Table 1.

The structure and composition of a typical functioning liposome

Size of a vesicle	1–100 fL (typically 4 fL) *
Lipid composition	POPC: cholesterol:DSPE-PEG5000 at a molar ratio of 58:39:3
List of proteins and RNAs in the liposome	Template RNA (70 nM), Ribosome (1.2 µM), translational initiation factors: IF1 (2.5 µM), IF2 (0.21 µM), IF3 (0.95 µM), elongation factors: EF-G (3.2 µM), EF-Tu (12 µM), EF-Ts(8.2 µM), releasing factors: RF1 (0.25 µM), RF2 (0.24 µM), RF3 (0.17 µM), RRF (0.48 µM), aminoacyl-tRNA synthetase: AlaRS (725 nM), ArgRS (31 nM), AsnRS (380 nM), AspRS (127 nM), CysRS (24 nM), GlnRS (60 nM), GluRS (233 nM), GlyRS (87 nM), HisRS (8 nM), IleRS (396 nM), LeuRS (42 nM), LysRS (113 nM), MetRS (27 nM), PheRS (676 nM), ProRS (165 nM), SerRS (39 nM), ThrRS (85 nM), TrpRS (28 nM), TyrRS (7 nM), ValRS (17 nM), methionyl-tRNA transformylase (588 nM), creatine kinase (0.47 µM), myokinase (0.93 µM), nucleoside-diphosphate kinase (1.3 µM), pyrophosphatase (0.62 µM), ribosomal protein S1 (4.6 µM), RNasin Plus RNase Inhibitor (1 U/μL), tRNA mix (48 A260 units)
List of substrates in the liposome †	ATP (2 mM), GTP (2 mM), CTP (1 mM), UTP (1 mM), 20 amino acids (0.3 mM each), creatine phosphate (20 mM), 10-formyl-5,6,7,8-tetrahydrofolic acid (10 ng/mL)
Other small compounds	Magnesium acetate (13 mM), potassium glutamate (100 mM), spermidine (2 mM), dithiothreitol (1 mM), 2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid (HEPES) (50 mM, pH 7.6),

*A liposome of a typical size (4 fL) is expected to carry 17 TyrSR molecules (the minimum among protein components), 28 800 EF-Tu molecules (the maximum among protein components), 72 RNA templates and 2 880 ribosomes. It is worth noting that all components that were required for the reactions were likely to be encapsulated into liposomes (>1 fL).

^†Our system does not have nutrient transport machinery such as α-hemolysin (Noireaux and Libchaber 2004). It works by consuming internal substrates that have been encapsulated.