All plasmids and strains in this work are listed in Table S1 . E. coli DH5α and C. crenatum were cultured at 37 °C and 30 °C, respectively, in Luria–Bertani (LB) medium (Oxoid, Hants, UK). According to the requirement of antibiotics, kanamycin and chloramphenicol (Solarbio, Beijing, China) were applied at doses of 20 and 12.5 mg/L for E. coli and C. crenatum, respectively. The strains harboring pXMJ19 were cultured with an additional 0.2 mM of isopropyl-β-D-thiogalactoside (IPTG) to induce protein expression. Brain–heart infusion medium (Solarbio, Beijing, China) was applied after electroporation to transform plasmids into C. crenatum. pK18mobsacB (a deletion vector contained the sacB-based suicide gene) was removed using sucrose medium [13 (link)].
Brain heart infusion medium
Manufactured by Solarbio
Sourced in China
Brain–heart infusion medium is a nutrient-rich growth medium used for the cultivation and isolation of a wide range of microorganisms, including fastidious bacteria and fungi. It provides essential nutrients and growth factors to support the growth of diverse microbial species.
Automatically generated - may contain errors
Lab products found in correlation
Chloramphenicol, by Solarbio (1 mentions)
Kanamycin, by Solarbio (1 mentions)
Luria bertani medium, by Thermo Fisher Scientific (1 mentions)
Sterile defibrinated sheep blood, by Solarbio (1 mentions)
Vitamin k1, by Solarbio (1 mentions)
Keracyanin chloride, by Merck Group (1 mentions)
Kuromanin chloride, by Merck Group (1 mentions)
Aluminum chloride hexahydrate, by Junsei (1 mentions)
Mannitol, by Daejung Chemicals (1 mentions)
Potassium acetate, by Junsei (1 mentions)
Folin ciocalteau, by Merck Group (1 mentions)
Sodium alginate, by Daejung Chemicals (1 mentions)
Escherichia coli e coli, by American Type Culture Collection (1 mentions)
Quercetin, by Merck Group (1 mentions)
Caffeic acid, by Merck Group (1 mentions)
Citric acid, by Daejung Chemicals (1 mentions)
Defibrinated sheep blood, by Hopebio (1 mentions)
Kanamycin, by Merck Group (1 mentions)
Luria bertani broth, by Solarbio (1 mentions)
Trans1 t1, by Transgene (1 mentions)
4 protocols using brain heart infusion medium
1
Genetic Manipulation of C. crenatum
2
Culturing Fusobacterium nucleatum for Infection
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Fn (Fn standard strain ATCC 25586, donated by the University of Louisville, USA) was cultured in a brain–heart infusion medium (Solarbio) containing 5% sterile defibrinated sheep blood (Solarbio), 1% haem chloride (Solarbio), and 0.1% vitamin K1 (Solarbio) at 37 °C in an anaerobic workstation (COY) containing 85% N2, 10% H2, and 5% CO2. The purity of Fn was detected by gram staining and culture on Columbia blood agar plates [12 (link)]. The Fn bacterial solution (OD600 = 1–2) with better viability was selected. When the cell confluence was 60–70%, Fn was added into the cell culture medium at an MOI of 10. Different times of infection (24 or 48 h) were used. A follow-up experiment was performed.
3
Probiotic Formulation Characterization and Evaluation
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The caffeic acid, keracyanin chloride, kuromanin chloride, Folin-Ciocalteau, and quercetin used in the experiment were purchased from Sigma-Aldrich (St. Louis, MO, USA). Aluminum chloride hexahydrate and potassium acetate were purchased from Junsei Chemical (Tokyo, Japan). Citric acid, sodium alginate, mannitol, and poloxamer 188 were purchased from Daejung (Seoul, Republic of Korea). Whey protein isolate, lecithin, and ascorbyl palmitate were purchased from ESfood (Gumpo, Republic of Korea). Lacticaseibacillus rhamnosus (L. rhamnosis), Pediococcus pentosaceus (P. pentosaceus), Enterococcus faecalis (E. faecalis). Escherichia coli (E. coli), and Streptococcus aureus (E. aureus) were purchased from ATCC (American Type Culture Collection). Man-Rogasa-shape (MRS) and brain heart infusion (BHI) medium was purchased from Solarbio (Shanghai, China).
4
Isolation and Cultivation of T. pyogenes
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T. pyogenes strain ATCC19411 was obtained from the American Type Culture Collection (Manassas, United States). T. pyogenes isolates (n = 19) were collected from dairy cows in Inner Mongolia and Liaoning, China and identified by 16S rRNA gene sequencing (Zhang et al., 2017 (link); Guo et al., 2020 (link)). T. pyogenes strains were cultured on Mueller-Hinton agar (MHA; Solarbio, Beijing, China) containing 5% (v/v) defibrinated sheep blood (Hopebio, Qingdao, China) in an incubator (5% CO2; Thermo Fisher, Shanghai, China) at 37°C for 36 h, and the colonies were inoculated in brain heart infusion (BHI) medium (Solarbio, Beijing, China) supplemented with 8% (v/v) fetal bovine serum (FBS; Gibco, Grand Island, United States). E. coli strains BL21 (DE3) (TransGen Biotech, Beijing, China) and Trans-1-T1 (TransGen Biotech, Beijing, China) were cultured in Luria-Bertani (LB) broth (Solarbio, Beijing, China) in a shaker at 200 rpm or on LB agar plates supplemented with kanamycin (50 μg/mL; Sigma, Shanghai, China) at 37°C.
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