E. coli BL21(DE3) cells containing pZM471 were grown in 12 L of LB containing 0.1 mg/mL ampicillin at 37 °C to an OD600nm of approximately 0.3 at which time ferric chloride was added to a final concentration of 50 μM and the flasks were cooled to 18 °C. The cells were grown further to an OD600nm of approximately 0.6 and protein expression was induced with 20 μL of 10 mg/mL anhydrotetracycline hydrochloride in dimethylformamide (Acros Organics) per 1 L of LB. Cells were grown overnight and harvested the next day by centrifugation (5,000 × g), frozen in liquid N2, and stored at −80 °C.
Anhydrotetracycline hydrochloride
Manufactured by Thermo Fisher Scientific
Sourced in Belgium
Anhydrotetracycline hydrochloride is an antibiotic compound used in research applications. It is a derivative of the tetracycline class of antibiotics. The compound inhibits bacterial protein synthesis, making it a useful tool for scientific studies.
Automatically generated - may contain errors
Lab products found in correlation
Mops ez rich defined medium, by Teknova (2 mentions)
Dimethylformamide, by Thermo Fisher Scientific (1 mentions)
Xhoi, by Thermo Fisher Scientific (1 mentions)
P nitrophenyl butyrate p npb, by Merck Group (1 mentions)
Mes sodium salt, by Thermo Fisher Scientific (1 mentions)
Deoxynucleotide dntp solution mix, by New England Biolabs (1 mentions)
Precision plus protein all blue standard, by Bio-Rad (1 mentions)
Oligonucleotide, by Integrated DNA Technologies (1 mentions)
Poly prep chromatography column, by Bio-Rad (1 mentions)
Mini protean electrophoresis cell, by Bio-Rad (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Precision plus protein dual color standard, by Bio-Rad (1 mentions)
Q5 hot start high fidelity dna polymerase, by New England Biolabs (1 mentions)
Shrimp alkaline phosphatase, by New England Biolabs (1 mentions)
4 protocols using anhydrotetracycline hydrochloride
1
Heterologous Protein Expression in E. coli
2
Genetic manipulation of E. coli and S. aureus
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E. coli MG1655 was routinely grown in MOPS EZ Rich defined medium (M2105, Teknova, Hollister, CA). Plasmid pBbE2A-RFP was a gift from Jay Keasling45 (link) (Addgene plasmid # 35322). RFP was induced with 20 nM anhydrotetracycline hydrochloride (233131000, Acros Organics, Geel, Belgium). GFP was expressed from plasmid prplN-GFP46 (link). Plasmid-containing MG1655 cells were grown in appropriate antibiotics (50 μg/mL kanamycin, 100 μg/mL carbenicillin). S. aureus USA30032 (link) was routinely grown in trypticase soy broth (TSB) medium (211825, BD, Franklin Lakes, NJ). All bacterial strains were grown at 37°C and shaken at 300 rpm.
3
Genetic manipulation of E. coli and S. aureus
4
Strep-Tag Protein Purification Protocol
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pASG-IBA2 Star Gate Acceptor Vector, Strep-Tactin Superflow (high capacity; 6% crosslinked agarose; 60–160 µm) resin and Strep-tag protein purification buffer set were purchased from IBA Life Sciences. p-nitrophenyl butyrate (p-NPB) was from Sigma. Oligonucleotides were from Integrated DNA Technologies. Fast digest restriction endonucleases (Esp3I, Hind III, NdeI, Xho I) and T4 DNA ligase enzymes were from Thermo Fisher Scientific. Q5 Hot Start High-Fidelity DNA Polymerase, Deoxynucleotide (dNTP) Solution Mix, Shrimp Alkaline phosphatase, NEB® 5-alpha Competent E. coli (Subcloning Efficiency) and NEB® Express Competent E. coli (High Efficiency) cells were purchased from New England Biolabs. Ampicillin was from G-Biosciences. Anhydrotetracycline hydrochloride, 4-morpholineethanesulfonic (MES) acid monohydrate and MES sodium salt were purchased from Acros Organics. Blue-Clean Protein Stain was purchased from IBI Scientific. Coomassie G-250 (Bradford) dye and bovine serum albumin were purchased from Thermo Scientific. 12% precast polyacrylamide gels for use with Mini-PROTEAN Electrophoresis Cells, Poly-Prep Chromatography Columns (2 ml bed volume and 10 ml reservoir), Precision Plus Protein All Blue Standards and Precision Plus Protein Dual Color Standards were from Bio-Rad. Commercial CALB was from Chiral Vision (CV-CALBY).
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