E. coli Strains
Host strains
The CSB has the following E. coli strains. Contact Noah Saracho 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. Only available from a glycerol stock. 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. |
| 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. Only available from a glycerol stock. |
| 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 100 uL. Can use as little as 20 uL 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 10 ng DNA per 100 ul of cells.
- Flick the tube several times to ensure even distribution of DNA.
- Ice for 20 min.
- Heat shock cells for 45 sec at 42 °C.
- Ice for 2 min.
- Add 900 ul of SOC medium and incubate for 1 hour at 37 °C with shaking.
- Plate 200 ul of transformation mix on antibiotic plates.