TITLE: Cell-free 3PGA energy solution
AUTHORS: Nadanai Laohakunakorn

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Energy solution for E. coli lysate based on 3PGA. Adapted from Sun 2013 and Cai 2015. Successfully implemented at the University of Edinburgh by Nadanai Laohakunakorn,  LBNC-EPFL by Zoe Swank.</div></div>

[STEPS]
?. [Amino acids stock solution]
Make stock solution of amino acids, 1000 uL at 50 mM.
?. [Amino acids stock solution]
Weigh each amino acid (excluding tyrosine) on parafilm paper, record the weight, and carefully add together in one tube. Alternative: carrying out at 10x quantities makes weighing much easier.  ABC1Amino acidweight (mg)added (mg)2Alanine4.53Arginine8.74Asparagine6.65Aspartate6.76Cysteine6.17Glutamate7.38Glutamine7.39Glycine3.810Histidine7.811Isoleucine6.612Leucine6.613Lysine9.114Methionine7.515Phenylalanine8.316Proline5.817Serine5.318Threonine6.019Tryptophan10.220Valine5.9
ABC1Amino acidweight (mg)added (mg)2Alanine4.53Arginine8.74Asparagine6.65Aspartate6.76Cysteine6.17Glutamate7.38Glutamine7.39Glycine3.810Histidine7.811Isoleucine6.612Leucine6.613Lysine9.114Methionine7.515Phenylalanine8.316Proline5.817Serine5.318Threonine6.019Tryptophan10.220Valine5.9
?. [Amino acids stock solution]
Weigh tyrosine and add to a separate tube  ABC1Amino acidweight (mg)added (mg)2Tyrosine9.1
ABC1Amino acidweight (mg)added (mg)2Tyrosine9.1
?. [Energy solution]
Preparation of final energy solution (4x)This energy solution will form 25% of the final reaction volume.
?. [Energy solution]
Measure and record pH of final solution using pH paper (should be ~8). Aliquot into storage tubes (25 uL recommended) and (optionally) flash freeze in liquid nitrogen.
?. [Energy solution]
Store at
-80 °C
?. [Energy solution]
Add the components together to produce the final energy solution, in the following order (not critical), vortexing after the addition of each one, and keeping tube   ABCD1ComponentStock (mM)Final conc (mM)Volume to add (uL)2HEPES20002001003Water114.24ATP1006605GTP1006606CTP1003.6367UTP1003.6368tRNA (in mg/ml)43.750.818.299CoA651.041610NAD1751.327.5411cAMP65034.6212Folinic acid33.90.278.0213Spermidine100044143-PGA140012085.715amino acids50612016tyrosine5036017PEG-800050%8%16018Mg-glutamate1000424219K-glutamate600040066.6720DTT10001121Total1000
on ice
ABCD1ComponentStock (mM)Final conc (mM)Volume to add (uL)2HEPES20002001003Water114.24ATP1006605GTP1006606CTP1003.6367UTP1003.6368tRNA (in mg/ml)43.750.818.299CoA651.041610NAD1751.327.5411cAMP65034.6212Folinic acid33.90.278.0213Spermidine100044143-PGA140012085.715amino acids50612016tyrosine5036017PEG-800050%8%16018Mg-glutamate1000424219K-glutamate600040066.6720DTT10001121Total1000
?. [Other components stock solution]
Make stock solution of other components
?. [Amino acids stock solution]
Add  of deionized water to the tube to make a 50 mM stock solution, vortex to mix, and adjust pH with KOH to ~5.2 (approximately 80 uL of 1M KOH and 920 uL dH20 required). pH can be measured roughly by spotting 1-2 uL of the solution on appropriate pH paper. If powder does not fully dissolve, add up to ~50 uL more of 1M KOH. pH will be ~8. Keep solution
1000 µl
on ice
?. [Amino acids stock solution]
Add  of  KOH, and dissolve as far as possible. The tyrosine powder will not be entirely soluble.
900 µl
?. [Amino acids stock solution]
Add  of 15% KOH, which should fully dissolve the powder. Then add  of deionized water. Vortex well, and measure pH, which should be around ~11-12. Keep solution
50 µl
50 µl
on ice
?. [Other components stock solution]
Weigh out and make the following stock solutions. Four species require titration; their pH can be approximately measured by spotting 1 uL on appropriate pH paper.  ABCDEF1ComponentMass to add (g)Water to add (uL)Tris to add (uL)Final concentrationNotes2L-glutamic acid monopotassium salt (K-glutamate)1.21910006 M3L-glutamic acid hemimagnesium salt (Mg-glutamate)0.388610001 M4DTT0.154310001 M5CoA0.0498100065 mM6NAD0.1161to 1000~90175 mMpH 7.5-8, titrate with 2M tris7Folinic acid0.0160100033.9 mM8cAMP0.2139to 1000~365650 mMpH 8, titrate with 2M tris93-PGA0.2604to 1000~5401.4 MpH 7.5, titrate with 2M tris10HEPES0.4766to 10002 MpH 8, titrate with 1M KOH (around 25 uL required)1112Volume to add (uL)Water to add (uL)Final concentration13spermidine23.55126.451 Mheat up stock solution in your hand14PEG-8000505050%
ABCDEF1ComponentMass to add (g)Water to add (uL)Tris to add (uL)Final concentrationNotes2L-glutamic acid monopotassium salt (K-glutamate)1.21910006 M3L-glutamic acid hemimagnesium salt (Mg-glutamate)0.388610001 M4DTT0.154310001 M5CoA0.0498100065 mM6NAD0.1161to 1000~90175 mMpH 7.5-8, titrate with 2M tris7Folinic acid0.0160100033.9 mM8cAMP0.2139to 1000~365650 mMpH 8, titrate with 2M tris93-PGA0.2604to 1000~5401.4 MpH 7.5, titrate with 2M tris10HEPES0.4766to 10002 MpH 8, titrate with 1M KOH (around 25 uL required)1112Volume to add (uL)Water to add (uL)Final concentration13spermidine23.55126.451 Mheat up stock solution in your hand14PEG-8000505050%
?. [Amino acids stock solution]
Keep the tyrosine and the rest of the amino acids separate. If storage is required keep at   (flash-freezing with liquid nitrogen is optional)
-80 °C
?. [Other components stock solution]
Prepare 2M stock solution of tris base.   ABCD1ComponentMass to add (g)Water to add (mL)Final concentration2Tris base60.572502 M
ABCD1ComponentMass to add (g)Water to add (mL)Final concentration2Tris base60.572502 M
?. [Energy solution]
It is possible to calibrate the energy solution for maximum yield, in which case the optimization proceeds sequentially by determining optimum concentrations for Mg-glutamate, then K-glutamate. More details are given in Sun 2013. Sun 2013 report final optimal concentrations of 4.5-10.5 mM Mg-glutamate, 40-160 mM K-glutamateKwon and Jewett 2015 report 12 mM Mg-glutamate, 130 mM K-glutamateThis step is not necessary if all that is required is functional extract.For optimisation of energy solution, add all components apart from PEG, Mg-glu, K-glu. This makes a solution of  and can be aliquoted into 10 tubes of  .
731.33 µl
73.13 µl
?. [Other components stock solution]
Prepare 1M KOH stock, and 15% KOH stock.