Supplemental Figure 1: Standard curve for converting fluorescence to protein concentration for sfGFP.
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Supplemental Table 1: Stock solution list for CFPS reactions
Stock Solutions
Salt Solution
Mg(Glu)
2
NH
4
(Glu)
2
K(Glu)
Stocks
1
2.4
3.5
Units
M
M
M
Nucleotide Mixture
ATP
GTP
UTP
CTP
Folinic acid tRNA
Energy Regeneration Mixture Stocks
20 amino acids 50 phosphoenolpyruvate (PEP) 1000
NAD 100
Acetyl-CoA
Sodium Oxalate
50
166.67
Putrescine
Spermidine
HEPES
250
250
1000
15x Stock Units
18 mM
12.75
12.75
12.75
0.51
2.559 mM mM mM mg/mL mg/mL
Units mM mM mM mM mM mM mM mM
2

Supplemental Table 2: CFPS reaction preparation. Each component of Solution 1 (Salt solution,
Nucleotide Mixture, and Energy Regeneration Mixture) is prepared and frozen separately at -
80˚C. These mixtures and water are combined the day of the experiment prior to mixing in equal ratios with the DNA solution and the Extract solution. The following table shows calculations for a 900 µ l reaction, but this mixture can be easily scaled up or down.
Volume of each component of the three starting solutions for a
900 µL reaction volume
Units Reaction Components
Solution 1
Salt Solution
Mg(Glu)
2
NH
4
(Glu)
2
K(Glu)
Water
Nucleotide Mixture
ATP
GTP
UTP
CTP
Folinic acid tRNA
Energy Regeneration Mixture
20 amino acids phosphoenolpyruvate (PEP)
NAD
Acetyl-CoA
Sodium Oxalate
Putrescine
Spermidine
HEPES
Water
Solution 2
Plasmid DNA (200 ng/µL)
Water
Solution 3
S30 Extract
Water
Total
155.73µL
36.00
30.00
2.97
4.86
21.60
3.60
5.40
51.30
24.27µL
300.00
60.00
240.00
Volume (µL) Final Concentration
300.00
60µL
10.80 12
3.75
33.43
12.02
60µL
10
130
1.2
0.85
0.85
0.85
0.034
0.171
2
33.33
0.33
0.27
4
1
1.5
57
13.33
300.00
240.00
60.00
900.00 µL mM mM mM mM mM mM mM mM mM mM mM mM mM mM mM mg/mL mg/mL ng/µL
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