Escherichia coli SURE 2 Supercompetent Cells [e14(McrA-) Δ(mcrCB-hsdSMR-mrr) 171 endA1 gyrA96 thi-1 supE44 relA1 lac recB recJ sbsC umuC::Tn5 (Kanr) uvrC [F’ proAB lacIqZΔM15 Tn10 (Tetr)]] (Agilent Technologies, Santa Clara, CA, USA) was used as a host strain in order to verify the gene sequences. For protein expression, recombinant pQE-80L (Qiagen, Hilden, Deutschland) vectors were transformed into E. coli BL21-CodonPlus(DE3)-RIPL [E. coli B F–ompT hsdS(rB– mB–) dcm+ Tetr gal λ(DE3) endA Hte [argU proL Camr] [argU ileY leuW Strep/Specr]] (Agilent Technologies). Bacteria were cultivated at 37 °C and 18 °C on Luria-Bertani solid medium (0.1% peptone, 0.05% yeast extract, 0.1% sodium chloride, 0.1% agar) or Luria-Bertani liquid medium (0.1% peptone, 0.05% yeast extract, 0.1% sodium chloride).
E coli bl21 codonplus de3 ripl
Manufactured by Agilent Technologies
Sourced in Japan
The E. coli BL21-CodonPlus(DE3)-RIPL is a competent bacterial cell line designed for protein expression. It is engineered to enhance the expression of recombinant proteins by providing tRNAs for rare codons found in many heterologous genes.
Automatically generated - may contain errors
Lab products found in correlation
Pqe 80l, by Qiagen (1 mentions)
F proab laciqzδm15 tn10 tetr, by Agilent Technologies (1 mentions)
One shot top10 chemically competent e coli, by Thermo Fisher Scientific (1 mentions)
Gibson assembly, by New England Biolabs (1 mentions)
Puc19, by Takara Bio (1 mentions)
Escherichia coli dh5α, by Takara Bio (1 mentions)
3 protocols using e coli bl21 codonplus de3 ripl
1
Protein Expression in E. coli
2
Clostridium scindens Bai gene cloning
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Genes encoding BaiA1/3, BaiA2, BaiCD, BaiH, BaiB, BaiF, BaiE, BaiJ, BaiO, BaiI, and BaiN from Clostridium scindens ATCC 35704 and BaiJ from C. scindens VPI 12708 (see Table S3 for gene loci and protein accession numbers) were amplified by PCR using primers listed in Table S4. Each PCR-amplified gene was cloned into pET28b+ or a modified pET28b+ vector (m-pET28b+) using Gibson Assembly (New England BioLabs), according to the manufacturer’s instructions. The modified pET28b+ carries both a His-Tag and a Strep-Tag sequence (K. Lau, unpublished). Restriction sites used for plasmid digestion are indicated in Table S4. The recombinant plasmids were transformed into E. coli TOP10 (One Shot™ TOP10 Chemically Competent E. coli, Invitrogen). All plasmid constructs were verified by sequencing and further transformed into the expression strain E. coli BL21-CodonPlus(DE3)-RIPL (Agilent).
3
Thermoadaptation of Tsr Enzyme in Geobacillus
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Bacterial strains, media, and plasmids.
Table 1 lists G. kaustophilus strains used. An error-prone strain, MK480, was used for thermoadaptation-directed evolution of tsr gene. Strain MK242 was used for gene characterization because it is genetically stable. These strains were grown at 60 °C in Luria-Bertani (LB) medium. However, G. kaustophilus [pGKE75 derivative], where square brackets denote the plasmid-carrier state, was grown in LB medium supplemented with 5 mg/L of kanamycin (LK5). Escherichia coli DH5α (Takara Bio, Otsu, Shiga, Japan) and E. coli BL21-CodonPlus(DE3)-RIPL (Agilent Technologies, Santa Clara, USA) were used for DNA manipulation and recombinant protein production, respectively. E. coli strains were grown at 37 °C in LB medium. Ampicillin (50 mg/L), kanamycin (50 mg/L), and chloramphenicol (13 mg/L) were added when necessary. The plasmid pGKE75 was constructed previously. 33) pUC19 and pET-16b were purchased from Takara Bio and Merck KGaA (Darmstadt, Germany), respectively.
Table 1 lists G. kaustophilus strains used. An error-prone strain, MK480, was used for thermoadaptation-directed evolution of tsr gene. Strain MK242 was used for gene characterization because it is genetically stable. These strains were grown at 60 °C in Luria-Bertani (LB) medium. However, G. kaustophilus [pGKE75 derivative], where square brackets denote the plasmid-carrier state, was grown in LB medium supplemented with 5 mg/L of kanamycin (LK5). Escherichia coli DH5α (Takara Bio, Otsu, Shiga, Japan) and E. coli BL21-CodonPlus(DE3)-RIPL (Agilent Technologies, Santa Clara, USA) were used for DNA manipulation and recombinant protein production, respectively. E. coli strains were grown at 37 °C in LB medium. Ampicillin (50 mg/L), kanamycin (50 mg/L), and chloramphenicol (13 mg/L) were added when necessary. The plasmid pGKE75 was constructed previously. 33) pUC19 and pET-16b were purchased from Takara Bio and Merck KGaA (Darmstadt, Germany), respectively.
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