Table 3. Recipe for making the S. venezuelae MES and SMM solutions.

MES	Stock (mM)	2.4X solution (mM)	Volume to pipette (uL)
HEPES pH 8	2000	60	30
NTP	25	7.2	288
Amino acids	6	2.4	400
Mg-glutamate	1000	9.6	9.6
K-glutamate	4000	360	90
PEG 6000	40	2.4	60
3PGA	1400	72	51.4
ddH2O			71
Total volume (uL)			1000
Note: Amino acids, K-glutamate and PEG 6000 can be omitted but expect reduced activity.
SMM	Stock (mM)	2.4X solution (mM)	Volume to pipette (uL)
HEPES pH 8	2000	60	30
NTP	25	2.4	96
Amino acids	6	2.4	400
Mg-glutamate	1000	9.6	9.6
K-glutamate	4000	360	90
PEG 6000	40	2.4	60
3-PGA	1400	72	51.4
G6P	1000	12	12
PVSA (mg/ml)	1000	12	12
ddH2O			239
Total volume (uL)			1000
Aliquot 50 mL aliquots and store at -80°C
Troubleshooting:
We have provided instructions for making 1 mL of MES/SMM, which minimises pipetting error and reduces freeze-thaw cycles of stocks.
100 uL (1/10 volume) MES/SMM aliquots can be prepared to initially validate the protocol.
If activity is low, optimisation of the Mg-glutamate and K-glutamate levels may be required between cell-extract batches. This is a guideline that existing TX-TL/CFPS protocols recommend performing.