Table 2.

Genes commonly modified in engineered cell-free strains. Citations indicate where more information about the gene in the context of cell-free can be found.

Gene	Description	Ref(s)
ackA	acetate kinase, added to increase yield	[64]
araC	transcriptional activator, removed to prevent interference with AraC-expressing circuits	[52,53]
csdA	cold shock degradosome protein, removed to prevent mRNA decay during preparation	[65]
dnaJ	chaperone protein, added to assist folding with dnaK, grpE	[63]
dnaK	chaperone protein, added to assist folding with dnaJ, grpE	[63]
dsbC	disulfide isomerase, added for disulfide bond formation	[66]
ef-tu	translation factor, added to increase yields (most abundant protein in cell, potentially rate-limiting)	[64,67]
endA	endonuclease, removed for plasmid stability	[56]
gamS	nuclease inhibitor from lambda phage, added to protect linear DNA	[60,62,68,69]
gorB	glutathione reductase, removed to prevent disulfide bond persistence	[59,63]
groEJ	chaperone protein, added to assist folding with groEL	[63]
groEL	chaperone protein, added to assist folding with groEJ	[63]
grpE	heat shock protein, added to assist folding with dnaJ, dnaK	[63]
gshA	glutamate-cysteine ligase, removed to stabilize cysteine	[57]
hchA	chaperone protein, added to increase solubility and yield	[64]
ibpA	small heat shock protein (chaperone), added to increase solubility and yield	[64]
ibpB	small heat shock protein (chaperone), added to increase solubility and yield	[64]
If-1	initiation factor 1, added to increase yield	[64]
If-2	initiation factor 2, added to increase yield	[64]
If-3	initiation factor 3, added to increase yield	[58,64,67,70]
lacI	transcriptional repressor, removed to prevent interference with LacI in circuits	[52,53]
lacZYA	lac operon, removed to eliminate background when using LacZ as a reporter	[14]
lysR	λ phage endolysin, added to disrupt the bacterial cell wall to facilitate lysis	[14]
mazF	MazF toxin, removed to prevent mRNA degradation at ‘ACA’ sites	[65]
met	P1 selection marker, engineering scar	[56]
pnp	PNPase, involved in mRNA degradation, removed or tagged for post-growth removal	[64,71]
recD	exonuclease subunit, removed to protect linear DNA (ineffective), or tagged and removed post-growth (successful)	[62,71]
rna	RNAse A, removed for RNA stability	[54,55]
rnb	RNAse II, removed for RNA stability	[65,67]
rpfA	release factor 1, removed to encourage noncanonical amino acid incorporation	[72]
sdaA	serine deaminase, removed to stabilize serine	[56]
sdaB	serine deaminase, removed to stabilize serine	[56]
speA	arginine decarboxylase, removed to stabilize arginine	[56]
tnaA	tryptophanase, removed to stabilize tryptophan	[56]
tonA	outer membrane protein, engineering scar	[56]
trxB	thioredoxin reductase, removed post-growth with HA tag to prevent disulfide bond persistence	[59,63]