma (Sigma-Aldrich)Catalog #N6522 
ReagentHEPESMerck MilliporeSigma (Sigma-Aldrich)Catalog #H6147 
ReagentGTP (Guanosine 5′-triphosphate sodium salt hydrate)Merck MilliporeSigma (Sigma-Aldrich)Catalog #10106399001 ROCHE 
ReagentMaltodextrin (4.0-7.0 dextrose equivalent)Merck MilliporeSigma (Sigma-Aldrich)Catalog #419672 
ReagentDTTMerck MilliporeSigma (Sigma-Aldrich)Catalog #D0632 
ReagentCoAMerck MilliporeSigma (Sigma-Aldrich)Catalog #C4282 
ReagentUTP (Uridine 5′-triphosphate trisodium salt dihydrate)Merck MilliporeSigma (Sigma-Aldrich)Catalog #94370  
ReagentFolinic AcidMerck MilliporeSigma (Sigma-Aldrich)Catalog #F7878 
ReagentATP (Adenosine 5′-triphosphate dipotassium salt hydrate)Merck MilliporeSigma (Sigma-Aldrich)Catalog #A8937 
Reagentsodium hexametaphosphateMerck MilliporeSigma (Sigma-Aldrich)Catalog #305553 

Prepare individual amino acid stock solutions

 
Dissolve the given amount of each amino acid with 1 ml of 5M KOH in a 3 ml screw-cap tube according to the following list.
 
 
AminoacidAmount of powder  needed
Alanine445.45 mg
Arginine644.6502 mg
Asparagine660.6 mg
Aspartic acid665.5 mg
Cysteine370.7496 mg
Glutamic acid735.65 mg
Glutamine447.219 mg
Glycine375.35 mg
Histidine775.75 mg
Isoleucine655.9 mg
Leucine401.4108 mg
Lysine447.3414 mg
Methionine456.5826 mg
Phenylalanine330.38 mg
Proline575.65 mg
Serine529.5 mg
Threonine595.6 mg
Tryptophan408.46 mg
Tyrosine554.4414 mg
Valine358.479 mg
 
 Vortex to dissolve.  Each solution can be stored at -20 °C.
ReagentL-aminoacidsMerck MilliporeSigma (Sigma-Aldrich)Catalog #LAA21–1KT 
ReagentL-ProlineMerck MilliporeSigma (Sigma-Aldrich)Catalog #81709–25G 
ReagentL-CysteineMerck MilliporeSigma (Sigma-Aldrich)Catalog #30089 
ReagentL-histidineCatalog #53319–25G 
ReagentL-lysineCatalog #L5501–5G 

Prepare amino acid mix solution (∼17 nM each)

 In a 50 mL Falcon tube add the following volumes of the individual amino acid stock solutions  
 
 
Aminoacid Stock solution in 5M KOHVolume needed
Alanine136 ul
Arginine222 ul
Asparagine136 ul
Aspartic acid136 ul
Cysteine222 ul
Glutamic acid136 ul
Glutamine222 ul
Glycine136 ul
Histidine136 ul
Isoleucine136 ul
Leucine222 ul
Lysine222 ul
Methionine222 ul
Phenylalanine340 ul
Proline136 ul
Serine136 ul
Threonine136 ul
Tryptophan340 ul
Tyrosine222 ul
Valine222 ul
TOTAL3816 µL
  
  
  
 

Prepare amino acid mix solution (∼14 mM each)

Add 35.084 µL of sterile water and 1.100 µl of Acetic acid (glacial) to the amino acid mix. Vortex well and aliquot in 2.0 ml Eppendorf tubes. Aliquots can be stored at -80°C

Prepare maltodextrin-based energy solution

    Prepare the following stock solutions:
  
 
 
 
 
  1M DTT: Put 2.31 g DTT in a 15 ml Falcon tube and fill with water to 15 ml. Sterilize using a 0.22 μM filter. Aliquot in 1.5 ml Eppendorf tubes. Store at -20 °C for later use.
2M HEPES pH 8:  Weight 19.1 g HEPES (MW 238.21). Dissolve with 30ml water. Adjust pH to 8.0 with KOH. Fill with water to 40 ml.
 5 mg/ml tRNA solution: Put 30 mg of tRNA in a 1.5 ml Eppendorf tube. Fill with water to 600 μl.
 CoA stock solution: Put 30 mg of CoA (MW 767.53) in a 1.5 ml Eppendorf tube and fill with water to 600 μl.
 38.3 mM NAD solution: Put 34.83 mg of NAD (MW 663.43) in a 1.5 ml Eppendorf tube, add 27 μl of Tris at 2 M (to bring the solution to pH 8.0). Finally, fill with water to 300 μl.
 23 mM cAMP solution: Put 42.80 mg of cAMP (MW 329.22) in a 1.5 ml Eppendorf tube, add 73 μl of Tris at 2 M (to bring the solution to pH 8.0). Finally, fill with water to 200 μl. 
 40 mM Folinic acid solution: To 20 mg of solid folinic acid calcium salt (MW 511.5), add 1.15 ml of water. 
 Spermidine stock solution: Briefly warm spermidine ( two minutes at 37°C) in order to melt it. Then, put 23.55 μl of spermidine (MW 145.25) into a 1.5 Eppendorf tube and fill with water to 150 μl.
 240 mg/ml maltodextrin solution: Put 2.4 g of maltodextrin in a 15 ml Falcon tube. Dissolve, and fill with water to 10 ml. 
 Nucleotide Mix solution: Put 145 mg of ATP dipotassium salt dihydrate (MW 619.4), 133 mg of GTP disodium salt dihydrate (MW 567.14), 79.4 mg of CTP disodium salt dihydrate (MW 563.16), 82.6 mg of UTP trisodium salt dihydrate (MW 586.12) in a 1.5 Eppendorf tube. Add 353 μl of KOH (15 w/v %) .Finally, fill with water to 1.5 ml. 
  
ReagentDTTMerck MilliporeSigma (Sigma-Aldrich)Catalog #D0632 
ReagentCoAMerck MilliporeSigma (Sigma-Aldrich)Catalog #C4282 
ReagentFolinic AcidMerck MilliporeSigma (Sigma-Aldrich)Catalog #F7878 
ReagentSpermidineMerck MilliporeSigma (Sigma-Aldrich)Catalog #85558 
ReagentNAD (β-Nicotinamide adenine dinucleotide hydrate)Merck MilliporeSigma (Sigma-Aldrich)Catalog #N6522 
ReagentHEPESMerck MilliporeSigma (Sigma-Aldrich)Catalog #H6147 
ReagentATP (Adenosine 5′-triphosphate dipotassium salt hydrate)Merck MilliporeSigma (Sigma-Aldrich)Catalog #A8937 
ReagentGTP (Guanosine 5′-triphosphate sodium salt hydrate)Merck MilliporeSigma (Sigma-Aldrich)Catalog #10106399001 ROCHE 
ReagentUTP (Uridine 5′-triphosphate trisodium salt dihydrate)Merck MilliporeSigma (Sigma-Aldrich)Catalog #94370  
ReagentMaltodextrin (4.0-7.0 dextrose equivalent)Merck MilliporeSigma (Sigma-Aldrich)Catalog #419672 

In a 15 mL Falcon tube add the following volumes of the stock solutions prepared before:
 
 
 
Stock solutionVolume needed
HEPES pH 81000 µl
Nucleotide mix396 µl
tRNA solution160 µl
NAD76.6 µl
CO-A160 µl
cAMP46 µl
Folinic acid80 µl
spermidine34 µl
Maltodextrin2000 µl
TOTAL 3952.6 µl
 
 
 

Dissolve well the maltodextrin-based energy solution using vortex.  Aliquot in 2 ml Eppendorf tubes and store at -80°C prior to use.

Prepare hexametaphophate solution

Weight 0.15 g of sodium hexametaphosphate and put it into a 15 ml Falcon tube. Dissolve well and fill with water to 5 ml. Put the solution in a boiling water bath for 5 minutes. Kepp in the solution in the lab bench until it reaches room temperature. Aliquot in 1.5 ml Eppendorf tubes and keep at -80°C before use. 
Reagentsodium hexametaphosphateMerck MilliporeSigma (Sigma-Aldrich)Catalog #305553 

	Aminoacid	Amount of powder needed
	Alanine	445.45 mg
	Arginine	644.6502 mg
	Asparagine	660.6 mg
	Aspartic acid	665.5 mg
	Cysteine	370.7496 mg
	Glutamic acid	735.65 mg
	Glutamine	447.219 mg
	Glycine	375.35 mg
	Histidine	775.75 mg
	Isoleucine	655.9 mg
	Leucine	401.4108 mg
	Lysine	447.3414 mg
	Methionine	456.5826 mg
	Phenylalanine	330.38 mg
	Proline	575.65 mg
	Serine	529.5 mg
	Threonine	595.6 mg
	Tryptophan	408.46 mg
	Tyrosine	554.4414 mg
	Valine	358.479 mg

	Aminoacid Stock solution in 5M KOH	Volume needed
	Alanine	136 ul
	Arginine	222 ul
	Asparagine	136 ul
	Aspartic acid	136 ul
	Cysteine	222 ul
	Glutamic acid	136 ul
	Glutamine	222 ul
	Glycine	136 ul
	Histidine	136 ul
	Isoleucine	136 ul
	Leucine	222 ul
	Lysine	222 ul
	Methionine	222 ul
	Phenylalanine	340 ul
	Proline	136 ul
	Serine	136 ul
	Threonine	136 ul
	Tryptophan	340 ul
	Tyrosine	222 ul
	Valine	222 ul
	TOTAL	3816 µL

	Stock solution	Volume needed
	HEPES pH 8	1000 µl
	Nucleotide mix	396 µl
	tRNA solution	160 µl
	NAD	76.6 µl
	CO-A	160 µl
	cAMP	46 µl
	Folinic acid	80 µl
	spermidine	34 µl
	Maltodextrin	2000 µl
	TOTAL	3952.6 µl

Public workspaceStep 2: Preparing amino acid, polyphosphates, and maltodextrin-based energy solutions for cell-free reactions V.3

Step 2: Preparing amino acid, polyphosphates, and maltodextrin-based energy solutions for cell-free reactions V.3