May 13, 2020
Cell-free 3PGA energy solution
- 1University of Edinburgh
Protocol Citation: Nadanai Laohakunakorn 2020. Cell-free 3PGA energy solution. protocols.io https://dx.doi.org/10.17504/protocols.io.8pshvne
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 25, 2019
Last Modified: May 13, 2020
Protocol Integer ID: 29138
Keywords: cell-free protein synthesis, synthetic biology, in vitro transcription translation
Abstract
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.
CITATION
CITATION
Materials
- Amino acids LAA21-1KT Sigma
- Mg-glutamate 49605-250G Sigma
- K-glutamate 49601-500G Sigma
- DTT 10708984001 Sigma
- NTP set R1481 ThermoFisher
- tRNA 10109541001 Sigma
- CoA C4282-10MG Sigma
- NAD 10127981001 Sigma
- cAMP A9501-1G Sigma
- folinic acid PHR1541-1G Sigma
- spermidine S2626-1G Sigma
- PEG-8000 89510 Sigma
- 3PGA P8877-1G Sigma
- HEPES H3375-100G Sigma
- Tris base T1503-100G Sigma
- KOH
- mass balance
Amino acids stock solution
Amino acids stock solution
Make stock solution of amino acids, 1000 uL at 50 mM.
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.
| Amino acid | weight (mg) | added (mg) | |
| Alanine | 4.5 | ||
| Arginine | 8.7 | ||
| Asparagine | 6.6 | ||
| Aspartate | 6.7 | ||
| Cysteine | 6.1 | ||
| Glutamate | 7.3 | ||
| Glutamine | 7.3 | ||
| Glycine | 3.8 | ||
| Histidine | 7.8 | ||
| Isoleucine | 6.6 | ||
| Leucine | 6.6 | ||
| Lysine | 9.1 | ||
| Methionine | 7.5 | ||
| Phenylalanine | 8.3 | ||
| Proline | 5.8 | ||
| Serine | 5.3 | ||
| Threonine | 6.0 | ||
| Tryptophan | 10.2 | ||
| Valine | 5.9 |
Add 1000 µL 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 On ice
Weigh tyrosine and add to a separate tube
| Amino acid | weight (mg) | added (mg) | |
| Tyrosine | 9.1 |
Add 900 µL of 1 millimolar (mM) KOH, and dissolve as far as possible. The tyrosine powder will not be entirely soluble.
Add 50 µL of 15% KOH, which should fully dissolve the powder. Then add 50 µL of deionized water. Vortex well, and measure pH, which should be around ~11-12. Keep solution On ice
Keep the tyrosine and the rest of the amino acids separate. If storage is required keep at -80 °C (flash-freezing with liquid nitrogen is optional)
Other components stock solution
Other components stock solution
Make stock solution of other components
Prepare 2M stock solution of tris base.
| Component | Mass to add (g) | Water to add (mL) | Final concentration | |
| Tris base | 60.57 | 250 | 2 M |
Prepare 1M KOH stock, and 15% KOH stock.
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.
| Component | Mass to add (g) | Water to add (uL) | Tris to add (uL) | Final concentration | Notes | |
| L-glutamic acid monopotassium salt (K-glutamate) | 1.219 | 1000 | 6 M | |||
| L-glutamic acid hemimagnesium salt (Mg-glutamate) | 0.3886 | 1000 | 1 M | |||
| DTT | 0.1543 | 1000 | 1 M | |||
| CoA | 0.0498 | 1000 | 65 mM | |||
| NAD | 0.1161 | to 1000 | ~90 | 175 mM | pH 7.5-8, titrate with 2M tris | |
| Folinic acid | 0.0160 | 1000 | 33.9 mM | |||
| cAMP | 0.2139 | to 1000 | ~365 | 650 mM | pH 8, titrate with 2M tris | |
| 3-PGA | 0.2604 | to 1000 | ~540 | 1.4 M | pH 7.5, titrate with 2M tris | |
| HEPES | 0.4766 | to 1000 | 2 M | pH 8, titrate with 1M KOH (around 25 uL required) | ||
| Volume to add (uL) | Water to add (uL) | Final concentration | ||||
| spermidine | 23.55 | 126.45 | 1 M | heat up stock solution in your hand | ||
| PEG-8000 | 50 | 50 | 50% |
Energy solution
Energy solution
Preparation of final energy solution (4x)
This energy solution will form 25% of the final reaction volume.
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 On ice
| Component | Stock (mM) | Final conc (mM) | Volume to add (uL) | |
| HEPES | 2000 | 200 | 100 | |
| Water | 114.2 | |||
| ATP | 100 | 6 | 60 | |
| GTP | 100 | 6 | 60 | |
| CTP | 100 | 3.6 | 36 | |
| UTP | 100 | 3.6 | 36 | |
| tRNA (in mg/ml) | 43.75 | 0.8 | 18.29 | |
| CoA | 65 | 1.04 | 16 | |
| NAD | 175 | 1.32 | 7.54 | |
| cAMP | 650 | 3 | 4.62 | |
| Folinic acid | 33.9 | 0.27 | 8.02 | |
| Spermidine | 1000 | 4 | 4 | |
| 3-PGA | 1400 | 120 | 85.7 | |
| amino acids | 50 | 6 | 120 | |
| tyrosine | 50 | 3 | 60 | |
| PEG-8000 | 50% | 8% | 160 | |
| Mg-glutamate | 1000 | 42 | 42 | |
| K-glutamate | 6000 | 400 | 66.67 | |
| DTT | 1000 | 1 | 1 | |
| Total | 1000 |
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.
Store at -80 °C
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-glutamate
- Kwon and Jewett 2015 report 12 mM Mg-glutamate, 130 mM K-glutamate
This 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 731.33 µL and can be aliquoted into 10 tubes of 73.13 µL .
Citations
Sun ZZ, Hayes CA, Shin J, Caschera F, Murray RM, Noireaux V. Protocols for implementing an Escherichia coli based TX-TL cell-free expression system for synthetic biology.
https://doi.org/10.3791/50762Cai Q, Hanson JA, Steiner AR, Tran C, Masikat MR, Chen R, Zawada JF, Sato AK, Hallam TJ, Yin G. A simplified and robust protocol for immunoglobulin expression in Escherichia coli cell-free protein synthesis systems.
https://doi.org/10.1002/btpr.2082