E coli dh5α

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E. coli DH5α is a strain of Escherichia coli bacteria commonly used in molecular biology laboratories. It is a non-pathogenic, genetically engineered strain with a well-characterized genome. E. coli DH5α is used as a host for the cloning and propagation of recombinant DNA plasmids.

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Escherichia coli (E. coli) DH5α (3 citations) E. coli MAX Efficiency DH5α (3 citations) E. coli DH5α strain (36 citations) DH5α competent E. coli strain (2 citations) DH5α strain of E. coli (2 citations) E. coli DH5α-T1R (2 citations) Competent E. coli DH5α (9 citations) E. coli DH5α competent cells (21 citations) E. coli DH5α cells (47 citations) DH5α™-T1R E. coli cells (2 citations)

180 protocols using e coli dh5α

Graphite-Based Nanocomposite Synthesis

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Graphite powder (G), potassium permanganate (KMnO₄), hydrogen peroxide (H₂O₂), potassium bromide (KBr), hoechst 333242 (HS), glutaraldehyde 50%, sodium borohydride (NaBH₄), 1% osmic acid, 2’,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), propidium iodide (PI), dimethyl sulfoxide (DMSO) and silver nitrate (AgNO₃) were purchased from Sigma-Aldrich, St. Louis, MO, USA. Sulfuric acid (H₂SO₄, 98%) was purchased from Lab Alley, Austin, Texas, USA. Hydrochloric acid (HCl) and nitric acid (HNO₃) were purchased from Showa Chemical Industry Co., Ltd., Honshu, Japan. Luria-Bertani (LB) broth, phosphate-buffered saline (PBS) and agar-agar powder were purchased from Sigma-Aldrich, St. Louis, MO, USA. E. coli (DH5α) was purchased from Thermo Fisher Scientific Inc., Taipei City, Taiwan.

Truong T.T., Kumar S.R., Huang Y.T., Chen D.W., Liu Y.K, & Lue S.J. (2020). Size-Dependent Antibacterial Activity of Silver Nanoparticle-Loaded Graphene Oxide Nanosheets. Nanomaterials, 10(6), 1207.

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Bacterial Strain Preparation for Metabolite Production

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E. coli DH5α (Thermofisher) was used for cloning experiments and E. coli BL21AI (Thermofisher) for metabolite production. Bacteria of strain DH5α were rendered chemically competent using the high efficiency transformation protocol described by Inoue et al.^{52 (link)}. Bacteria of strain BL21AI were rendered electrocompetent using the method of Sambrook et al.⁵³. LB medium was used for standard protocols. Minimal medium, used for metabolite production, consisted of a base of M9 minimal salts supplemented with oligo-elements and vitamins as previously described^{29 (link)}. Carbon sources consisted of 0.5% glucose for starter cultures and 0.05% glucose, 0.5% glycerol, 0.2% lactose, and 0.2% arabinose for DKP production cultures. Ampicillin (200 µg/ml), kanamycin (50 mg/ml), and chloramphenicol (25 µg/ml) were added as required.

Dubois P., Correia I., Le Chevalier F., Dubois S., Jacques I., Canu N., Moutiez M., Thai R., Gondry M., Lequin O, & Belin P. (2019). Reprogramming Escherichia coli for the production of prenylated indole diketopiperazine alkaloids. Scientific Reports, 9, 9208.

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Bacterial Strains and Culturing Conditions

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Salmonella enterica serovar Typhimurium strain SL1344, E. coli strain K12 were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA), E. coli DH5α was obtained from ThermoFischer Scientific, E. coli strain Nissle 1917 was obtained from Ardeypharm, and the SL1344 sifA mutant strain was obtained from Dr Olivia Steele-Mortimer.^{36 (link),45 (link)} All bacteria were maintained as described previously.^{9 (link),46 (link)} Briefly, a single colony was inoculated into LB broth and grown for 8 h under aerobic conditions and then under oxygen-limiting conditions. For the SL1344 sifA mutant, streptomycin with a final conc of 100 μg/mL was added to LB broth. Cells were infected with a multiplicity of infection (moi) of 10.

Sayed I.M., Ibeawuchi S.R., Lie D., Anandachar M.S., Pranadinata R., Raffatellu M, & Das S. (2021). The interaction of enteric bacterial effectors with the host engulfment pathway control innate immune responses. Gut Microbes, 13(1), 1991776.

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Cloning and Expression of Fhf1 and Fhf1Δ470

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The constructs containing the gene encoding Fhf1 and Fhf1Δ470 were designed to harbor a C-terminal 10xhistidine tag. The synthetic codon-optimized genes, optimized for E. coli expression and all devoid of their original signal peptides, were synthesized by GenScript (Piscataway, NJ, USA) and inserted in the pET-31b(+) vector between the NdeI and XhoI restriction sites.
E. coli DH5α (Thermo Fisher Scientific, Waltham, MA, USA) was used as plasmid propagation host. E. coli BL21 (DE3) cells (Thermo Fisher Scientific, Waltham, MA, USA) were used as an expression host.

Vuillemin M., Silchenko A.S., Cao H.T., Kokoulin M.S., Trang V.T., Holck J., Ermakova S.P., Meyer A.S, & Mikkelsen M.D. (2020). Functional Characterization of a New GH107 Endo-α-(1,4)-Fucoidanase from the Marine Bacterium Formosa haliotis. Marine Drugs, 18(11), 562.

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Plasmid Rescue for Tns Mapping in C. jejuni

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Plasmid rescue was used to assess the chromosomal location of Tns within C. jejuni M1 STMs. The technique was based on the method described in Grant et al.23 (link) but was amended to make use of vector cloning. BglII-digested STM genomic DNA was ligated to BamHI-digested and dephosphorylated pUC19. Ligations were transformed into E. coli DH5α (Thermo Scientific) according to manufacturer’s instructions and transferred onto LB agar supplemented with chloramphenicol (Cm). Colonies present after O/N incubation were used to inoculate 5 ml LB broth with Cm. After O/N incubaction of LB cultures, plasmid DNA was extracted and analysed by Sanger sequencing using primers directed out of the Tn (AJG227 and CC1, Table S6).

Esson D., Mather A.E., Scanlan E., Gupta S., de Vries S.P., Bailey D., Harris S.R., McKinley T.J., Méric G., Berry S.K., Mastroeni P., Sheppard S.K., Christie G., Thomson N.R., Parkhill J., Maskell D.J, & Grant A.J. (2016). Genomic variations leading to alterations in cell morphology of Campylobacter spp. Scientific Reports, 6, 38303.

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Cell Culture and Bacterial Growth Protocols

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This study used the following cell lines: HEK293A (Thermo Fisher Scientific), E. coli DH5α (Thermo Fisher Scientific), and E. coli OverExpress C43(DE3) (Sigma-Aldrich). All HEK293A cells were grown following the standard protocols. Briefly, they were grown in Dulbecco’s Modified Eagle Medium (DMEM 2, Nissui Pharmaceutical) supplemented with 10% fetal bovine serum (Sigma-Aldrich), penicillin (Sigma-Aldrich), streptomycin (Thermo Fisher Scientific), and glutamine (Thermo Fisher Scientific) at 37°C with 5% CO₂. For culturing E. coli, general LB media (Thermo Fisher Scientific), ampicillin (Sigma-Aldrich), and IPTG (Goldbio) were used at 37°C at 250 rpm in a shaker incubator.

Bumbak F., Pons M., Inoue A., Paniagua J.C., Yan F., Wu H., Robson S.A., Bathgate R.A., Scott D.J., Gooley P.R, & Ziarek J.J. (2023). Ligands selectively tune the local and global motions of neurotensin receptor 1 (NTS1). Cell reports, 42(1), 112015.

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Recombinant Protein Expression Workflow

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Restriction endonucleases (HindIII, XhoI, NcoI, SnaBI, NheI), T4 DNA ligase, Phusion High-Fidelity PCR Master Mix, PageRulerTM Prestained Protein Ladder, and Pierce™ Coomassie Plus Assay Reagent were purchased from Thermo Fisher Scientific, Vilnius, Lithuania. pET21d and pET28b vectors were purchased from Novagen, Madison, WI, USA. Nutrient medium was purchased from Roth, Karlsruhe, Germany. Sucrose and salts were purchased from Sigma-Aldrich, Buchs, Switzerland. TSKgel G4000SWXL was obtained from Tosoh Bioscience, Tokyo, Japan. E. coli DH5α (Thermo Fisher Scientific, Vilnius, Lithuania) and BL21 (DE3) (Novagene, Madison, WI, USA) were used for cloning and expression experiments.

Labutytė G., Povilonienė S., Šimoliūnas E., Gabrielaitis D., Skapas M., Noreika A., Meškys R, & Časaitė V. (2021). Functionalized Protein Nanotubes Based on the Bacteriophage vB_KleM-RaK2 Tail Sheath Protein. Nanomaterials, 11(11), 3031.

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Cultivation and Storage of P. aeruginosa and E. coli

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P. aeruginosa and E. coli isolates were grown in LB medium (10 g/L tryptone, 10 g/L NaCl, 5 g/L yeast extract (pH 7.2). All strains used in this study are summarized in Supplementary Table S3. Strains were routinely grown at 37°C, with shaking at 180 rpm. Plasmids were propagated in E. coli DH5α (ThermoFisher Scientific, U.S.A.) and E. coli BL21 (DE3) pLysS (Agilent Technologies, U.S.A.) was used for the production of pyocins. Media were supplemented with ampicillin at 100 µg/ml (Sigma–Aldrich, U.S.A.), kanamycin at 50 µg/ml (Sigma–Aldrich, U.S.A.), chloramphenicol at 34 µg/ml (Sigma–Aldrich, U.S.A.) or gentamicin at 50 µg/ml (Gibco, U.S.A.) when required. Bacterial strains used in this study were stored in 50% glycerol at −80°C until used.

Premsuriya J., Mosbahi K., Atanaskovic I., Kleanthous C, & Walker D. (2023). Outer membrane translocation of pyocins via the copper regulated TonB-dependent transporter CrtA. Biochemical Journal, 480(14), 1035-1049.

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Anaerobic Bacteroides and E. coli Culturing

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Detailed information of the strains used in this study is provided in Supplementary Data 4. All anaerobes were cultured in an anaerobic chamber with an atmosphere of 83% N₂, 2% H₂ and 15% CO₂ at 37 °C. For most experiments, Bacteroides strains, AC, CC were grown in ABB media, ER was grown in ABB media with the addition of 3.3 mM sodium acetate (Sigma) and RI was grown in Brain Heart Infusion Broth (BHI, Sigma). We used E. coli pir2 (Invitrogen) for cloning and maintenance of plasmids with R6K origin (pNBU2-ermGb derivatives and pFW1000 derivatives, Supplementary Data 4). E. coli DH5α (Thermo Fisher Scientific) was used for cloning and maintenance of plasmids with p15A, pSC101ts and ColE1 origins (pFW2000 derivatives, pFW3000 and pFW4000). We used E. coli BW29427 (E. coli Genetic Stock Center, CGSC) for E. coli-Bacteroides conjugations. All E. coli strains were grown aerobically in Luria Bertoni (LB, Sigma) media. To support the growth of E. coli BW29427, we supplemented LB media with 25 μM of 2,6-Diaminopimelic acid (DAP, Sigma). We used the following antibiotics when required including 100 μg mL⁻¹ carbenicillin (Carb, IBI Scientific), 25 μg mL⁻¹ erythromycin (Erm, Sigma) and 200 μg mL⁻¹ gentamicin (Gm, Sigma). We used 1 mM of Isopropyl β-D-1-thiogalactopyranoside (IPTG, Gold Biotechnology) for the induction of FnCpf1.

Feng J., Qian Y., Zhou Z., Ertmer S., Vivas E.I., Lan F., Hamilton J.J., Rey F.E., Anantharaman K, & Venturelli O.S. (2022). Polysaccharide utilization loci in Bacteroides determine population fitness and community-level interactions. Cell host & microbe, 30(2), 200-215.e12.

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Recombinant protein expression in E. coli and P. pastoris

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E. coli DH5α and P. pastoris Mut^s strain KM71H were procured from Thermo Fisher Scientific Corporation. The P. pastoris integrative plasmids pD912 and pD915 were obtained from Atom Inc. (previously DNA 2.0). Both the vectors contain Saccharomyces cerevisiae derived α-prepro signal sequence, alcohol oxidase 1 (AOX1) terminator, zeocin resistance marker and pUC ori sequence for propagation in E. coli. Vector pD912 has methanol inducible AOX1 promoter, while constitutive glyceraldehye-3-phosphate dehydrogenase (GAP) promoter is present in pD915.

Kaushik N., Lamminmäki U., Khanna N, & Batra G. (2020). Enhanced cell density cultivation and rapid expression-screening of recombinant Pichia pastoris clones in microscale. Scientific Reports, 10, 7458.

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