The ruvCgfp and ruvCDefgfp genes were subcloned into pET28a expression vectors and then transformed into the BL21 derivative strain (T7 Express lysY/Iq Competent E. coli, New England Biolabs). After induction with 0.1 mM IPTG, the proteins with an N-terminal His6-tag were produced at 20°C for 16 hours and pelleted by centrifugation at 7000 rpm for 30 min. The cell pellet was resuspended in lysis buffer [50 mM Hepes (pH 8.0), 300 mM NaCl, 5 mM imidazole, and 1 mM phenylmethylsulfonyl fluoride] and disrupted by sonication (10 times for 30 s, on/off at output 5; XL-2000, Misonix). The total cell lysate was centrifuged at 30,000g for 40 min, and the soluble recombinant protein was purified by immobilized metal ion chromatography with a Ni-NTA column (Qiagen). After washing with wash buffer (lysis buffer with 50 mM imidazole added), the protein was eluted with elution buffer (lysis buffer with 500 mM imidazole) and further desalted against the storage buffer [20 mM Hepes (pH 8.0) and 20 mM NaCl]. The protein was concentrated to about 2 mg/ml and stored at −80°C. The Flp protein was purified to near homogeneity using DNA affinity enrichment as the final step (81 (link)).
T7 express lysy iq competent e coli
Manufactured by New England Biolabs
T7 Express lysY/Iq Competent E. coli is a bacterial strain engineered for high-level protein expression. It contains the T7 RNA polymerase gene under the control of the lacUV5 promoter, which can be induced by IPTG. The strain also carries the lysY and Iq mutations, which help reduce basal expression of T7 RNA polymerase and improve protein yields.
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Lab products found in correlation
Ni nta column, by Qiagen (1 mentions)
Xl 2000, by Bioventus (1 mentions)
His 1, by Merck Group (1 mentions)
Isopropyl β d thiogalactopyranoside, by Thermo Fisher Scientific (1 mentions)
Hispur ni nitrilotriacetic acid nta resin, by Thermo Fisher Scientific (1 mentions)
Complete mini edta, by Roche (1 mentions)
Pet15b, by Merck Group (1 mentions)
4 protocols using t7 express lysy iq competent e coli
1
Purification of Recombinant Proteins
2
Purification and Binding of Recombinant HIS-Tagged Proteins
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Recombinant HIS-tagged proteins were produced by IPTG induction (0.4 mM) of T7 Express lysY/Iq Competent E.coli (New England Biolabs C3013I) transformed with HIS-tag expressing control vector, pET-30a+ (Novagen) or HIS-tagged KRT76 domains in pDEST17 gateway backbone (Life Technologies), grown for 6–8 hours at 37°C in low salt LB, supplemented with 100 µg/ml ampicillin or 50 µg/mL kanamycin (as required). Recombinant protein was purified using 0.1 ml per 1 ml of culture of PopCulture lysis reagent (Novagen), 1 µl per mL of culture of 40 U/ml of Lysonase bioprocessing reagent (Novagen), protease inhibitors (Sigma P8849), and His-Mag beads (Novagen) according to manufacturer's protocols. Bound recombinant HIS and HIS-KRT76 protein were washed and stored at 4°C as a 1∶2 resin slurry in Tris-saline pH 7.4 containing protease inhibitors. Paw pad skin of adult was collected in RIPA lysis buffer and incubated with HIS or HIS-KRT76 overnight at 4°C. HisMag bead-bound HIS and HIS-KRT76 + lysates were then washed four times in Tris-saline pH 7.4 including 1% Triton X-100 and immunoblotted for mCLDN1 (Santa Cruz Biotechnology, SC-81796) and the HIS tag (Sigma-Aldrich, clone HIS-1).
3
Modular Expression Vector System Design
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Three expression vectors were designed to have distinct antibiotic resistance markers and compatible origins of replication. pZFN1 contained the ampicillin resistance gene, the low-copy p15A origin of replication, and ZFN1 under control of the inducible arabinose promoter. Expression from the arabinose promoter is proportional to the level of arabinose in the culture medium allowing the level of ZFN expression to be varied as needed42 (link). pZFN2 contained the spectinomycin resistance gene, the low-copy ColE1 origin of replication, and ZFN2 under control of the arabinose promoter. pTox contained the kanamycin resistance gene, the high-copy pUC origin of replication, and the toxic topoisomerase inhibitor ccdB gene under control of the T7 promoter.
The T7 RNA polymerase gene, which is required for expression from the T7 promoter70 (link), is inserted into the lac operon of the Escherichia coli genome. Induction of T7 RNA polymerase is achieved by addition of IPTG70 (link),71 (link). The system also used T7 Express lysY/Iq Competent E. coli (NEB, Ipswich, MA) for the selection. These cells express the lac inhibitor to suppress expression from the lac promoter and also T7 lysozyme, which inactivates any T7 RNA polymerase that may be produced due to leaky expression. These repression mechanisms are then overcome upon the addition of IPTG to the medium.
The T7 RNA polymerase gene, which is required for expression from the T7 promoter70 (link), is inserted into the lac operon of the Escherichia coli genome. Induction of T7 RNA polymerase is achieved by addition of IPTG70 (link),71 (link). The system also used T7 Express lysY/Iq Competent E. coli (NEB, Ipswich, MA) for the selection. These cells express the lac inhibitor to suppress expression from the lac promoter and also T7 lysozyme, which inactivates any T7 RNA polymerase that may be produced due to leaky expression. These repression mechanisms are then overcome upon the addition of IPTG to the medium.
4
Recombinant Giardia lamblia Protein Production
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Sequences of ENO and OCT were obtained from the genome of G. lamblia GS/M [17 (link)]. Sequence comparison between different G. lamblia assemblages and humans were done with BlastP (National Center for Biotechnology Information). Coding DNAs were generated by total gene synthesis with codon optimization for Escherichia coli (GenScript) and inserted into NdeI-XhoI cloning sites of the bacterial expression vector pET-15b (Millipore Sigma). Vectors were transformed into T7 Express lysY/Iq Competent E. coli (NEB) per the manufacturer’s protocol. Cells were grown at 37°C in LB medium with carbenicillin until they reached an optical density at 600 nm (OD600) of 0.4 to 0.6, and protein expression was induced by addition of 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) for 2 to 3 h. Recombinant proteins were purified from bacterial lysates using HisPur Ni-nitrilotriacetic acid (NTA) resin (Thermo Scientific). Production and purification of α1-giardin and uridine phosphorylase-like protein-1 were described before [26 (link),27 (link)]. Recombinant proteins were stored at a concentration of >1 mg/ml at 4°C in a buffer of 300 mM NaCl, 25 mM HEPES, 100 mM arginine, 20 mM imidazole, 10% glycerol, 0.1% Tween 20, and protease inhibitors (Complete Mini-EDTA, Roche). Purity of the soluble recombinant proteins was analyzed by SDS-PAGE and Coomassie Blue staining.
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