B834
B834 is the parental strain for BL21 (1). These protease-deficient hosts are methionine auxotrophs and allow high specific-activity labeling of target proteins with 35S-methionine and selenomethionine for crystallography (2).
BL21
BL21 is the most widely used host background and has the advantage of being deficient in both lon (3) and ompT proteases.
BLR
BLR is a recA– derivative of BL21 (4) that improves plasmid monomer yields and may help stabilize target plasmids containing repetitive sequences or whose products may cause the loss of the DE3 prophage (5). These strains are also deficient in the lon and ompT proteases.
HMS174
HMS174 strains provide the recA mutation in a K-12 background. Like BLR, these strains may stabilize certain target genes whose products may cause the loss of the DE3 prophage.
NovaBlue
NovaBlue is a K-12 strain ideally suited as an initial cloning host due to its high transformation efficiency, blue/white screening capability (with appropriate plasmids) and recA endA mutations, which result in high yields of excellent quality plasmid DNA. The NovaBlue T1R strain has the added benefit of resistance to T1 and T5 phage. The DE3 lysogen of NovaBlue is potentially useful as a stringent host due to the presence of the lacIq repressor encoded by the F episome. Blue/white screening is not possible with NovaBlue(DE3) due to the presence of the lacZ α-peptide coding sequences in the lysogenic phage.
NovaF-
NovaF- is a K-12 strain ideally suited as a cloning host due to its high transformation efficiency and recA endA mutations, which result in high yields of excellent quality plasmid DNA. In contrast to NovaBlue, NovaF- lacks the F episome that encodes lacZΔM15 and lacIq mutations. Therefore this strain is not appropriate for blue/white screening by α-complementation nor for propagation of expression vectors that contain an E. coli promoter controlled by a lac operator sequence, i.e. tac, trc, T5, etc. in the absence of an additional source of lac repressor. NovaF- is recommended for the preparation of pETcoco™ recombinants.
Origami™
Origami host strains are K-12 derivatives that have mutations in both the thioredoxin reductase (trxB) and glutathione reductase (gor) genes, which greatly enhance disulfide bond formation in the cytoplasm (6). Studies have shown that expression in Origami(DE3) yielded 10-fold more active protein than in another host even though overall expression levels were similar (7). Origami hosts are compatible with ampicillin-resistant plasmids and are ideal for use with pET-32 vectors, since the thioredoxin fusion tag further enhances the formation of disulfide bonds in the cytoplasm. The trxB and gor mutations are selectable on kanamycin and tetracycline, respectively; therefore these strains cannot be used with plasmids carrying kanamycin- or tetracycline-resistance genes. To reduce the possibilty of disulfide bond formation between molecules, hosts containing the trxB/gor mutations are only recommended for the expression of proteins that required disulfide bond formation for proper folding.
Origami 2
Origami 2 host strains are K-12 derivatives that have mutations in both the thioredoxin reductase (trxB) and glutathione reductase (gor) genes, which greatly enhance disulfide bond formation in the cytoplasm (6). Unlike the original Origami strains, the Origami 2 strains are kanamycin sensitive, making these host strains compatible with many Novagen expression vectors. The gor mutation is still selected for by tetracycline, as are the original strains.
Origami B
Origami B host strains carry the same trxB/gor mutations as the original Origami strains, except that they are derived from a lacZY mutant of BL21. Thus the Origami B strains combine the desirable characteristics of BL21, Tuner™ and Origami hosts in one strain background. The trxB and gor mutations are selectable on kanamycin and tetracycline, respectively; therefore, these strains are not compatible with kanamycin- or tetracycline-resistant plasmids.
Rosetta™ and Rosetta 2
Rosetta and Rosetta 2 host strains are BL21 derivatives designed to enhance the expression of eukaryotic proteins that contain codons rarely used in E. coli (8-12). The original Rosetta strains supply tRNAs for the codons AGG, AGA, AUA, CUA, CCC, and GGA on a compatible chloramphenicol-resistant plasmid, pRARE (13). The Rosetta 2 strains supply a tRNA for a seventh rare codon (CGG) in addition to the six found in the original Rosetta strains (14). By supplying tRNAs for rare codons, the Rosetta strains provide for “universal” translation, where translation would otherwise be limited by the codon usage of E. coli (10, 11, 15). The tRNA genes are driven by their native promoters (13). In the pLysS and pLacI derivatives of these strains, the rare tRNA genes are present on the same plasmids that carry the T7 lysozyme and lac repressor genes, respectively.
RosettaBlue™
RosettaBlue host strains are NovaBlue derivatives that combine high transformation efficiency and recA endA lacIq mutations with enhanced expression of eukaryotic proteins that contain codons rarely used in E. coli. These strains supply tRNAs for AGG, AGA, AUA, CUA, CCC, GGA codons on a compatible chloramphenicol-resistant plasmid. The tRNA genes are driven by their native promoters. In RosettaBlue(DE3)pLysS and RosettaBlue(DE3)pLacI, the rare tRNA genes are present on the same plasmids that carry the T7 lysozyme and lac repressor genes, respectively. Blue/white screening is not possible with RosettaBlue(DE3) strains due to the presence of lacZ α-peptide coding sequences in the DE3 lysogenic phage.
Rosetta-gami™
Rosetta-gami host strains are Origami derivatives that combine the enhanced disulfide bond formation resulting from trxB/gor mutations with enhanced expression of eukaryotic proteins that contain codons rarely used in E. coli. These strains supply tRNAs for the codons AGG, AGA, AUA, CUA, CCC, and GGA, on a compatible chloramphenicol-resistant plasmid. In the pLysS and pLacI derivatives of these strains, the rare tRNA genes are present on the same plasmids that carry the T7 lysozyme and lac repressor genes, respectively. The Rosetta-gami strains are resistant to kanamycin, tetracycline, streptomycin, and chloramphenicol. These strains are recommended for use with expression plamsids carrying the ampicillin resistance marker bla. If using pCDF vectors, spectinomycin must be used for antibiotic selection because the rpsL mutation in pCDF vectors confers streptomycin resistance.
Rosetta-gami 2
Rosetta-gami 2 host strains combine features of Origami 2 and Rosetta 2, allowing for enhanced expression of eukaryotic proteins that contain codons rarely used in E. coli. These strains are derived from Origami 2, a kanamycin-sensitive K-12 strain carrying the trxB/gor mutations for enhanced disulfide bond formation in the cytoplasm. These strains carry the chloramphenicol-resistant plasmid, pRARE-2, which supplies tRNAs for seven rare codons, AGG, AGA, AUA, CUA, CCC, GGA and CGG under the control of their native promoters. The gor mutation is selectable on tetracycline.
Rosetta-gami B
The Rosetta-gami B strains combine the key features of BL21 (and its Tuner™ derivative), Origami, and Rosetta strains to enhance both the expression of eukaryotic proteins and the formation of target protein disulfide bonds in the bacterial cytoplasm. These strains are compatible with ampicillin- or spectinomycin-resistant vectors.
Tuner™
Tuner strains are lacZY deletion mutants of BL21 and enable adjustable levels of protein expression throughout all cells in a culture. The lac permease (lacY) mutation allows uniform entry of IPTG into all cells in the population, which produces a concentration-dependent, homogeneous level of induction. By adjusting the concentration of IPTG, expression can be regulated from very low level expression up to the robust, fully induced expression levels commonly associated with pET vectors. Lower level expression may enhance the solubility and activity of difficult target proteins. These strains are also deficient in the lon and ompT proteases.
References
1. Wood, W.B. (1966) J. Mol. Biol. 16, 118–133.
2. Leahy, D.J., Hendrickson, W.A., Aukhil, I. and Erickson, H.P. (1992) Science 258, 987–991.
3. Phillips, T.A., VanBogelen, R.A. and Neidhardt, F.C. (1984) J. Bacteriol. 159, 283–287.
4. Roca, A. (U. of Wisconsin, PhD thesis).
5. Stewart, E.J., Aslund, F. and Beckwith, J. (1998) EMBO J. 17, 5543–5550.
6. Bessette, P.H., Aslund, F., Beckwith, J. and Georgiou, G. (1999) Proc. Natl. Acad. Sci. USA 96, 13703–13708.
7. Prinz, W.A., Aslund, F., Holmgren, A. and Beckwith, J. (1997) J. Biol. Chem. 272, 15661–15667.
8. Kane, J.F. (1995) Curr. Opin. Biotechnol. 6, 494–500.
9. Kurland, C. and Gallant, J. (1996) Curr. Opin. Biotechnol. 7, 489–493.
10. Brinkmann, U., Mattes, R.E. and Buckel, P. (1989) Gene 85, 109–114.
11. Seidel, H.M., Pompliano, D.L. and Knowles, J.R. (1992) Biochemistry 31, 2598–2608.
12. Baca, A.M. and Hol, W.G. (2000) Int. J. Parasitol. 30, 113–118.
13. Novy, R., Drott, D., Yaeger, K. and Mierendorf, R. (2001) inNovations 12, 1–3.
14. Rosetta 2(DE3) Competent Cells for enhanced coverage of codon bias in E. coli (2003) inNovations 18, 28.
15. Del Tito, B.J., Jr., Ward, J.M., Hodgson, J., Gershater, C.J., Edwards, H., Wysocki, L.A., Watson, F.A., Sathe, G. and Kane, J.F. (1995) J. Bacteriol. 177, 7086–7091.