Escherichia coli krx

Manufactured by Promega

Sourced in United States

Escherichia coli KRX is a bacterial strain designed for high-level protein expression. It is a genetically engineered derivative of the E. coli K-12 host strain.

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Lab products found in correlation

Pd 10 desalting column, by GE Healthcare (1 mentions)

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Escherichia coli strain KRX Escherichia coli

4 protocols using escherichia coli krx

Recombinant PfAgo Protein Purification

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Example 1

PfAgo Expression and Purification

A strep(II)-tagged (N-terminal) codon-optimized version of PfAgo gene was ordered from GeneScript (Piscataway, N.J.). The codon-optimized gene was cloned into pET28a plasmid to yield the expression plasmid pHZ-PfAgo. The expression plasmid was transformed into Escherichia coli KRX (Promega) according to manufacturer's protocol. The strain was cultivated overnight at 37° C. in LB medium supplemented with 0.4% (w/v) glucose and 50 μg/ml kanamycin. Following overnight incubation, the culture was centrifuged at 3220×g for 5 min and the supernatant was removed. Cell pellets were resuspended in Terrific Broth containing 50 μg/ml kanamycin and incubated at 37° C. until the OD₆₀₀of 1.2-1.5 was reached. The culture was cold shocked by incubation in ice bath for 15 min and protein expression was induced by addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) and L-rhamnose to final concentrations of 1 mM and 0.1% (w/v), respectively. Expression was continued by incubation at 30° C. for 20 h. Purification was performed using previously mentioned protocol (10) with minor modification in the sonication step (twenty 30 sec pulses at 30% power with 30 sec pause between pulses). The purified protein was stored in storage buffer (20 mM Tris-HCl, pH 8.0, 300 mM NaCl, 0.5 mM MnCl₂, 15% (v/v) glycerol) and the aliquots were stored at −80° C.

US11261483B2. Programmable DNA-guided artificial restriction enzymes (2022-03-01). The Board of Trustees of the University of Illinois [US]. Inventors: Huimin Zhao [US], Behnam Enghiad [US].

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Cultivation of E. coli, P. pastoris, and S. cerevisiae

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Escherichia coli KRX (Promega, USA) was used for plasmid construction and propagation work. KRX was cultivated in LB (Lysogeny Broth) medium supplemented with 100 µg/mL ampicillin. For experiments involving P. pastoris CBS 7435 (ΔHIS4), obtained from ACIB (Austrian Center of Industrial Biotechnology, Austria) as well as the wild type CBS 7435 (CECT 11047 at Spanish Type Culture Collection, Spain), were used. Saccharomyces cerevisiae wild type strain LBG H620 was provided by the Institute for Agricultural Bacteriology and Fermentation Biology, ETH Zurich, Switzerland. Yeast shake flask cultivations were carried out in BMD (Buffered Minimal Dextrose) [49 ] or YPD (Yeast Peptone Dextrose) medium, supplemented with 4 mg/L l-histidine when necessary. Experiments in 96-deep-well plates with 2.4 mL total volume (Eppendorf, Germany) used BMD, BMM2 (Buffered Minimal Methanol) and BMM10 as previously described by Weis et al. [30 (link)] and Hartner et al. [31 (link)]. In brief, BMD is used for the growth phase while BMM2 and BMM10 induce expression of the target gene by maintaining a 0.5 % (v/v) methanol content in the culture medium. The 96-deep-well plates contained up to 500 µL of culture media, were sealed with sterile Breathseal film (Greiner, Germany) and were shaken at 340 rpm at 28 °C.

Schwarzhans J.P., Wibberg D., Winkler A., Luttermann T., Kalinowski J, & Friehs K. (2016). Integration event induced changes in recombinant protein productivity in Pichia pastoris discovered by whole genome sequencing and derived vector optimization. Microbial Cell Factories, 15, 84.

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Purification and Use of PfAgo Protein

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PfAgo protein was purified using the established protocol^{11 (link)}. Briefly, a strep(II)-tagged (N-terminal) codon-optimized version of PfAgo gene was cloned into pET28a plasmid. The expression plasmid was transformed into Escherichia coli KRX (Promega) followed by induction and purification. The purified protein was stored in storage buffer (20 mM Tris-HCl, pH 8.0, 300 mM NaCl, 0.5 mM MnCl₂, 15% (v/v) glycerol) and the aliquots were stored at −80 °C.
The length and GC content optimized reporter is a key component in SPOT detection as cleavage of the reporter will generate a detectable fluorescent output signal. In order to get significant net fluorescence value increases in the detection of SARS-CoV-2 using the SPOT assay, we used a 17 nt reporter probe (N gene: /56-FAM/XXX/3BHQ-1/, E gene: /6-ROX/XXX/3BHQ-2/) for PfAgo recognition and cleavage. 5’ phosphorylated 16 nt gDNAs were used for primary cleavage to generate the secondary gDNA and trigger the secondary cleavage reaction. All the primers, gDNAs, and reporters were ordered from IDT, and sequences are listed in Supplementary Data 4.

Xun G., Lane S.T., Petrov V.A., Pepa B.E, & Zhao H. (2021). A rapid, accurate, scalable, and portable testing system for COVID-19 diagnosis. Nature Communications, 12, 2905.

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Recombinant Protein Expression and Purification

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Escherichia coli KRX (Promega) was transformed with plasmid pBK01. An overnight pre-culture (5 mL LB, 100 mg/mL kanamycin, 37 C, 210 rpm) was grown to saturation and used to inoculate the production culture (400 mL LB, identical conditions). With an optical density of OD 600 = 0.7 reached, gene expression was induced by supplementation of L-rhamnose to a final concentration of 0.1% (m/v) and carried out for 18 hours at 16 C. Cells were harvested by centrifugation (4 C, 3,200 3 g, 30 min) and suspended in lysis buffer (50 mM NaH 2 PO 4 $H 2 O, 300 mM NaCl, pH 8.0) amended with 20 mM imidazole. Disruption was carried out with a Sonopuls ultrasonic sonifier (Bandelin) and was followed by centrifugation (4 C, 17,000 3 g, 20 min) for the removal of cell debris. Protein purification was performed by nickel affinity chromatography on a Protino nickel-nitrilo acetic resin (Macherey-Nagel) subjecting the sample to a stepwise imidazole gradient (20-500 mM) in lysis buffer. The purified Pys N-His 6 C-A didomain was desalted on a PD-10 desalting column (GE Healthcare) and eluted with reaction buffer (100 mM Tris, pH 7.5, 25 mM MgCl 2 , 2.5 mM EDTA). Protein purification and concentration were verified and determined by SDS-PAGE and Bradford's assay (Bradford, 1976) , respectively (c.f. Figure S1).

Klapper M., Braga D., Lackner G., Herbst R, & Stallforth P. (2018). Bacterial Alkaloid Biosynthesis: Structural Diversity via a Minimalistic Nonribosomal Peptide Synthetase. Cell chemical biology, 25(6).

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