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E. coli Strains

Host strains

The CSB has the following E. coli strains. Contact Walter Chazin for more information.


Strain Source Resistance Comments
58F3 Genentech None Used with tphac promoter vectors for expression of membrane proteins. W3110 background. Genotype: Δfhu Δlon galE rpoHt ΔclpP lacIq ΔompTDΔslyD.
See: Kim et al. PLoS ONE (2012) 7(4) e35844.
ArcticExpress(DE3) Agilent Technologies (formerly Stratagene) None* Contains genes for cold-adapted chaperonins (cpn60, cpn10) to enhance folding/growth at low temperatures and increase soluble protein yields. See the expression protocol instruction manual. Co-purification of cpn60/10 with the protein of interest is a common problem. Incubation with MgCl2/ATP/KCl facilitates separation. See: R. E. Joseph; A. H. Andreotti, Protein Expr. Purif. 2008, 60, 194–197.
ArcticExpress(DE3)RIL Agilent Technologies (formerly Stratagene) None** Contains genes for cold-adapted chaperones and tRNAs for rare Arg/Ile/Leu codons. See the expression protocol instruction manual. See above note on purification strategy.
BL21(DE3) Agilent Technologies (formerly Stratagene) None General purpose T7 expression strain.
BL21(DE3)pLysS Agilent Technologies (formerly Stratagene) Cap Contains gene for T7 lysozyme. T7 lysozyme inhibits T7 RNA polymerase and allows tighter expression control.
BL21(DE3)Star Life Technologies (formerly Invitrogen) None Contains mutation in RNaseE to improve stability of mRNA transcripts and increase yield.
BL21CodonPlus(DE3)RIL Agilent Technologies (formerly Stratagene) Cap Contains tRNAs for rare Arg (AGA, AGG), Ile (AUA), and Leu (CUA) codons that often restrict translation from AT-rich genomes.
BL21CodonPlus(DE3)RP Agilent Technologies (formerly Stratagene) Cap Contains tRNAs for rare Arg (AGA, AGG) and Pro (CCC) codons that often restrict translation from GC-rich genomes.
C41(DE3) Sanders lab None Mutant BL21 strain shown to be effective for expression of toxic and membrane proteins.
C41(DE3)pLysS Made in-house from C41(DE3) Cap Mutant BL21 strain shown to be effective for expression of toxic and membrane proteins. Contains pLysS for tighter expression control.
C43(DE3) Sanders lab None Mutant BL21 strain shown to be effective for expression of toxic and membrane proteins.
Origami B(DE3) EMD Millipore (formerly Novagen) Kan/Tet BL21 derivative with mutant thioredoxin reductase (trxB) and glutathione reductase (gor) genes to enhance cytosolic disulfide bond formation. Use of a thioredoxin fusion tag further enhances the formation of disulfide bonds in the cytoplasm.
Origami B(DE3) EMD Millipore (formerly Novagen) Kan/Tet BL21 derivative with mutant thioredoxin reductase (trxB) and glutathione reductase (gor) genes to enhance cytosolic disulfide bond formation. Use of a thioredoxin fusion tag further enhances the formation of disulfide bonds in the cytoplasm.
Rosetta2(DE3) EMD Millipore (formerly Novagen) Cap Enhances the expression of eukaryotic proteins that contain codons rarely used in E. coli. Contains tRNAs for 7 rare codons (AUA, AGG, AGA, CUA, CCC, GGA, CGG).
Rosetta2(DE3)pLysS EMD Millipore (formerly Novagen) Cap Enhances the expression of eukaryotic proteins that contain codons rarely used in E. coli. Contains tRNAs for 7 rare codons and pLysS for tighter expression control.
SoluBL21(DE3) Genlantis None Improves expression of toxic clones and proteins in soluble form.
Tuner(DE3) EMD Millipore (formerly Novagen) None BL21 lacZY deletion that enables tuning expression levels by adjusting IPTG concentration.

Cap=Chloramphenicol; Kan=Kanamycin; Tet=Tetracycline
*Chaperonin-encoding genes are on a gentamycin (Gent) plasmid. You don’t need to add Gent to transformation plates, but vendor recommends selection when expressing protein.
**Chaperonin-encoding genes are on a Gent plasmid (see above). Rare tRNA genes are on a streptomycin (Strep) plasmid with a functional partitioning locus that prevents plasmid loss even in the absence of Strep.
***Rare Arg, Ile, Leu tRNA genes are on a streptomycin (Strep) plasmid with a functional partitioning locus that prevents plasmid loss even in the absence of Strep.

Transformation Protocol:
Use for all strains except SoluBL21(DE3). For SoluBL21 protocol, click on the link in table above.
Cell aliquots are 100uL. Can use as little as 20uL per transformation.
NOTE: The 45 sec. heat shock was found to be optimal. Using LB in lieu of SOC often results in fewer colonies.

 

  • Thaw cells on ice.
  • Add 10ng DNA per 100ul of cells.
  • Flick the tube several times to ensure even distribution of DNA.
  • Ice 20 minutes.
  • Heat shock cells for 45 sec. at 42°C.
  • Ice for 2 min.
  • Add 900ul of SOC medium and incubate for 1 hour at 37°C shaking.

 

  • Plate 200ul of transformation mix on antibiotic plates.