The plasmid pGEM-T Easy was purchased from Promega (Madison, WI, USA), whereas the Escherichia coli cloning strain TOP10F′ was from Invitrogen (Carlsbad, CA, USA). All other reagents were of analytical grade.
Top10f
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
TOP10F' is a chemically competent Escherichia coli strain commonly used in molecular biology applications. It is designed to provide high transformation efficiency for the introduction of plasmid DNA into bacterial cells.
Automatically generated - may contain errors
Lab products found in correlation
Dna modifying enzymes, by New England Biolabs (3 mentions)
Quikchange, by Agilent Technologies (3 mentions)
Pgem t easy, by Promega (1 mentions)
Er2566, by New England Biolabs (1 mentions)
Pet30a, by Thermo Fisher Scientific (1 mentions)
Puc19, by Thermo Fisher Scientific (1 mentions)
Pzero 2, by Thermo Fisher Scientific (1 mentions)
Pet15b, by Thermo Fisher Scientific (1 mentions)
Bradford reagent, by Bio-Rad (1 mentions)
Galangin, by Merck Group (1 mentions)
Ca9 22, by Thermo Fisher Scientific (1 mentions)
Cal27, by Thermo Fisher Scientific (1 mentions)
Quercetin, by Merck Group (1 mentions)
Kaempferol, by Merck Group (1 mentions)
Myricetin, by Merck Group (1 mentions)
Restriction enzyme, by New England Biolabs (1 mentions)
Nickel column, by Qiagen (1 mentions)
Anti flag magnetic beads, by Merck Group (1 mentions)
Topo xl pcr cloning kit, by Thermo Fisher Scientific (1 mentions)
Restriction enzyme, by Thermo Fisher Scientific (1 mentions)
35 protocols using top10f
1
Cloning with pGEM-T Easy Vector
2
Bacterial Genetic Construction and Protein Expression
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All restriction enzymes and DNA-modifying enzymes were purchased from New England Biolabs (Ipswich, MA, USA) unless explicitly stated otherwise. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless explicitly stated otherwise. The E. coli strains TOP10F′ (Invitrogen, USA) and BL21(DE3)pLysS (Novagen, UK) were used for genetic construction and protein expression, respectively.
3
Isolation and Cultivation of Serratia Phage Hosts
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The bacterial host of investigated phages Serratia sp. OS31 was isolated from rock biofilms of the Zloty Stok gold and arsenic mine (SW Poland) using the methods described previously [93 (link)] and was kindly provided by Lukasz Drewniak. The strain was grown under aerobic conditions in lysogeny broth (LB) or R2A media [94 ] at 20 °C. Using the same methodology [93 (link)], we isolated two other Serratia spp. strains (BZSmr3 and BZSmr6) from biofilms of the walls of the Gertruda Adit in the Zloty Stok in April 2018. They were used in host range testing assay. Bacteria used in host range testing (Table S8 ) were grown under aerobic conditions in LB at 30 °C, except Yersinia eneterolitica 2/O:9 (at room temperature, RT) and environmental Serratia spp. strains from Zloty Stok (at 20 °C).
The following Escherichia coli strains were used in this study: TOP10F’ (Invitrogen, Waltham, MA, USA), ER2566 (New England BioLabs, Ipswich, MA, USA), and ER2929 Dam− strain lysogenized with λDE3 element, which carried the gene for T7 RNA polymerase under control of the lacUV5 promoter [95 (link)]. These were cultured under standard conditions in LB medium at 37 °C. When required, the media were supplemented with kanamycin at 50 μg·mL−1, and ampicillin at 100 μg·mL−1. Plasmids pUC19 (Thermo Fisher Scientific, Waltham, MA, USA) and pET30a (Invitrogen) were used as cloning or expression vectors.
The following Escherichia coli strains were used in this study: TOP10F’ (Invitrogen, Waltham, MA, USA), ER2566 (New England BioLabs, Ipswich, MA, USA), and ER2929 Dam− strain lysogenized with λDE3 element, which carried the gene for T7 RNA polymerase under control of the lacUV5 promoter [95 (link)]. These were cultured under standard conditions in LB medium at 37 °C. When required, the media were supplemented with kanamycin at 50 μg·mL−1, and ampicillin at 100 μg·mL−1. Plasmids pUC19 (Thermo Fisher Scientific, Waltham, MA, USA) and pET30a (Invitrogen) were used as cloning or expression vectors.
4
E. coli Protein Expression Protocol
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Escherichia coli K-12 strain TOP10F′ (Invitrogen, UK) and MC4100 (Casadaban, 1976 (link)) were used for plasmid-based protein expression. MC4100ΔtatA/E (Sargent et al., 1998 (link)) was used when indicated. When grown in culture, cells were grown in LB medium in a shake incubator in the presence of ampicillin (100 μg/ml). Cultures of cells containing pOFX-tac (derivative) plasmids also contained kanamycin (50 μg/ml) and glucose (0.2%). Cultures were grown at 37°C in tubes or flasks in a shake incubator (200 rpm) using a 5:1 tube/flask:culture volume ratio. Growth on solid medium was performed at 37°C making use of LB-agar (1.5%) plates supplemented with ampicillin (100 μg/ml). Plates also contained chloramphenicol (30 μg/ml) if indicated.
5
Cloning and Expression of β-Galactosidases
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B. circulans ATCC 31382, obtained from LGC Standards (Wesel, Germany), was grown at 30 °C in 3% (w/v) beef extract and 5% (w/v) peptone. E. coli strain TOP10F (Invitrogen, Carlsbad, CA) was used for cloning purposes and grown at 37 °C in Luria–Bertani medium. For plasmid selection the appropriate antibiotic was added at the following concentrations: 50 μg ml−1 for kanamycin and 100 μg ml−1 for ampicillin. E. coli BL21 (DE3) grown at 30 °C was used for high-level expression of β-galactosidases of strain ATCC 31382. Plasmids pZErO-2 and pET15b (both Invitrogen) were used for cloning and expression purposes, respectively. Protein concentrations were determined using the Bradford reagent (Bio-Rad, Munich, Germany) with bovine serum albumin as standard.
6
Generating T4 motB Amber Mutant Phage
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E. coli strains TOP10F’ (Invitrogen, Carlsbad, CA, USA), BL21(DE3), and BL21(DE3)/pLysE [20 (link)] were used for expression studies. NapIV suppressing (NapIV S) and NapIV wt (non-suppressing, NapIV NS) [21 (link)] E. coli were used for T4 infections. Unless otherwise noted, cells were grown at 37 °C with shaking at 250 rpm.
Wild-type T4D+ (T4 wt), T4 amG1 (T4 motAam) [22 (link)], and T4 motBam were used for infections. T4 motBam was obtained by recombination of pTE103-motBam into the T4 genome. BL21(DE3)/pTE103-motBam was infected with T4 wt at a multiplicity of infection (MOI) of 1 during exponential phase and incubated for 75 min. Cells were lysed with chloroform and resulting phage were titered on NapIV S. Plaques were screened for the presence of the amber mutation by hybridization of a 32P-labeled probe containing either the wt or mutant sequence as described [23 (link)]. Potential mutant plaques were used to generate phage stocks by infecting NapIV S. Phage stocks containing the amber mutation were subjected to a subsequent round of single plaque selection to ensure a homogenous stock of T4 motBam. To confirm the presence of the amber mutation, the motB gene was amplified by PCR then sequenced by Macrogen (Rockville, MD, USA).
Wild-type T4D+ (T4 wt), T4 amG1 (T4 motAam) [22 (link)], and T4 motBam were used for infections. T4 motBam was obtained by recombination of pTE103-motBam into the T4 genome. BL21(DE3)/pTE103-motBam was infected with T4 wt at a multiplicity of infection (MOI) of 1 during exponential phase and incubated for 75 min. Cells were lysed with chloroform and resulting phage were titered on NapIV S. Plaques were screened for the presence of the amber mutation by hybridization of a 32P-labeled probe containing either the wt or mutant sequence as described [23 (link)]. Potential mutant plaques were used to generate phage stocks by infecting NapIV S. Phage stocks containing the amber mutation were subjected to a subsequent round of single plaque selection to ensure a homogenous stock of T4 motBam. To confirm the presence of the amber mutation, the motB gene was amplified by PCR then sequenced by Macrogen (Rockville, MD, USA).
7
Cloning of Hydrophobin-Alpha Fusion Protein
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Escherichia coli (E. coli) TOP10 F′ (Invitrogen, Darmstadt, Germany) was used for molecular cloning purposes. All constructs were obtained using standard molecular techniques [35 ]. The DNA sequence encoding the EAS hydrophobin without its N-terminal secretion signal was amplified and integrated into pET28b vector (Novagen, Darmstadt, Germany) 3′ of the sequence encoding the (His)6-tag, thus generating pET28b-EAS. To obtain a fusion protein of EAS and the yeast α-factor, the DNA sequences encoding a (GGGGS)3 linker element and the α-factor peptide were codon-optimized for expression in E. coli [36 ] and inserted 3′ of the EAS sequence in pET28b-EAS, resulting in pET28b-EAS-α.
8
Cultivation and Selection of E. coli and Hfx. volcanii
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The Escherichia coli strain Top10F’ (Invitrogen, Carlsbad, United States) was grown at 37°C overnight in Luria-Bertani broth. For selection of ampicillin-resistant clones, the medium was supplemented with 100 μg/ml ampicillin. The Hfx. volcanii strains used are listed in Supplementary Table S1 . Hfx. volcanii WFD11 and WR340 were cultivated in medium containing 3 M NaCl, 150 mM MgSO4, 50 mM KCl, 10 nM MnCl2, 25 mM Tris/HCl pH 7.2, 0.5% (w/v) tryptone, 0.3% (w/v) yeast extract, and 0.02% (w/v) histidine. In case of Hfx. volcanii H1424, Hv-Ca medium (Allers et al., 2004 (link)) was used, supplemented with thymidine (40 μg/ml) and uracil (50 μg/ml). Hfx. volcanii transformants were selected by 6 mg/ml lovastatin (Lam and Doolittle, 1989 (link)). Plates with solid media (containing 1.8% [w/v] agar) were incubated in plastic bags at 42°C under humid conditions for 4–5 days.
9
Bacterial Strain Cultivation for Cloning and Expression
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E. coli strain Top 10F’ (Invitrogen, UK) was used for cloning and BL21(DE3) (Novagen, Germany) was used for expression experiments. Both were grown in lysogeny broth (LB; 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl). For expression experiments the LB was supplemented with 0.2% (w/v) glycerol. To select for plasmids (Table 2 ) antibiotics were added to the following concentrations: ampicillin, 100 µg/mL; kanamycin, 50 µg/mL; and chloramphenicol 30 µg/mL. Unless stated otherwise, cultures were incubated at 37 °C with shaking.
10
Plasmid Construction Using Standard Techniques
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