All TxTl experiments were
performed using the E. coli cell extract
prepared, according to the published protocol.26 (link) We used Rosetta 2 (DE3) E. coli strain to prepare all extracts.
For cell extract preparation,
cells were grown in 2xYTPG media at 30 °C to OD of 0.5. For each
TxTl reaction, the final concentration of reagents was from the energy
mix: 500 mM HEPES pH 8, 15 mM ATP and GTP, 9 mM CTP and UTP, 2 mg/mL
of E. coli tRNA mixture, 0.68 mM folinic
acid, 3.3 mM nicotinamide adenine dinucleotide (NAD), 2.6 mM coenzyme-A
(CoA), 15 mM spermidine, 40 mM sodium oxalate, 7.5 mM cAMP, 300 mM
3-PGA; from the amino acid mix: 2 mM each of alanine, arginine, asparagine,
aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine,
isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,
threonine, tryptophan, tyrosine and valine; and from the salt mix:
130 mM potassium glutamate, 10 mM ammonium acetate, and 10 mM magnesium
glutamate.27 (link)Due to the batch-to-batch
variability of TxTl preparation yields,
we performed all of the directly comparable experiments (experiments
shown on the same figure) using the same batch of the extract.