E. coli strain BL21 (DE3) pLysS (Invitrogen) containing pET3a-qscR (Oinuma and Greenberg, 2011 (link))(gift from the Peter E. Greenberg, University of Washington) was precultured in LB broth with 100 μg/mL ampicillin and 34 μg/mL chloramphenicol at 37°C overnight. Three 500 mL flasks with 100 mL LB and 100 μg/mL ampicillin were inoculated with 100 μL of this 6 mL overnight starter culture. The cells were grown at 37°C to an OD600 of 0.89. Either 3OC12-HSL, agonist or antagonist was added to each flask at a final concentration of at 50 μM and the flasks were cooled on ice for 2 min. Expression was induced with a final concentration of 0.5 mM IPTG (GoldBio), and the cells grown for 17 hr at 17°C. Cells were harvested and lysed in 25 mM sodium phosphate, pH 7.0, 1 mM DTT, 1 mM EDTA, 10% glycerol, 150 mM NaCl, 0.01% Tween20, with a protease inhibitor cocktail tablet (Roche). In addition, each compound was added to a final concentration of 10 μM to the lysis buffer. The insoluble pellets were resuspended in 5 mL of lysis buffer. SDS-PAGE was used to evaluate 10 μL of each sample of the cleared lysate and resuspended pellet.
Bl21 de3 plys
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, Japan
BL21(DE3)pLysS is a competent E. coli strain commonly used in protein expression experiments. It is designed for high-level protein expression from T7-based expression systems.
Automatically generated - may contain errors
Lab products found in correlation
Gst trap column, by GE Healthcare (3 mentions)
Glutathione sepharose 4b beads, by GE Healthcare (3 mentions)
Dna retardation gel, by Thermo Fisher Scientific (2 mentions)
Pgex 6p 1 vector, by GE Healthcare (2 mentions)
Chemiluminescent nucleic acid detection module, by Thermo Fisher Scientific (2 mentions)
M15prep4, by Qiagen (2 mentions)
Chloramphenicol, by Merck Group (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Pgex 6p 1, by GE Healthcare (2 mentions)
Glutathione sepharose 4b, by GE Healthcare (2 mentions)
Escherichia coli dh5α, by New England Biolabs (2 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by GoldBio (1 mentions)
Protease inhibitor cocktail tablet, by Roche (1 mentions)
Mbptrap column, by GE Healthcare (1 mentions)
Ni nta, by Qiagen (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Bradford assay kit, by Bio-Rad (1 mentions)
Neb5α competent cells, by New England Biolabs (1 mentions)
Jm109, by New England Biolabs (1 mentions)
Bl21 de3 cells, by Thermo Fisher Scientific (1 mentions)
66 protocols using bl21 de3 plys
1
Induction and Purification of QscR Protein
2
Cloning and Expression of ptmO Gene
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The ptmO gene was amplified using the primers ptmO-BglII and ptmO-EcoRI (Supplementary Table 5 ). The PCR product was cloned into the BamHI and EcoRI sites of the pRSET B to generate the expression vector pTMM080 (pRSET B-ptmO), which was then introduced into E. coli BL21(DE3)pLysS (Invitrogen). The empty vector pRSET B was also introduced into E. coli BL21(DE3)pLysS and used as a control. For the expression of ptmO, the bacteria were grown in LB medium supplemented with chloramphenicol (50 μg/mL) and ampicillin (100 μg/mL) at 37 °C for 16 h. An aliquot (2 mL) of the overnight grown culture was used to inoculate a fresh LB medium (200 mL in 1000 mL flask), supplemented with the same antibiotics, at 37 °C with shaking (200 rpm) and was grown until an OD600 of 0.5 was reached. Then, the temperature was reduced to 30 °C and isopropyl β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.3 mM to induce protein expression. After further growth for 8 h, the cells were harvested by centrifugation (4200 rpm, 10 min, 4 °C) and stored at −80 °C until used.
3
Recombinant MBP-Fused Protein Expression
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The expression of NPP-10N N-terminally fused to maltose-binding protein (MBP) was induced by the addition of 1 mM IPTG to 1 liter of LB cultures of exponentially growing E. coli BL21 DE3 pLysS (Invitrogen) strain (OD = 0.6) before incubation for 3 hours at 25°C. After pelleting by centrifugation, the bacteria were resuspended in lysis buffer [0.5 M NaCl, 5% glycerol, 50 mM tris-HCl (pH 8), and 1× protease inhibitors] before lysis by sonication. The soluble portion of the lysate was loaded on a 1-ml MBP-Trap Column (GE, healthcare). The column was washed with 10 volumes of lysis buffer, and bound proteins were eluted in a lysis buffer containing 20 mM maltose (pH 8). Proteins were aliquoted and flash-frozen in liquid nitrogen and stored at −80°C.
4
Expression and Purification of CMV Polyepitope
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The expression and purification of the polyepitope protein was performed as previously described.52 (link),53 (link) Briefly, following transformation of the CMV polyepitope expression vectors (CMVpoly or CMVpoly-L) into E. coli BL21 (DE3) pLysS (Invitrogen, Grand Island, NY), the expression of CMV polyepitope protein was induced for 4 h with 1 mM/mL of IPTG. The E. coli cultures were harvested by centrifugation, the cell pellet was resuspended in 80 mL of lysis buffer (25 mM Tris pH 7.4, 0.5% TritonX100, 150 mM NaCl, 0.5 mg/mL lysozyme) supplemented with a protease inhibitor cocktail (Roche) and the CMV polyepitope proteins were purified using a 5 mL of Ni-NTA (QIAGEN) metal-affinity chromatography matrix. Following analysis of the eluted fractions using SDS-PAGE, positive fractions were pooled and protein was dialysed against 25 mM MES (2-[N-morpholino] ethanesulfonic acid) buffer pH 5.6. Protein concentration was determined using a Bradford assay kit (Bio-Rad).
5
Cloning and Expression of E. coli Phytase
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The gene encoding the Escherichia coli periplasmic phytase, AppA, was amplified from the genome of strain BL21(DE3) pLysS (Invitrogen, Waltham, MA, USA) and cloned into the expression vector pOPINB [61 (link)]. pOPINB was a gift from Ray Owens (Addgene plasmid # 41142; http://n2t.net/addgene:41142 ; accessed on 1 January 2019). The construct was designed for the isopropylthio-β-D-galactoside (IPTG) inducible expression of an N-terminal cleavable His-tag recombinant protein in the cytoplasm of a suitable E. coli host strain. The sequence was cloned in a truncated form lacking the periplasmic signal peptide (residues 1–22) and fused to an N-terminal 3C-protease cleavable His6-tag (residues MAHHHHHHSSGLEVLFQ|GP, where | indicates the 3C-protease cleavage site). The sequence of this construct (named 6H3C-AppA) was confirmed through nucleotide sequencing. Site-directed variants were generated from this construct using the method reported by Liu and Naismith [62 (link)]. Note that the residue numbering used in this report refers to the amino acid sequence of the mature enzyme lacking the signal peptide, i.e., the catalytic histidine residue becomes H17 and the proton donor residue, H304.
6
Cholesterolicum Strain Characterization
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N. cholesterolicum NRRL 5767 was a gift from USDA ARS Culture and Patent Culture Collections, Peoria, Illinois. JM109 and NEB 5-α competent cells were from New England Biolabs. BL21(DE3)pLysS was from Invitrogen. Protein expression vectors pET-15b and pET-28a were from Novagen. pCRISPomyces-2 was a gift from Huimin Zhao [59 (link)] (Addgene plasmid # 61737; http://n2t.net/addgene:61737 ). Various recombinant clones obtained throughout this research were suspended in 20% glycerol for long term storage at -70°C.
7
Purification of Hcp1 and Hcp2 Proteins
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Hcp1 and Hcp2 wild-type and mutant proteins were purified using an identical protocol. Briefly, E. coli BL21(DE3) pLys S (Invitrogen) cells were transformed with the pDEST-14 derivatives. Overnight cultures grown on Terrific Broth (TB; 1.2% peptone, 2.4% yeast extract, 72 mM K2HPO4, 17 mM KH2PO4, and 0.4% glycerol) supplemented with ampicillin (100 µg/ml) and chloramphenicol (35 µg/ml) at 37°C were diluted in TB medium and grown at 37°C to an OD600 = 0.6. The temperature was then decreased to 25°C and the expression of the hcp genes was induced by IPTG (500 µM) for 18 hours. Cells were harvested, resuspended in buffer A (50 mM Tris pH 8.0, 300 mM NaCl) supplemented with EDTA (1 mM), lysozyme (0.5 mg/ml), and phenylmethylsulfonyl fluoride (PMSF), submitted to three freeze-thawing cycles and sonicated after the addition of DNase (20 µg/ml) and MgCl2 (20 mM). Insoluble material was discarded by centrifugation for 30 min at 16000×g. All the subsequent purification steps were performed using an AKTA FPLC system. First, the soluble fraction was loaded into a 5-mL HisTrap FF colomn (GE Health Sciences). After extensive washing, the Hcp proteins were eluted in one step gradient of Imidazole 250 mM in Buffer A. The second purification step consisted to a gel filtration on a Sephadex 200 26/60 column (GE Health Sciences) in Tris 20 mM, NaCl 100 mM at pH 8.
8
Heterologous Expression of hRpn13 Pru and Ubiquitin
9
Cloning and Expression of PtmS Protein
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The ptmS gene was amplified using the primers ptmS-EcoRI and ptmS-HindIII (Supplementary Table 5 ). The PCR product was cloned into the EcoRI and HindIII sites of pMBP-28 B to generate the expression vector pMBP-28 B-ptmS, which was then transferred into E. coli BL21(DE3)pLysS (Invitrogen). The empty vector pMBP-28 B was introduced into E. coli BL21(DE3)pLysS to be used as a control. For expression of MBP-PtmS fusion protein, the bacteria were grown in LB medium supplemented with kanamycin (50 μg/mL) and chloramphenicol (50 μg/mL) at 37 °C for 16 h. An aliquot (2 mL) of overnight grown culture was used to inoculate a fresh LB medium (200 mL in 1000 mL flask), supplemented with the same antibiotics, at 37 °C with shaking (200 rpm) until OD600 reached 0.5. Recombinant protein expression was induced with isopropyl β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.3 mM and the culture was further cultivated for 8 h. The cells were harvested by centrifugation (4200 rpm, 10 min, 4 °C) and stored at −80 °C until used.
10
Protein-DNA Binding Assay Protocol
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The archetypic PtrEPSP‐TF, BESC‐35 PtrEPSP‐TF, and BESC‐876 PtrEPSP‐TF were cloned into the pGEX‐6P‐1 vector (GE Healthcare) by BamHI and EcoRI for GST fusion constructs. The constructs were transformed into E. coli strain BL21(DE3)pLysS (Invitrogen) for protein expression. GST fusion proteins were extracted and purified as previously described using Glutathione Sepharose 4B beads (GE Healthcare) (Xie, Ren, Costa‐Nunes, Pontes, & Yu, 2012 ). To perform EMSA, proteins were then eluted from beads by incubating with Elution Buffer (50 mM Tris‐HCl pH 8.0, 10 mM reduced glutathione) at 4°C for 30 min. For DNA probes, PtrhAT promoter DNA was PCR amplified, gel purified, and end labeled with biotin as described previously (Xie, Muchero, et al., 2018 ). The DNA‐binding reaction included 0.25 nM Biotin‐labeled probe, 0.4 µg of purified protein, 10 mM Tris‐HCl (pH 7.5), 50 mM KCl, 1 mM DTT, 2.5% Glycerol, 5 mM MgCl2, 1 µg Poly (dI‐dC), 0.05% NP‐40. Reactions were incubated at room temperature for 20 min. The reaction mixtures were then resolved in 6% DNA retardation gel (Novex) by electrophoresis at 100 V for 1–2 hr and electrophoretically transferred to Nylon membrane. Signals of biotin were detected using Chemiluminescent Nucleic Acid Detection Module (Thermo Scientific) as suggested by the manufacturer.
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