RED20 is a so-called "fail-safe genetic code" introduced by Calles and colleagues in 2019 [1]. RED20 encodes each of the 20 canonical amino acids with exactly one sense codon and terminates translation with the canonical three stop codons. RED20 therefore leaves 41 of the 64 triplet codons unassociated with any translation machinery, meaning they are "null codons". Recoding an organism in RED20 requires recoding all of its translated genes into RED20 as well as deleting its tRNA genes associated with RED20 null codons. The Free Genes E. coli RED20 essential gene distribution represents RED20 recoded versions of 245 of the 299 translated genes in the Keio collection, which represents the set of genes essential for life in E. coli as determined by single gene deletion viability [2].**What can it be used for**

The E. coli RED20 gene set can be used to research the engineering of RED20 encoded organisms and genetic systems. The E. coli RED20 gene set can also be used in E. coli based expression systems incorporating non-canonical amino acids. Because every gene in this gene set is encoded using only 20 sense codons and one of three stop codons, users of this gene set can reprogram any of the other 41 null codons using orthogonal translation machinery.**Where can I find more information**

- [1] Calles, J., Justice, I., Brinkley, D., Garcia, A. & Endy, D. Fail-safe genetic codes designed to intrinsically contain engineered organisms. Nucleic Acids Res. (2019). doi:10.1093/nar/gkz745
- [2] Baba, T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2, 2006.0008 (2006).

This product is made available under the unilateral OpenMTA.

245 unique parts

**Instructions for Use**

Culture at 37C overnight, with shaking at around 300rpm. The selection marker for all parts is ampicillin.

PlateSet: ecolired20-1 ecolired20-2 ecolired20-3

#### Genes

Gene | Name | NCBI ID |
---|---|---|

minD | inhibitor of FtsZ ring polymerization;chromosome-membrane tethering protein; membrane ATPase ofthe MinCDEE system | NP_415693.1 |

pth | peptidyl-tRNA hydrolase | NP_415722.1 |

prs | phosphoribosylpyrophosphate synthase | NP_415725.1 |

ispE | 4-diphosphocytidyl-2-C-methylerythritol kinase | NP_415726.1 |

lolB | lipoprotein localization factor | NP_415727.1 |

hemA | glutamyl tRNA reductase | NP_415728.1 |

prfA | peptide chain release factor RF-1 | NP_415729.1 |

kdsA | 3-deoxy-D-manno-octulosonate 8-phosphatesynthase | NP_415733.1 |

topA | DNA topoisomerase 1 | NP_415790.1 |

fabI | Enoyl-[acyl-carrier-protein] reductase [NADH] FabI | NP_415804.1 |

dicA | Qin prophage; putative regulator for DicB | NP_416088.1 |

ydfB | Qin prophage; uncharacterized protein | NP_416090.2 |

tyrS | tyrosyl-tRNA synthetase | NP_416154.1 |

ydiL | putative HTH domain DNA-binding protein | NP_416204.2 |

pheS | Phenylalanine--tRNA ligase alpha subunit | NP_416229.1 |

rplT | 50S ribosomal subunit protein L20 | NP_416231.1 |

infC | translation initiation factor IF-3 | NP_416233.1 |

nadE | NH(3)-dependent NAD(+) synthetase | NP_416254.1 |

tsaB | tRNA threonylcarbamoyladenosine biosynthesis protein TsaB | NP_416321.1 |

aspS | aspartyl-tRNA synthetase | NP_416380.1 |

argS | arginyl-tRNA synthetase | NP_416390.1 |

pgsA | phosphatidylglycerophosphate synthetase | NP_416422.1 |

yefM | antitoxin of the YoeB-YefM toxin-antitoxinsystem | NP_416521.2 |

yejM | essential inner membrane DUF3413domain-containing protein; lipid A production and membranepermeability factor | NP_416693.1 |

gyrA | DNA gyrase subunit A | NP_416734.1 |

folC | bifunctional folylpolyglutamate synthase/dihydrofolate synthase | NP_416818.1 |

accD | Acetyl-coenzyme A carboxylase carboxyl transferase subunit beta | NP_416819.1 |

fabB | 3-oxoacyl-[acyl-carrier-protein] synthase I | NP_416826.1 |

gltX | glutamyl-tRNA synthetase | NP_416899.1 |

ligA | DNA ligase | NP_416906.1 |

hisS | histidyl tRNA synthetase | NP_417009.1 |

ispG | 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (flavodoxin) | NP_417010.1 |

suhB | inositol monophosphatase | NP_417028.1 |

tadA | tRNA-specific adenosine deaminase | NP_417054.2 |

era | GTPase Era | NP_417061.1 |

lepB | Signal peptidase I | NP_417063.1 |

rpoE | RNA polymerase sigma E factor | NP_417068.1 |

pssA | phosphatidylserine synthase;CDP-diacylglycerol-serine O-phosphatidyltransferase | NP_417080.4 |

bamD | BamABCDE complex OM biogenesis lipoprotein | NP_417086.1 |

rplS | 50S ribosomal subunit protein L19 | NP_417097.1 |

trmD | tRNA (guanine-N(1)-)-methyltransferase | NP_417098.1 |

rpsP | 30S ribosomal subunit protein S16 | NP_417100.1 |

ffh | Signal recognition particle protein | NP_417101.1 |

grpE | heat shock protein | NP_417104.1 |

csrA | pleiotropic regulatory protein for carbon sourcemetabolism | NP_417176.1 |

alaS | alanyl-tRNA synthetase | NP_417177.1 |

ispF | 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase | NP_417226.1 |

ispD | 4-diphosphocytidyl-2C-methyl-D-erythritolsynthase | NP_417227.1 |

ftsB | cell division protein | NP_417228.1 |

eno | enolase | NP_417259.1 |

pyrG | CTP synthetase | NP_417260.1 |

mazE | antitoxin of the ChpA-ChpR toxin-antitoxinsystem | NP_417263.1 |

lgt | phosphatidylglycerol-prolipoproteindiacylglyceryl transferase | NP_417305.1 |

prfB | peptide chain release factor RF-2 | NP_417367.1 |

fbaA | Fructose-bisphosphate aldolase | NP_417400.1 |

pgk | phosphoglycerate kinase | NP_417401.1 |

metK | S-adenosylmethionine synthetase | NP_417417.1 |

yqgF | putative Holliday junction resolvase | NP_417424.1 |

plsC | 1-acyl-sn-glycerol-3-phosphate acyltransferase | NP_417490.1 |

mqsA | antitoxin for MqsR toxin; transcriptionalrepressor | NP_417493.1 |

parE | DNA topoisomerase 4 subunit B | NP_417502.1 |

ribB | 3,4-dihydroxy-2-butanone 4-phosphate synthase | NP_417513.1 |

cca | Multifunctional CCA protein | NP_417528.1 |

tsaD | tRNA N6-adenosine threonylcarbamoyltransferase | NP_417536.1 |

rpsU | 30S ribosomal subunit protein S21 | NP_417537.1 |

dnaG | DNA primase | NP_417538.1 |

rsmI | Ribosomal RNA small subunit methyltransferase I | NP_417615.1 |

yhbV | U32 peptidase family protein | NP_417628.2 |

ftsH | ATP-dependent zinc metalloprotease FtsH | NP_417645.1 |

rpmA | 50S ribosomal subunit protein L27 | NP_417652.1 |

rplU | 50S ribosomal subunit protein L21 | NP_417653.1 |

ispB | octaprenyl diphosphate synthase | NP_417654.1 |

murA | UDP-N-acetylglucosamine1-carboxyvinyltransferase | NP_417656.1 |

lptC | periplasmic membrane-anchored LPS-bindingprotein; LPS export protein | NP_417666.1 |

lptA | lipopolysaccharide export ABC transporterperiplasmic binding protein; Lipid A binding protein; LPSexport and assembly protein | NP_417667.1 |

rplM | 50S ribosomal subunit protein L13 | NP_417698.1 |

degS | Serine endoprotease DegS | NP_417702.1 |

mreD | cell wall structural complex MreBCDtransmembrane component MreD | NP_417715.1 |

mreC | cell wall structural complex MreBCDtransmembrane component MreC | NP_417716.1 |

mreB | Cell shape-determining protein MreB | NP_417717.2 |

accB | Biotin carboxyl carrier protein of acetyl-CoA carboxylase | NP_417721.1 |

accC | Biotin carboxylase | NP_417722.1 |

tsaC | Threonylcarbamoyl-AMP synthase | NP_417741.1 |

def | peptide deformylase | NP_417745.1 |

fmt | Methionyl-tRNA formyltransferase | NP_417746.1 |

rplQ | 50S ribosomal subunit protein L17 | NP_417753.1 |

rpoA | DNA-directed RNA polymerase subunit alpha | NP_417754.1 |

rpsD | 30S ribosomal subunit protein S4 | NP_417755.1 |

rpsK | 30S ribosomal subunit protein S11 | NP_417756.1 |

secY | preprotein translocase membrane subunit | NP_417759.1 |

rplO | 50S ribosomal subunit protein L15 | NP_417760.1 |

rpsE | 30S ribosomal subunit protein S5 | NP_417762.1 |

rplR | 50S ribosomal subunit protein L18 | NP_417763.1 |

rpsH | 30S ribosomal subunit protein S8 | NP_417765.1 |

rpsN | 30S ribosomal subunit protein S14 | NP_417766.1 |

rplE | 50S ribosomal subunit protein L5 | NP_417767.1 |

rplX | 50S ribosomal subunit protein L24 | NP_417768.1 |

rplN | 50S ribosomal subunit protein L14 | NP_417769.1 |

rpmC | 50S ribosomal subunit protein L29 | NP_417771.1 |

rplP | 50S ribosomal subunit protein L16 | NP_417772.1 |

rpsC | 30S ribosomal subunit protein S3 | NP_417773.1 |

rplV | 50S ribosomal subunit protein L22 | NP_417774.1 |

rplB | 50S ribosomal subunit protein L2 | NP_417776.1 |

rplW | 50S ribosomal subunit protein L23 | NP_417777.1 |

rplD | 50S ribosomal subunit protein L4 | NP_417778.1 |

rpsL | 30S ribosomal subunit protein S12 | NP_417801.1 |

trpS | tryptophanyl-tRNA synthetase | NP_417843.1 |

yrfF | Putative membrane protein IgaA homolog | NP_417857.1 |

asd | Aspartate-semialdehyde dehydrogenase | NP_417891.1 |

ftsX | putative ABC transporter permease | NP_417919.1 |

ftsE | cell division ATP-binding protein | NP_417920.1 |

yhhQ | DUF165 family inner membrane protein | NP_417928.1 |

glyQ | Glycine--tRNA ligase alpha subunit | NP_418017.1 |

gpsA | NAD(P)H-dependent glycerol-3-phosphate dehydrogenase | NP_418065.1 |

waaU | lipopolysaccharide core biosynthesis | NP_418080.1 |

waaA | 3-deoxy-D-manno-octulosonic acid transferase | NP_418090.1 |

coaD | pantetheine-phosphate adenylyltransferase | NP_418091.1 |

rpmB | 50S ribosomal subunit protein L28 | NP_418094.1 |

dfp | Coenzyme A biosynthesis bifunctional protein CoaBC | NP_418096.4 |

dut | deoxyuridinetriphosphatase | NP_418097.1 |

gmk | guanylate kinase | NP_418105.1 |

gyrB | DNA gyrase subunit B | YP_026241.1 |

dnaN | Beta sliding clamp | NP_418156.1 |

dnaA | Chromosomal replication initiator protein DnaA | NP_418157.1 |

rpmH | 50S ribosomal subunit protein L34 | NP_418158.1 |

rnpA | protein C5 component of RNase P | NP_418159.1 |

tnaB | tryptophan transporter of low affinity | NP_418165.1 |

glmS | L-glutamine:D-fructose-6-phosphateaminotransferase | NP_418185.1 |

glmU | fused N-acetyl glucosamine-1-phosphateuridyltransferase/glucosamine-1-phosphate acetyltransferase | NP_418186.1 |

rho | transcription termination factor | NP_418230.1 |

wzyE | putative ECA polysaccharide chain elongationprotein | NP_418241.1 |

hemD | uroporphyrinogen III synthase | NP_418248.1 |

hemC | hydroxymethylbilane synthase | YP_026260.1 |

ubiB | Probable protein kinase UbiB | NP_418279.1 |

hemG | Protoporphyrinogen IX dehydrogenase [menaquinone] | NP_418292.1 |

yihA | cell division GTP-binding protein | NP_418301.3 |

ftsN | essential cell division protein | NP_418368.1 |

murI | glutamate racemase | NP_418402.2 |

murB | UDP-N-acetylenolpyruvoylglucosamine reductase | NP_418403.1 |

birA | Bifunctional ligase/repressor BirA | NP_418404.1 |

coaA | pantothenate kinase | NP_418405.1 |

secE | preprotein translocase membrane subunit | NP_418408.1 |

nusG | transcription termination factor | NP_418409.1 |

rplJ | 50S ribosomal subunit protein L10 | NP_418412.1 |

rplL | 50S ribosomal subunit protein L7/L12 | NP_418413.1 |

rpoB | DNA-directed RNA polymerase subunit beta | NP_418414.1 |

ubiA | p-hydroxybenzoate octaprenyltransferase | NP_418464.1 |

plsB | glycerol-3-phosphate O-acyltransferase | NP_418465.4 |

lexA | transcriptional repressor of SOS regulon | NP_418467.1 |

dnaB | replicative DNA helicase | NP_418476.1 |

ssb | single-stranded DNA-binding protein | NP_418483.1 |

alsK | D-allose kinase | NP_418508.1 |

groS | 10 kDa chaperonin | NP_418566.1 |

orn | oligoribonuclease | NP_418586.4 |

tsaE | tRNA threonylcarbamoyladenosine biosynthesis protein TsaE | NP_418589.1 |

rpsR | 30S ribosomal subunit protein S18 | NP_418623.1 |

chpS | antitoxin of the ChpBS toxin-antitoxin system | NP_418645.2 |

ppa | inorganic pyrophosphatase | NP_418647.1 |

valS | valyl-tRNA synthetase | NP_418679.1 |

lptF | lipopolysaccharide export ABC permease | NP_418682.1 |

lptG | lipopolysaccharide export ABC permease | NP_418683.4 |

dnaC | DNA biosynthesis protein | NP_418781.1 |

ribF | bifunctional riboflavin kinase/FAD synthetase | NP_414566.1 |

ileS | isoleucyl-tRNA synthetase | NP_414567.1 |

lspA | Lipoprotein signal peptidase | NP_414568.1 |

ispH | 4-hydroxy-3-methylbut-2-enyl diphosphate reductase | NP_414570.1 |

folA | dihydrofolate reductase | NP_414590.1 |

lptD | LPS-assembly protein LptD | NP_414596.1 |

ftsL | membrane bound cell division leucine zipperseptum protein | NP_414625.1 |

ftsI | transpeptidase involved in septal peptidoglycansynthesis; penicillin-binding protein 3 | NP_414626.1 |

murF | UDP-N-acetylmuramoyl-tripeptide:D-alanyl-D-alanine ligase | NP_414628.1 |

mraY | phospho-N-acetylmuramoyl-pentapeptidetransferase | NP_414629.1 |

murD | UDP-N-acetylmuramoyl-L-alanine:D-glutamateligase | NP_414630.1 |

ftsW | putative lipid II flippase; integral membraneprotein; FtsZ ring stabilizer | NP_414631.1 |

murG | N-acetylglucosaminyl transferase | NP_414632.1 |

ftsQ | Cell division protein FtsQ | NP_414635.1 |

ftsA | ATP-binding cell division FtsK recruitmentprotein | NP_414636.1 |

ftsZ | GTP-binding tubulin-like cell division protein | NP_414637.1 |

lpxC | UDP-3-O-acyl N-acetylglucosamine deacetylase | NP_414638.1 |

secM | regulator of secA translation | NP_414639.2 |

secA | Protein translocase subunit SecA | NP_414640.1 |

can | carbonic anhydrase | NP_414668.1 |

folK | 2-amino-4-hydroxy-6-hydroxymethyldihyropteridinepyrophosphokinase | NP_414684.1 |

hemL | Glutamate-1-semialdehyde 2,1-aminomutase | NP_414696.1 |

erpA | iron-sulfur cluster insertion protein | NP_414698.1 |

rpsB | 30S ribosomal subunit protein S2 | NP_414711.1 |

tsf | translation elongation factor EF-Ts | NP_414712.1 |

pyrH | uridylate kinase | NP_414713.1 |

frr | ribosome recycling factor | NP_414714.1 |

dxr | 1-deoxy-D-xylulose 5-phosphate reductoisomerase | NP_414715.1 |

ispU | undecaprenyl pyrophosphate synthase | NP_414716.1 |

cdsA | CDP-diglyceride synthase | NP_414717.2 |

rseP | Regulator of sigma-E protease RseP | NP_414718.1 |

bamA | BamABCDE complex OM biogenesis outer membranepore-forming assembly factor | NP_414719.1 |

lpxD | UDP-3-O-(3-hydroxymyristoyl)glucosamine N-acyltransferase | NP_414721.1 |

lpxA | UDP-N-acetylglucosamine acetyltransferase | NP_414723.1 |

lpxB | tetraacyldisaccharide-1-P synthase | NP_414724.1 |

accA | Acetyl-coenzyme A carboxylase carboxyl transferase subunit alpha | NP_414727.1 |

tilS | tRNA(Ile)-lysidine synthase | NP_414730.1 |

proS | prolyl-tRNA synthetase | NP_414736.1 |

yafN | antitoxin of the YafO-YafN toxin-antitoxinsystem | NP_414767.1 |

yagG | CP4-6 prophage; putative sugar transporter | NP_414804.1 |

hemB | Delta-aminolevulinic acid dehydratase | NP_414903.4 |

secD | SecYEG protein translocase auxillary subunit | NP_414942.1 |

secF | SecYEG protein translocase auxillary subunit | NP_414943.1 |

ribD | Riboflavin biosynthesis protein RibD | NP_414948.1 |

ribE | riboflavin synthase beta chain | NP_414949.1 |

thiL | thiamine monophosphate kinase | NP_414951.1 |

dxs | 1-deoxy-D-xylulose-5-phosphate synthase | NP_414954.1 |

ispA | geranyltranstransferase | NP_414955.1 |

dnaX | DNA polymerase III subunit tau | NP_415003.1 |

adk | adenylate kinase | NP_415007.1 |

hemH | ferrochelatase | NP_415008.1 |

lpxH | UDP-2,3-diacylglucosamine hydrolase | NP_415057.1 |

cysS | cysteinyl-tRNA synthetase | NP_415059.1 |

folD | Bifunctional protein FolD | NP_415062.1 |

entD | phosphopantetheinyltransferase component ofenterobactin synthase multienzyme complex | NP_415115.2 |

mrdB | cell wall shape-determining protein | NP_415167.1 |

mrdA | Peptidoglycan D,D-transpeptidase MrdA | NP_415168.1 |

nadD | Nicotinate-nucleotide adenylyltransferase | NP_415172.1 |

holA | DNA polymerase III subunit delta | NP_415173.1 |

lptE | LPS-assembly lipoprotein LptE | NP_415174.1 |

lnt | apolipoprotein N-acyltransferase | NP_415190.1 |

glnS | glutamyl-tRNA synthetase | NP_415206.1 |

fldA | flavodoxin 1 | NP_415210.1 |

cydA | Cytochrome bd-I ubiquinol oxidase subunit 1 | NP_415261.2 |

infA | translation initiation factor IF-1 | NP_415404.1 |

serS | seryl-tRNA synthetase | NP_415413.1 |

msbA | lipid ABC transporter permease/ATPase | NP_415434.1 |

lpxK | Tetraacyldisaccharide 4'-kinase | NP_415435.1 |

kdsB | 3-deoxy-manno-octulosonate cytidylyltransferase | NP_415438.1 |

mukF | Chromosome partition protein MukF | NP_415442.1 |

mukE | Chromosome partition protein MukE | NP_415443.2 |

asnS | asparaginyl tRNA synthetase | NP_415450.1 |

fabA | beta-hydroxydecanoyl thioester dehydrase | NP_415474.1 |

yceQ | uncharacterized protein | NP_415603.2 |

fabD | malonyl-CoA-[acyl-carrier-protein] transacylase | NP_415610.1 |

fabG | 3-oxoacyl-[acyl-carrier-protein] reductase | NP_415611.1 |

acpP | Acyl carrier protein | NP_415612.1 |

tmk | thymidylate kinase | NP_415616.1 |

holB | DNA polymerase III subunit delta' | NP_415617.1 |

lolC | lipoprotein-releasing system transmembraneprotein | NP_415634.1 |

lolE | lipoprotein-releasing system transmembraneprotein | NP_415636.1 |

purB | adenylosuccinate lyase | NP_415649.1 |

minE | cell division topological specificity factor | NP_415692.1 |