IP-S photoresist was purchased from Nanoscribe GmbH (Germany); SU-8 developer, from Microchem (USA); Novec 1720, from 3 M (USA); isopropyl alcohol, from VWR Analytical (USA); epoxy (#04001) from Hardman Inc. (USA); perfluorodecalin (PFD), from Sigma-Aldrich (USA); 1H,1H,2H,2H-perfluorooctanol (PFO), from Alfa Aesar Co. Inc. (USA); KDO8PS genes, from GenScript Inc. (USA); E. Coli, from New England Biolabs (USA); Sigma Fast tablets, β-mercaptoethanol, Tris, HCl, ethylenediaminetetraacetic acid (EDTA), protamine sulfate, KCl, and protease inhibitor cocktail, from Sigma-Aldrich (USA); and poly(ethylene glycol) 5000 methyl ether (PEG5000 MME), from Hampton Research (USA). Fused silica capillaries were obtained from Molex LLC (USA); tubing and capillary union connectors, from IDEX Health and Science LLC (USA); PicoClear unions, from New Objective, Inc. (USA); PEEK tubing, from Zeus (USA); double sided tape, from 3 M (USA); and 10 kDa cutoff filters, from Centricon, Millipore (USA).
E coli
Manufactured by New England Biolabs
Sourced in United States
E. coli is a common laboratory bacteria strain used for various research and application purposes. It serves as a model organism for studying cellular processes, genetic engineering, and protein expression. E. coli can be easily cultured and genetically manipulated, making it a valuable tool in the field of biology and biotechnology.
Automatically generated - may contain errors
Lab products found in correlation
Shrna oligos of kpnb1, by Integrated DNA Technologies (2 mentions)
Psuper retro puro vector, by Oligoengine (2 mentions)
Plasmid miniprep kit, by Thermo Fisher Scientific (2 mentions)
Polybrene, by Merck Group (2 mentions)
Hifi dna assembly master mix, by New England Biolabs (2 mentions)
Nanodrop one, by Thermo Fisher Scientific (2 mentions)
Ip s photoresist, by Nanoscribe (1 mentions)
Novec 1720, by 3M (1 mentions)
Sigmafast tablet, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Potassium chloride (kcl), by Merck Group (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
Double sided tape, by 3M (1 mentions)
Su 8 developer, by MicroChem (1 mentions)
E coli cells, by New England Biolabs (1 mentions)
Pcdna3.1 myc hisa, by GenScript (1 mentions)
Genejet gel extraction kit, by Thermo Fisher Scientific (1 mentions)
Maxi plasmid dna prep kit, by Qiagen (1 mentions)
Gene pulser, by Bio-Rad (1 mentions)
Calcium phosphate transfection kit, by Merck Group (1 mentions)
18 protocols using e coli
1
Microfluidic Protein Purification Protocol
2
Recombinant Protein Expression in E. coli
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MnmE, MnmG, SHMT, and MTHFR plasmids are all transformed into E. coli BL21-DE3 phage T1 resistant electrocompetent E. coli cells (New England Biolabs) and inoculated onto Lennox broth (LB) agar plates containing kanamycin (34 μg/ml). The plates were incubated at 37 °C overnight to allow for the growth of the colonies. A single colony was picked from each plate and inoculated into ∼120 to 130 ml LB media and subsequently grown at 37 °C overnight by shaking at 200 rpm.
Large scale expression was carried out in 6 to 12 2.8 l Fernbach flasks containing 1 l of LB media. An aliquot of the overnight cultured bacteria (10 ml) was added to each flask containing media and kanamycin (34 μg/ml). The cells were grown at 37 °C by shaking at 175 to 200 rpm until the A600 of the culture reached ∼0.5 to 0.6, at which point the cells were induced by the addition of IPTG to a final concentration of 100 μM. Flasks containing the bacteria expressing the MnmG were further supplemented with solid riboflavin (∼50 mg/l) after the IPTG induction to support the overexpression of the flavoprotein. The induced cells were shaken at 150 to 175 rpm overnight (∼12–16 h) at 37 °C (for MnmE and MnmG) or 18 °C (for SHMT or MTHFR). The cells were harvested by centrifugation at 5000g for 10 min, flash frozen in liquid nitrogen, and stored at –80 °C until further use.
Large scale expression was carried out in 6 to 12 2.8 l Fernbach flasks containing 1 l of LB media. An aliquot of the overnight cultured bacteria (10 ml) was added to each flask containing media and kanamycin (34 μg/ml). The cells were grown at 37 °C by shaking at 175 to 200 rpm until the A600 of the culture reached ∼0.5 to 0.6, at which point the cells were induced by the addition of IPTG to a final concentration of 100 μM. Flasks containing the bacteria expressing the MnmG were further supplemented with solid riboflavin (∼50 mg/l) after the IPTG induction to support the overexpression of the flavoprotein. The induced cells were shaken at 150 to 175 rpm overnight (∼12–16 h) at 37 °C (for MnmE and MnmG) or 18 °C (for SHMT or MTHFR). The cells were harvested by centrifugation at 5000g for 10 min, flash frozen in liquid nitrogen, and stored at –80 °C until further use.
3
Codon-Optimized Expression of dcm1 and dam2 in E. coli
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Open reading frames of dcm1 (isolate FBG) and dam2 (isolate 8958) were codon optimized for best expression in E. coli and cloned into BamHI-HinDIII sites of pcDNA3.1(-)-myc-HisA (GenScript Biotech B.V., Leiden, The Netherlands). Plasmids were propagated into dam/dcm-deficient E. coli (New England Biolabs GmbH, Frankfurt am Main, Germany) and cultivated in LB medium supplemented with 100 mg/L ampicillin and 25 mg/L chloramphenicol. Heterologous expression of His6-tagged proteins was documented in His4-immunostaining as formerly published [35 (link)].
4
Comprehensive E3 Ligase CRISPR Library
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sgRNAs targeting 606 Ubiquitin E3 ligases, as well as 243 non‐targeting control (NTC) sequences, were designed by Azimuth2 (Doench et al, 2016 (link)) and the top four picks (on‐target scores > 0.6) were chosen per gene, extended by 5 and 3 prime 3Cs homology and obtained as a pool from Twist Bioscience (Dataset EV1 ). The E3‐targeting sgRNA library was made by 3Cs, as described previously (Wegner et al, 2019 (link), 2020 ; Diehl et al, 2021 (link)). In brief, 3Cs‐DNA was generated by mixing phosphorylated pre‐annealed oligonucleotides (sgRNA‐encoding) with ssDNA (library template plasmid). 3Cs‐DNA was purified and desalted using the GeneJET Gel Extraction Kit (Thermo Fisher) and parts of it were analysed by gel electrophoresis alongside the dU‐ssDNA template. The remaining 3Cs‐DNA was electroporated into 10‐beta electrocompetent E. coli (New England Biolabs) by using a Bio‐Rad Gene Pulser and incubated overnight. The final sgRNA library plasmid DNA was purified using the Maxi Plasmid DNA Prep Kit (Qiagen).
5
Lentiviral Vector Production and Titration
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Lentiviral Gene Ontology (LeGO) vectors LeGO-Cer2, LeGO-V2 and LeGO-T2 were generous gifts from Drs. Kristoffer Riecken (né Weber) and Boris Fehse at the University Medical Center Hamburg-Eppendorf. Plasmid replications were performed via transformation of NEB 5-alpha competent E. Coli (New England Biolabs, Ipswich, MA) and DNA purification with the QIAfilter Plasmid Midi Kit (Qiagen, Valencia, CA), according to manufacturer’s instructions.
Lentivirus generation and titer calculation followed previously published protocol38 , scaled up 2.75×. Briefly, 1.375E7 HEK-293T cells were seeded in a T175 flask for an 8-hour transfection, for which 27.5 μg LeGO (Cer2, V2 or T2), 27.5 μg pMDLg/RRE, 13.75 μg rPSV-Rev and 5.50 μg pHCMV-VSV-G plasmids were added. Cells were subsequently filter-removed with 100 μm nylon mesh cell strainer and 0.45 μm PVDF syringe filters. For titer determination, HEK-293 T cells were seeded in 24-well plates at 5E4 cells per well, and transduced for 13 hours with 0.1, 0.5, 1.0, 5.0, 10.0, or 100 μL of viral supernatant. Three wells were transduced per supernatant volume. Percent fluorescent cells were determined three days later with flow cytometry. Calcium phosphate transfection kit was purchased from Sigma-Aldrich.
Lentivirus generation and titer calculation followed previously published protocol38 , scaled up 2.75×. Briefly, 1.375E7 HEK-293T cells were seeded in a T175 flask for an 8-hour transfection, for which 27.5 μg LeGO (Cer2, V2 or T2), 27.5 μg pMDLg/RRE, 13.75 μg rPSV-Rev and 5.50 μg pHCMV-VSV-G plasmids were added. Cells were subsequently filter-removed with 100 μm nylon mesh cell strainer and 0.45 μm PVDF syringe filters. For titer determination, HEK-293 T cells were seeded in 24-well plates at 5E4 cells per well, and transduced for 13 hours with 0.1, 0.5, 1.0, 5.0, 10.0, or 100 μL of viral supernatant. Three wells were transduced per supernatant volume. Percent fluorescent cells were determined three days later with flow cytometry. Calcium phosphate transfection kit was purchased from Sigma-Aldrich.
6
Knockdown of KPNB1 Using shRNA
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ShRNA oligos of KPNB1 are purchased from Integrated DNA Technologies, Inc. (Coralville, IA,USA). Annealed double strand oligos were cloned into pSUPER.retro.puro vector (OligoEngine, Seattle, WA, USA) according to the provided manual for the expression of shRNA. The reconstructed plasmids were amplified in E.coli (New England Biolabs, Ipswich, MA, USA) and purified using plasmid miniprep kit (Thermo fisher scientific).
Virus was produced using Gryphon retroviral packaging cell lines (Allele Biotechnology, San Diego, CA, USA). In brief, 10 μg of plasmid was transfected to Grypho cell lines in a 6-cm dish. In 48 hours, supernatant that contains virus particles was collected. For virus infection, 1 ml of the collected virus supernatant supplemented with 3 μl of polybrene (EMD Millipore, Billerica, MA, USA) at 5 mg/ml was added to the culture medium of C42B in a 6-cm plate. In 24 hours, virus supernatant was replaced with fresh medium. Infected cells were selected using puromycin (Sigma-Aldrich-Aldrich, St. Louis, MO, USA).
Virus was produced using Gryphon retroviral packaging cell lines (Allele Biotechnology, San Diego, CA, USA). In brief, 10 μg of plasmid was transfected to Grypho cell lines in a 6-cm dish. In 48 hours, supernatant that contains virus particles was collected. For virus infection, 1 ml of the collected virus supernatant supplemented with 3 μl of polybrene (EMD Millipore, Billerica, MA, USA) at 5 mg/ml was added to the culture medium of C42B in a 6-cm plate. In 24 hours, virus supernatant was replaced with fresh medium. Infected cells were selected using puromycin (Sigma-Aldrich-Aldrich, St. Louis, MO, USA).
7
Genetically Engineered Protein for Biomaterials
8
Purification and Characterization of Recombinant Human LCN-2
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Recombinant human LCN-2 was produced by transformation of E. coli (New England Biolabs, C2527H) with either a pGEX-4T3-LCN-2 or the pGEX-4T3_LCN-2_mutant plasmid, both containing an insert for human LCN-2 tagged with glutathione-S-transferase (GST) for purification. LCN-2 expression of bacterial culture was initiated by supplementing Isopropyl-β-D-thiogalactopyranosid (IPTG, Sigma Aldrich, I6758). LCN-2 was purified by using Pierce Glutathione Agarose (Thermo Fisher, 16100). Detoxi-Gel Endotoxin Removing Resin (Thermo Fisher, 20339) was utilised for eliminating bacterial endotoxins.
LCN-2 iron–catechol complex formation was monitored by UV–Vis spectrometry in the range of 300–800 nm. The iron-free (apo-) protein spectrum was obtained from a 6.25 µM LCN-2 solution. For acquisition of spectra of both iron-loaded (holo-) LCN-2 and mutant LCN-2, the protein solution was mixed with an aliquot of 5 mM iron–catechol (3:1 mol:mol) solution, resulting in equimolar concentration of LCN-2 and iron–catechol.
LCN-2 iron–catechol complex formation was monitored by UV–Vis spectrometry in the range of 300–800 nm. The iron-free (apo-) protein spectrum was obtained from a 6.25 µM LCN-2 solution. For acquisition of spectra of both iron-loaded (holo-) LCN-2 and mutant LCN-2, the protein solution was mixed with an aliquot of 5 mM iron–catechol (3:1 mol:mol) solution, resulting in equimolar concentration of LCN-2 and iron–catechol.
9
Optogenetic control of Rho GTPases
10
Knockdown of KPNB1 Using shRNA
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