Strains and media used for this study were essentially the same as previously reported (Graf et al. 2015 (link); Krondorfer et al. 2014b (link)). In short, Escherichia coli strain NEB5α was obtained from New England Biolabs (Ipswich, MA, USA) and Pichia pastoris strain X33 from Invitrogen. For fermenter cultivations, the basal salts medium with 4.35 mL/L PTM1 trace salts was used, which is described in detail by Invitrogen or in Krondorfer et al. (2014b (link)).
Escherichia coli strain neb5α
Manufactured by New England Biolabs
Sourced in United States
Escherichia coli strain NEB5α is a competent bacterial strain used for the transformation and propagation of plasmid DNA. This strain is derived from the common laboratory strain DH5α and is optimized for efficient transformation and high-fidelity plasmid replication.
Automatically generated - may contain errors
Lab products found in correlation
Restriction enzyme, by New England Biolabs (3 mentions)
Pfuultra high fidelity dna polymerase, by Agilent Technologies (3 mentions)
Pichia pastoris strain x 33, by Thermo Fisher Scientific (2 mentions)
Ptm1 trace salts, by Thermo Fisher Scientific (2 mentions)
Hc toxin, by Cayman Chemical (2 mentions)
S cerevisiae bj5465 strain, by LGC (1 mentions)
E coli strain bl21, by Merck Group (1 mentions)
Ppicz b, by Thermo Fisher Scientific (1 mentions)
P pastoris strain x 33, by Thermo Fisher Scientific (1 mentions)
N8 acetylspermidine dihydrochloride, by Merck Group (1 mentions)
Trichostatin a tsa, by Apexbio (1 mentions)
8 protocols using escherichia coli strain neb5α
1
Escherichia coli and Pichia pastoris Cultivation
2
Biochemical Assay for HDAC Inhibitors
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PCR reactions were performed using the PfuUltra High-Fidelity DNA polymerase (Agilent Technologies). Restriction enzymes were purchased from New England Biolabs and used according to the manufacturer’s instructions. Custom oligonucleotides were synthesized by Integrated DNA Technologies. Escherichia coli strain NEB5α (New England Biolabs) was used for cloning procedures. Assay substrates 1–2 and 7–13 were purchased from GenScript®, and assay substrates 3–6 were purchased from Enzo® Life Sciences. HC toxin was purchased from Cayman Chemicals. A sample of 7-[(3-aminopropyl)amino]-1,1,1-trifluoroheptan-2-one was synthesized according to published procedures35 (link) and determined to be >95% pure by mass spectrometry, NMR spectroscopy, and X-ray crystallographic structure validation. All other HDAC inhibitors were purchased from ApexBio®. All substrates and inhibitors were purchased as >95% pure preparations and used without further purification.
3
Recombinant Protein Production in P. pastoris
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P. pastoris strain X33 was purchased from Invitrogen, the protease-deficient S. cerevisiae strain BJ5465 from LGC Promochem (Barcelona, Spain) and Escherichia coli strain NEB5α from New England Biolabs (Ipswich, MA). YPD plates contained 10 g L−1 peptone, 20 g L−1 yeast extract, 10 g L−1 D-glucose, 20 g L−1 agar and 100 mg L−1 Zeocin. LB low salt medium contained 10 g L−1 peptone from casein, 5 g L−1 yeast extract, 5 g L−1 NaCl and 25 mg L−1 Zeocin. Basal salts medium (Invitrogen) was used for fermentation, containing 0.93 g L−1 calcium sulfate, 18.2 g L−1 potassium sulfate, 14.9 g L−1 magnesium sulfate heptahydrate, 4.13 g L−1 potassium hydroxide, 40 g L−1 glycerol, 26.7 mL 85% phosphoric acid and 4.35 mL PTM1 trace salts per liter. SC-dropout plates contained 1.92 g L−1 Y1501 (synthetic dropout medium without uracil), 20 g L−1 agar, 100 mL L−1 20% D-glucose, 100 mL L−1 10× YNB and 1 mL L−1 chloramphenicol. For the preparation of the liquid medium (without agar), D-glucose was replaced by raffinose. SG/R-CAA expression medium [27] contained 5 g L−1 casein hydrolysate, 9.67 g L−1 NaH2PO4.2H2O, 6.77 g L−1 Na2HPO4.2H2O, 50 mL L−1 10× YNB, 100 mL L−1 20% D-galactose, 100 mL L−1 20% raffinose and 5 mL L−1 20% D-glucose.
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P. pastoris strain X33 was purchased from Invitrogen, the protease-deficient S. cerevisiae strain BJ5465 from LGC Promochem (Barcelona, Spain) and Escherichia coli strain NEB5α from New England Biolabs (Ipswich, MA). YPD plates contained 10 g L−1 peptone, 20 g L−1 yeast extract, 10 g L−1 D-glucose, 20 g L−1 agar and 100 mg L−1 Zeocin. LB low salt medium contained 10 g L−1 peptone from casein, 5 g L−1 yeast extract, 5 g L−1 NaCl and 25 mg L−1 Zeocin. Basal salts medium (Invitrogen) was used for fermentation, containing 0.93 g L−1 calcium sulfate, 18.2 g L−1 potassium sulfate, 14.9 g L−1 magnesium sulfate heptahydrate, 4.13 g L−1 potassium hydroxide, 40 g L−1 glycerol, 26.7 mL 85% phosphoric acid and 4.35 mL PTM1 trace salts per liter. SC-dropout plates contained 1.92 g L−1 Y1501 (synthetic dropout medium without uracil), 20 g L−1 agar, 100 mL L−1 20% D-glucose, 100 mL L−1 10× YNB and 1 mL L−1 chloramphenicol. For the preparation of the liquid medium (without agar), D-glucose was replaced by raffinose. SG/R-CAA expression medium [27] contained 5 g L−1 casein hydrolysate, 9.67 g L−1 NaH2PO4.2H2O, 6.77 g L−1 Na2HPO4.2H2O, 50 mL L−1 10× YNB, 100 mL L−1 20% D-galactose, 100 mL L−1 20% raffinose and 5 mL L−1 20% D-glucose.
4
Isolation and Cultivation of GAS Strains
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GAS 854 is an M-type 1 strain, isolated from a retroperitoneal abscess (42 (link)). The GAS strains used in this study are listed in Table 1 . GAS was grown at 37°C in L3 or THY medium (Todd-Hewitt broth supplemented with yeast extract) as previously described or on Trypticase soy agar supplemented with 5% defibrinated sheep blood (Remel). Escherichia coli strain NEB5α (New England Biolabs) was used for cloning, and E. coli strain BL21 (Sigma) was used for protein expression unless otherwise noted. When necessary, antibiotics were added at the following concentrations: erythromycin was used at 1 µg/ml and 150 µg/ml for GAS and E. coli, respectively, and spectinomycin was used at 50 µg/ml in GAS or E. coli. For infection assays, penicillin was used at 20 µg/ml and gentamicin was used at 200 µg/ml. Ampicillin or carbenicillin was used at 100 µg/ml for E. coli.
5
Recombinant Protein Expression in Pichia
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All chemicals were purchased from Sigma Aldrich (St. Louis, MO), Roth (Karlsruhe, Germany) or VWR (Radnor, PA). Pichia pastoris strain X-33 and Zeocin-resistance encoding shuttle vector pPICZB were from Invitrogen (Carlsbad, CA). Escherichia coli strain NEB5α was from New England Biolabs (Ipswich, MA).
6
Escherichia coli and Pichia pastoris Cultivation
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Strains and media used for the present study were essentially the same as reported previously 19 . In short, Escherichia coli strain NEB5α was obtained from New England Biolabs (Ipswich, MA, USA) and P. pastoris strain X33 was obtained from Invitrogen. YPD plates contained 20 g·L−1 peptone from casein, 10 g·L−1 yeast extract, 4 g·L−1 GLC, 15 g·L−1 agar and 100 mg·L−1 Zeocin. LB low‐salt (LB‐LS) plates contained 10 g·L−1 peptone from casein, 5 g·L−1 yeast extract, 10 g·L−1 NaCl, 15 g·L−1 agar and 25 mg·L−1 Zeocin. For fermenter cultivations, the basal salts medium with 4.35 mL·L−1 PTM1 trace salts was used, in accordance with the manufacturer's instructions (Invitrogen) and as described in detail previously 19 .
7
Biochemical Assay for HDAC Inhibitors
8
Acetylated Polyamine Synthesis and Characterization
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No statistical methods were used to predetermine sample size. PfuUltra High-Fidelity DNA polymerase (Agilent Technologies) was used for PCR analysis. Restriction enzymes (New England Biolabs) were used according to the manufacturer's specifications. Primers were synthesized by Integrated DNA Technologies. Escherichia coli strain NEB5α (New England Biolabs) was used for cloning procedures. Peptides were synthesized by Genscript. N-(3-aminopropyl)acetamide was purchased from Chem-Implex International, Inc. Acetylcadaverine was purchased from TCI. N1-acetylspermidine dihydrochloride was purchased from Cayman Chemicals, Inc. Acetylputrescine, N8-acetylspermidine dihydrochloride, N1-acetylspermine trihydrochloride, N-butylacetamide and N-(aminooctyl)acetamide were purchased form Sigma-Aldrich. N1,N8-diacetylspermidine was purchased from Toronto Research Chemicals. Inhibitors trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA) were purchased from ApexBio. All substrates and ligands are used without further purification (>95% purity). The transition state analogue inhibitor AAT was synthesized as previously described24 (link).
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