Escherichia coli strain top10

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Escherichia coli strain TOP10 is a chemically competent bacterial strain commonly used in molecular biology applications. It is designed for high-efficiency transformation and plasmid propagation.

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Bl21 de3 plyss, by Merck Group (3 mentions) Pet 20b, by Merck Group (3 mentions) Chloramphenicol, by Merck Group (2 mentions) Todd hewitt broth, by BD (2 mentions) Erythromycin, by Merck Group (2 mentions) Ampicillin, by Fujifilm (2 mentions) Dam dcm e coli strain, by New England Biolabs (2 mentions) E coli bl21 star de3 strain, by Thermo Fisher Scientific (2 mentions) Groel groes chaperones, by Takara Bio (2 mentions) Tryptic soy agar plates, by Merck Group (1 mentions) L arabinose, by Merck Group (1 mentions) Tryptic soy broth tsb, by Merck Group (1 mentions) Pichia expression manual, by Thermo Fisher Scientific (1 mentions) Pichia pastoris, by Thermo Fisher Scientific (1 mentions) Manno oligosaccharides, by Megazyme (1 mentions) Pichia pastoris gs115, by Thermo Fisher Scientific (1 mentions) Ba plates, by Thermo Fisher Scientific (1 mentions) Microfluidizer, by Microfluidics (1 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions) Yeast extract, by BD (1 mentions)

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44 protocols using escherichia coli strain top10

Staphylococcus Diversity Protocols for Expression

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Different strains of Staphylococcus belonging to nine species were used: 11 Staphylococcus epidermidis (RP62A, ATCC 12228, S06-011, S06-013, S06-022, S04-028, S04-036, S04-038, S04-056, S04-057, S04-058) isolated from medical samples [25 (link),26 (link)], Staphylococcus xylosus C2a [27 (link)], Staphylococcus aureus (MW2, UAMS-1, Coch, SA113, S30) isolated from medical samples [25 (link),28 (link),29 (link)], Staphylococcus simulans DSM20273, Staphylococcus saprophyticus CIP 76.125^T, Staphylococcus hominis CIP 81.57^T, Staphylococcus haemolyticus CIP 81.56^T, Staphylococcus hyicus DSM 20459^T, and Staphylococcus sciuri CIP 81.62^T. One strain of Micrococcus aurantiacus ATCC 11731 was also used. They were grown at 37 °C in Tryptic Soy Broth (TSB, Sigma-Aldrich, St. Louis, MO, USA) with orbital shaking (150 rpm) or on Tryptic Soy Agar plates (TSA, Sigma-Aldrich).
Escherichia coli strain TOP10 (Invitrogen, Carlsbad, CA, USA) was used as the cloning host for propagation of expression vector and used for protein expression. It was grown at 37 °C in lysogeny broth (LB) with orbital shaking (200 rpm) or on agar medium supplemented with ampicillin (100 µg/mL) when appropriate. Protein expression was induced by adding L-arabinose (0.2% w/v) (Sigma-Aldrich).

Vermassen A., Talon R., Andant C., Provot C., Desvaux M, & Leroy S. (2019). Cell-Wall Hydrolases as Antimicrobials against Staphylococcus Species: Focus on Sle1. Microorganisms, 7(11), 559.

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Heterologous Expression of P. oxalicum GZ-2 Enzyme

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The P. oxalicum GZ-2 referred to in this study was isolated and identified as previously reported [13 (link)] and has been deposited at the China General Microbiological Culture Collection Center (CGMCC 7527). Escherichia coli strain TOP 10 (Invitrogen, San Diego, CA) grown in Luria Bertani (LB) medium at 37°C was used as the host cell for plasmid cloning and propagation of the recombinant expression pPICZαA vector. Pichia pastoris GS115 (Invitrogen, San Diego, CA) was used for heterologous protein expression. All media and protocols for Pichia pastoris are described in the Pichia expression manual (Invitrogen, San Diego, CA). Manno-oligosaccharides were purchased from Megazyme (Bray, Ireland). P. oxalicum GZ-2 was maintained and cultured according previously study [32 (link)]. Pichia pastoris GS115 was cultivated at 30°C in yeast extract peptone dextrose (YPD) medium (1% yeast extract, 2% tryptone, and 2% dextrose).

Liao H., Li S., Zheng H., Wei Z., Liu D., Raza W., Shen Q, & Xu Y. (2014). A new acidophilic thermostable endo-1,4-β-mannanase from Penicillium oxalicum GZ-2: cloning, characterization and functional expression in Pichia pastoris. BMC Biotechnology, 14, 90.

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Bacterial Strains for Rhizobia and Agrobacteria Transformations

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All rhizobial strains were maintained on TY medium. Escherichia coli strain TOP10 (Invitrogen, Stockholm, Sweden) was used for plasmid propagation and manipulation. Strains TOP10 and GJ23 helper (Van Haute et al., 1983 (link)) were used for triparental mating. E. coli bacteria were grown in Luria–Bertani medium (LB; Miller, 1972 ) at 37°C. Agrobacterium rhizogenes strains R1000 (Moore et al., 1979 (link)) and Arqua1 (Quandt et al., 1993 (link)) were used for hairy root transformation of M. truncatula, and LBA1334 (Offringa et al., 1986 (link)) and AR1193 (Stougaard et al., 1987 (link)) for hairy root transformation of D. glomerata. Agrobacteria were grown in YEB medium (Van Larebeke et al., 1977 (link)) at 28°C. M. truncatula plants were inoculated with the strain S. meliloti Sm1021 (Meade and Signer, 1977 (link)). D. glomerata plants were inoculated with the strain Candidatus Frankia datiscae Dg1, originating from nodules of Coriaria nepalensis from Pakistan (Mirza et al., 1994 (link); Persson et al., 2011 (link); Persson et al., 2015 (link)). Crushed D. glomerata nodules were used for the inoculation of root systems.

Demina I.V., Maity P.J., Nagchowdhury A., Ng J.L., van der Graaff E., Demchenko K.N., Roitsch T., Mathesius U, & Pawlowski K. (2019). Accumulation of and Response to Auxins in Roots and Nodules of the Actinorhizal Plant Datisca glomerata Compared to the Model Legume Medicago truncatula. Frontiers in Plant Science, 10, 1085.

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Culturing M. pneumoniae and E. coli Strains

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Wild‐type M. pneumoniae strain M129 and its derivatives (Reagents and Tools Table, Appendix Table S1) were grown in modified Hayflick medium (Yus et al, 2009) at 37°C under 5% CO₂ in tissue culture flasks, unless otherwise indicated. When needed, Hayflick medium was supplemented with 0.8% agar, puromycin (3 µg/ml), chloramphenicol (20 µg/ml), or tetracycline (2 µg/ml) for selection of transformants. Escherichia coli strain TOP10 (Invitrogen) was used for vector cloning. This strain was grown at 37°C in LB broth or LB agar plates containing ampicillin (100 µg/ml) and X‐Gal (40 µg/ml) as needed.

Burgos R., Weber M., Martinez S., Lluch‐Senar M, & Serrano L. (2020). Protein quality control and regulated proteolysis in the genome‐reduced organism Mycoplasma pneumoniae. Molecular Systems Biology, 16(12), e9530.

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Cultivation and Glycolipid Production in U. maydis

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U. maydis strains were grown at 28°C in liquid YEPS (YEPS-L) medium (1% yeast extract, 0.4% peptone, 0.4% sucrose) or on solid potato dextrose broth containing 1.5% Bacto agar. All U. maydis strains used and generated in this study are listed in Table S1 in the supplemental material and are derivatives of the U. maydis wild-type strain MB215 (39 (link)). To induce glycolipid production, U. maydis strains were inoculated into nitrogen starvation medium (optical density [OD] = 0.1) containing 0.17% YNB (yeast nitrogen base without ammonium sulfate) and 1% glucose as a carbon source and grown for 3 to 6 days at 23°C or 30°C. Escherichia coli strain Top10 (Invitrogen) was used for the transformation and amplification of plasmid DNA (40 (link)).

Becker F., Linne U., Xie X., Hemer A.L., Bölker M., Freitag J, & Sandrock B. (2022). Import and Export of Mannosylerythritol Lipids by Ustilago maydis. mBio, 13(5), e02123-22.

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Propagation of Bacteriophage CP_F1 on Campylobacter

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Bacteriophage CP_F1 was originally isolated from a pig manure sample and was propagated on C. jejuni PT14 (Brathwaite et al., 2013 (link)). All strains, associated plasmids and oligonucleotide PCR primers used in this study are listed in Table 1. The C. jejuni strains were routinely grown on blood agar base No. 2 plates (BA plates; Oxoid) supplemented with 5% horse blood (TCS Biosciences) under microaerobic conditions in either a modular atmosphere controlled cabinet (5% CO₂, 5% O₂, 2% H₂, 88% N₂) or in anaerobic jars using gas replacement (7.3% CO₂, 5.6% O₂, 3.6% H₂, 83.5% N₂) at 42°C. Escherichia coli strain TOP10 (Invitrogen) was used for cloning and cultured aerobically at 37°C on Luria agar plates or in Luria broth containing appropriate antibiotic as required.

Lis L, & Connerton I.F. (2016). The Minor Flagellin of Campylobacter jejuni (FlaB) Confers Defensive Properties against Bacteriophage Infection. Frontiers in Microbiology, 7, 1908.

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Cultivation and Glycolipid Production in Fungal Strains

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Fungal strains were grown in liquid YEPSL (1% yeast extract, 0.4% peptone, 0.4% sucrose) or on solid potato dextrose broth containing 1.5% Bacto agar at 28°C and 23°C, respectively. The U. maydis strains used and generated in this study are listed in Supplementary Table S1 and are derivatives of the U. maydis strain MB215 (Hewald et al., 2005 (link)). U. hordei (Uh4857-4) and Sporisorium scitamineum (RK109) were kind gifts from Regine Kahmann (MPI Marburg, Germany). Moesziomyces aphidis (formerly Pseudozyma aphidis) (DSM70725) was received from Michael Günther (Fraunhofer Institute Stuttgart, Germany). To induce glycolipid production U. maydis strains were grown to stationary phase in YEPSL and then transferred to nitrogen starvation medium (OD = 0.1) containing 0.17% YNB (yeast nitrogen base without ammonia) and 1% glucose as carbon source. Glycolipid production was then allowed for 6 days at 23°C under rotation. Escherichia coli strain Top10 (Invitrogen) was used for transformation according to Hanahan et al., and amplification of plasmid DNA (Hanahan et al., 1991 (link)). Bacillus subtilis wildtype strain was a kind gift of Erhard Bremer (Philipps-University Marburg, Germany).

Becker F., Stehlik T., Linne U., Bölker M., Freitag J, & Sandrock B. (2021). Engineering Ustilago maydis for production of tailor-made mannosylerythritol lipids. Metabolic Engineering Communications, 12, e00165.

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Microbial Metabolite Uptake Assay

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Proteus mirabilis (ATCC 29906™), Escherichia fergusonii (ATCC 35469™), Lactobacillus acidophilus (ATCC 4356), and Escherichia coli strain Top10 (Invitrogen) were grown in LB medium in an incubator shaking at 250 rpm, 37 °C overnight. The bacterial suspension was spun down at 6000× g, 4 °C for 12 min and resuspended in ¼ volume of cold PBS for incubation with d₉-GPC and d₆-choline at a final concentration of 100 µM. P. mirabilis lysate was prepared by Microfluidizer (M110Y, Microfluidics Corporation, Newton, MA, USA), with lysozyme added in advance for incubation with d₉-GPC and d₉-choline.

Wang Z., Hazen J., Jia X., Org E., Zhao Y., Osborn L.J., Nimer N., Buffa J., Culley M.K., Krajcik D., van den Born B.J., Zwinderman K., Levison B.S., Nieuwdorp M., Lusis A.J., DiDonato J.A, & Hazen S.L. (2021). The Nutritional Supplement L-Alpha Glycerylphosphorylcholine Promotes Atherosclerosis. International Journal of Molecular Sciences, 22(24), 13477.

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Culturing Streptococcal and E. coli Strains

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Streptococcal strains listed in Supplementary Table 5 were cultured in Todd-Hewitt broth (BD Biosciences) supplemented with or without 0.2% yeast extract (BD Biosciences) (THY or TH medium) at 37 °C. Streptococcus pseudopneumoniae ATCC BAA-960 (also called as SK1069 or CCUG 49455)47 (link) was kindly provided by Dr. T. Hoshino, Nagasaki University, Japan. The Escherichia coli strain TOP10 (Invitrogen) was used as a host for derivatives of plasmids pSET4s, pDCerm, and pDESTerm. All E. coli strains were cultured in Luria-Bertani (LB) broth at 37 °C with agitation. For selection and maintenance of mutants, antibiotics were added to the media at the following concentrations: ampicillin (Wako), 100 μg/ml for E. coli; kanamycin (Sigma-Aldrich), 50 μg/ml for E. coli; chloramphenicol (Sigma-Aldrich), 10 μg/ml for E. coli; spectinomycin (Wako), 100 μg/ml for E. coli and 150 μg/ml for GBS; and erythromycin (Sigma-Aldrich), 400 μg/ml for E. coli and 5 μg/ml for GBS. Human brain endothelial cell line (hBMEC) was maintained in RPMI 1640 medium (Invitrogen) supplemented with 10% FBS, 10% NuSerum (BD), and 1% MEM nonessential amino acids, and were incubated at 37 °C in 5% CO₂.

Yamaguchi M., Hirose Y., Nakata M., Uchiyama S., Yamaguchi Y., Goto K., Sumitomo T., Lewis A.L., Kawabata S, & Nizet V. (2016). Evolutionary inactivation of a sialidase in group B Streptococcus. Scientific Reports, 6, 28852.

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Expression of Recombinant Proteins

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The K. phaffii strain X33 and Escherichia coli strain TOP10 were purchased from Invitrogen (Carlsbad, CA). The expression vector pGAPzα-rDNA was constructed and preserved in our lab. HRP conjugated-anti-His-antibody (A00612) was purchased from Genscript.

Li Y., Xie S., Chen M., Li H., Wang Y., Fan Y., An K., Wu Y, & Xiao W. (2023). Development of an antibody-ligand fusion protein scFvCD16A-sc4-1BBL in Komagataella phaffii with stimulatory activity for Natural Killer cells. Microbial Cell Factories, 22, 67.

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