Escherichia coli hst08

Manufactured by Takara Bio

Sourced in China, Japan

Escherichia coli HST08 is a competent bacterial strain commonly used in molecular biology laboratories. It is designed for the transformation of plasmid DNA into bacterial cells.

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Lab products found in correlation

Pcdna3, by Thermo Fisher Scientific (2 mentions) Endofree plasmid maxi kit, by Qiagen (2 mentions) Puc19, by Takara Bio (1 mentions) Ampicillin, by Fujifilm (1 mentions) Pmd19, by Takara Bio (1 mentions) Pbe s vector, by Takara Bio (1 mentions) Pgem t easy cloning vector, by Promega (1 mentions) Chloromycetin, by Merck Group (1 mentions) Pet32a, by Takara Bio (1 mentions) Luria bertani medium, by Merck Group (1 mentions) Escherichia coli bl21, by Takara Bio (1 mentions) Ampicillin, by Merck Group (1 mentions)

8 protocols using escherichia coli hst08

MERS-CoV Spike Protein DNA Vaccine

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The gene sequence encoding amino acid 1-1353 (S) of the spike protein of the Al-Hasa_15_2013 strain of MERS-CoV (GenBank accession No. KF600645.1) was synthesized by Sangon Biotech Company (Shanghai, China). The synthetic full-length S, SΔCD (S without the entire cytoplasmic domain), and S1 fragment were respectively subcloned into the mammalian expression vector pcDNA3.1 (+) (Invitrogen, San Diego, CA, USA) to generate recombinant plasmid pcDNA3.1-S, pcDNA3.1-SΔCD, and pcDNA3.1-S1 (Fig.1A). The recombinant plasmid was then amplified in Escherichia coli HST08 (TaKaRa, Dalian, China) and purified using the EndoFree Plasmid Maxi Kit (QIAGEN GmbH, Shanghai, China). The recombinant plasmid was dissolved in PBS at a final concentration of 1 μg/μL for in vitro transfection and in vivo animal immunization.Fig. 1

Construction and verification of DNA vaccine. Schematic diagrams of the construction of DNA vaccines encoding different fragments of MERS-CoV spike protein (A). Western blot analyses of MERS-CoV spike protein expression in vitro. Lysates from pcDNA3.1-S, pcDNA3.1-SΔCD, pcDNA3.1-S1 transfected 293T cells (lane 1–3) and lysates from pcDNA3.1-Empty transfected 293T cells (lane 4) were incubated with mouse anti-MERS-S1 monoclonal antibodies and mouse anti-β-tubulin monoclonal antibodies (B). The schematic of the experiment (C).

Chi H., Zheng X., Wang X., Wang C., Wang H., Gai W., Perlman S., Yang S., Zhao J, & Xia X. (2017). DNA vaccine encoding Middle East respiratory syndrome coronavirus S1 protein induces protective immune responses in mice. Vaccine, 35(16), 2069-2075.

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Acetic Acid Bacteria Cultivation Protocols

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Acetic acid bacteria (AAB) used in this study are listed in Table S2, and were obtained from culture collections including JCM, NBRC, IAM, NRIC, ATCC, and DSM. Escherichia coli HST08 for general cloning host, pUC19 as a cloning vector, and pMD19 as a TA cloning vector were purchased from Takara Bio Inc. (Shiga, Japan). An ampicillin-resistant pMV24 plasmid was used as an Acetobacter spp.-E. coli shuttle vector^{25 (link)}. Oligonucleotide primers used for PCR are summarized in Table S5. AABs were grown at 30 °C in YPG medium [containing (per liter): yeast extract, 5 g; hipolypeptone, 3 g; glucose, 30 g]. E. coli was grown in Luria–Bertani (LB) medium. For preparation of solid medium, 1.5% agar was added. To select transformants of E. coli and Acetobacter spp., ampicillin was added at 40

μ

g/mL. All chemicals and enzymes used were obtained from Wako Pure Chemical (Osaka, Japan) and Takara Bio Inc., respectively, unless otherwise indicated.

Omata K., Hibi N., Nakano S., Komoto S., Sato K., Nunokawa K., Amano S., Ueda K, & Takano H. (2021). Distribution and genome structures of temperate phages in acetic acid bacteria. Scientific Reports, 11, 21567.

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Recombinant Protein Production Protocol

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C. cellulovorans (ATCC 35296) was used as the source of chromosomal DNA. Escherichia coli HST08 (TaKaRa) and origami (Novagene) were used for the construction of plasmids and cloning host for the production of recombinant proteins, respectively.

Yamamoto K, & Tamaru Y. (2016). Synergistic properties of cellulases from Clostridium cellulovorans in the presence of cellobiose. AMB Express, 6, 1.

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MERS-CoV Spike Protein Expression

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The gene sequence encoding amino acid 1-1353 (S) of the spike protein of the Al-Hasa_15_2013 strain of MERS-CoV (GenBank accession No. KF600645.1) was synthesized by Sangon Biotech Company (Shanghai, China). The synthetic full-length S, S ΔCD (S without the entire cytoplasmic domain), and S1 fragment were respectively subcloned into the mammalian expression vector pcDNA3.1 (+) (Invitrogen, San Diego, CA, USA) to generate recombinant plasmid pcDNA3.1-S, pcDNA3.1-SΔCD, and pcDNA3.1-S1 (Fig. 1A). The recombinant plasmid was then amplified in Escherichia coli HST08 (TaKaRa, Dalian, China) and purified using the EndoFree Plasmid Maxi Kit (QIAGEN GmbH, Shanghai, China). The recombinant plasmid was dissolved in PBS at a final concentration of 1 μg/μL for in vitro transfection and in vivo animal immunization.

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Cloning of hepT gene in S. aureus

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The hepT gene from S. aureus was amplified using the primer sets BamF vs. SalR1 and BamF vs. SalR2 for Smith and RN4220 strains, respectively (Table 2). The BamHI SalI digested PCR product was then ligated to pND50-pfbaA vector (Paudel et al., 2020 (link)) digested with the same enzymes to construct pND50-pfbaA-hepT_Smith and pND50-pfbaA-hepT_RN4220, respectively. The ligated plasmid was then transformed to Escherichia coli HST08 (Takara Bio) and selected on chloramphenicol plates. The strains with correct sequences were selected for transformation into electrocompetent S. aureus RN4220. Insertion in the RN4220 strain was then confirmed by PCR.

Panthee S., Paudel A., Hamamoto H., Uhlemann A.C, & Sekimizu K. (2020). The Role of Amino Acid Substitution in HepT Toward Menaquinone Isoprenoid Chain Length Definition and Lysocin E Sensitivity in Staphylococcus aureus. Frontiers in Microbiology, 11, 2076.

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Tannase-producing Lactobacillus Species Identification

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Bacterial strains used in the study and their respective sources are listed in supplementary Additional file 1: Table S1. A total of 27 tannase-producing closely related Lactobacillus species isolates, consisting of 8 isolates of L. plantarum, 9 isolates of L. paraplantarum, 10 isolates of L. pentosus were used to study the identification of their tannase encoding genes and the determination of those sequences. The taxonomic identity of L. plantarum, L. paraplantarum, and L. pentosus were determined by a 16S/23S rRNA gene spacer region targeted PCR assay [6 (link)]. Among them, L. plantarum ATCC 14917^T, L. paraplantarum NOS120 and L. pentosus 21A-3 were used as DNA donor strains for the gene cloning and expression of each of the tannases. Escherichia coli HST08 (TaKaRa Bio Inc., Shiga, Japan) strain was employed for recombinant plasmid construction. Bacillus subtilis RIK 1285 (TaKaRa) was used as the host for gene expression and enzyme purification. The pGEM^®-T Easy cloning vector (Promega, Madison, USA) and pBE-S vector (TaKaRa) were utilized for the gene cloning for DNA sequencing and heterologous expression of tannase encoding genes in B. subtilis, respectively. The lactobacilli strains were cultured statically at 37°C in MRS broth (Difco, Detroit, USA) or on MRS supplemented with 1.5% agar before the experiment.

Ueda S., Nomoto R., Yoshida K.I, & Osawa R. (2014). Comparison of three tannases cloned from closely related lactobacillus species: L. Plantarum, L. Paraplantarum, and L. Pentosus. BMC Microbiology, 14, 87.

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Bacillus coagulans T242 β-Galactosidase Expression

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Bacillus coagulans T242 is a bacterium that was isolated from a fermentation pond of a condiment factory in Dalian; preserved in the Dalian Key Laboratory of Functional Probiotics, Dalian Polytechnic University, China; and used for genomic DNA extraction.
Escherichia coli HST08 (TaKaRa Biotechnology, Dalian, China) and pET-32a(+) (TaKaRa Biotechnology) were used as host vector systems for gene cloning and DNA sequencing. Escherichia coli BL21, pET-32a(+) (TaKaRa Biotechnology), and pGro7 (molecular chaperone; Novagen, Beijing, China) were used for construction and sequencing of the recombinant expression plasmid.
Bacillus coagulans T242 was grown in fermentation medium (2% lactose, 1.5% peptone, 0.5% yeast extract, 0.5% MgSO 4 , natural pH). The expression of the β-galactosidase gene was performed in LB/Amp me-dium [Luria-Bertani medium, 100 μg•mL -1 ampicillin (Sigma, St. Louis, MO), pH 7.0]. Soluble expression of the β-galactosidase gene was achieved in LB/Amp/Cm medium (Luria-Bertani medium, 100 μg•mL -1 ampicillin, 34 μg•mL -1 chloromycetin; Sigma, pH 7.0; Samiee et al., 2016) .

Xu Y., Wu Q., Bai L., Mu G., Tuo Y., Jiang S., Zhu X, & Qian F. (2021). Cloning, expression, and bioinformatics analysis and characterization of a β-galactosidase from Bacillus coagulans T242. Journal of dairy science, 104(3).

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Cloning and Transformation of hepT in S. aureus

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The hepT gene from S. aureus was amplified using the primer sets BamF vs SalR1 and BamF vs SalR2 for Smith and RN4220 strains, respectively (Table 2). The BamHI SalI digested PCR product was then ligated to pND50-pfbaA vector 23 digested with the same enzymes to construct pND50-pfbaA-hepT Smith and pND50-pfbaA-hepT RN4220 , respectively. The ligated plasmid was then transformed to Escherichia coli HST08 (Takara Bio) and selected on chloramphenicol plates. The strains with correct sequences were selected for transformation into electrocompetent S. aureus RN4220. Insertion in the RN4220 strain was then confirmed by PCR.

Panthee S., Paudel A., Hamamoto H., Uhlemann A., & Sekimizu K. (2020). Alteration of menaquinone isoprenoid chain length and antibiotic sensitivity by single amino acid substitution in HepT.

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