M17 medium

Manufactured by Merck Group

Sourced in Germany, United States

M17 medium is a microbiological culture medium used for the isolation and cultivation of streptococcus species. It provides the necessary nutrients and growth factors to support the growth of these bacteria.

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Lab products found in correlation

Erythromycin, by Merck Group (6 mentions) Chloramphenicol, by Merck Group (6 mentions) Mrs medium, by Merck Group (5 mentions) Biliverdin hcl, by Merck Group (4 mentions) 5 bromo 4 chloro 3 indolyl β d galacto pyranoside x gal, by Thermo Fisher Scientific (3 mentions) Anaerocult a, by Merck Group (3 mentions) Luria bertani medium, by Acumedia (2 mentions) Glycerol, by Merck Group (2 mentions) Fatty acid, by Merck Group (2 mentions) Agmatine sulfate salt, by Merck Group (2 mentions) Gibson assembly kit, by New England Biolabs (2 mentions) Dna purification kit, by Qiagen (2 mentions) Dna polymerase, by New England Biolabs (2 mentions) Ni nta resin, by Qiagen (2 mentions) T4 ligase, by New England Biolabs (2 mentions) Nadph, by Merck Group (2 mentions) Malonyl coa, by Merck Group (2 mentions) Restriction endonuclease, by New England Biolabs (2 mentions) Oligonucleotide primers, by Integrated DNA Technologies (2 mentions) Gene pulser 2, by Bio-Rad (2 mentions)

41 protocols using m17 medium

Culturing and Preserving E. coli and L. lactis

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Bacteria and plasmids used in this study are listed in Table 1. E. coli Top10 strains were aerobically grown in Luria-Bertani (LB) medium (Acumedia), incubated at 37°C with vigorous shaking. L. lactis subsp. cremoris MG1363 was grown in M17 medium (Sigma-Aldrich) containing 0.5% glucose (Synth) (GM17) at 30°C without agitation. Antibiotics were added at the indicated concentrations as necessary: kanamycin (Sigma-Aldrich) 50 μg/mL for E. coli and erythromycin (Sigma-Aldrich) 500 μg/mL for both E. coli and L. lactis.
All pure cultures of bacteria were kept as stock cultures in 40% glycerol (Sigma-Aldrich) for E. coli and 25% glycerol for L. lactis at −80°C.

Mancha-Agresti P., Drumond M.M., Carmo F.L., Santos M.M., Santos J.S., Venanzi F., Chatel J.M., Leclercq S.Y, & Azevedo V. (2016). A New Broad Range Plasmid for DNA Delivery in Eukaryotic Cells Using Lactic Acid Bacteria: In Vitro and In Vivo Assays. Molecular Therapy. Methods & Clinical Development, 4, 83-91.

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Bacterial Strain Cultivation Protocols

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The bacterial strains used in this study are shown in Table S1. Lactococcus lactis NZ9000 (de Ruyter et al., 1996; Kuipers et al., 1998; Mierau and Kleerebezem, 2005) and Lactococcus lactis NZ9000ΔhtrA (Lindholm et al., 2004) were grown at 30 °C in M17 medium (Sigma) supplemented with 0.5% glucose (GM‐17) without agitation or in the same medium solidified with 1.5% agar. To maintain selection pressure on transformation, 10 μg ml⁻¹ chloramphenicol or erythromycin, or both, was added to the growth medium of L. lactis NZ9000 and L. lactis NZ9000ΔhtrA. Lactobacillus salivarius ATCC 11741 was grown in De Man, Rogosa and Sharpe (MRS) medium (Merck) at 37 °C without aeration. E. coli strain DH5α was grown at 37 °C with agitation in lysogeny broth (LB) medium supplemented with 100 μg ml⁻¹ ampicillin.

Škrlec K., Pucer Janež A., Rogelj B., Štrukelj B, & Berlec A. (2017). Evasin‐displaying lactic acid bacteria bind different chemokines and neutralize CXCL8 production in Caco‐2 cells. Microbial Biotechnology, 10(6), 1732-1743.

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Bacterial Strain Cultivation and Storage

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Bacterial strains and plasmids used in this study are listed in Table 1. E. coli Top10 strain was grown in Luria-Bertani (LB) medium (Acumedia, Lansing, MI, United States) at 37°C with vigorous shaking. L. lactis ssp. cremoris MG1363 was grown statically at 30°C in M17 medium (Sigma-Aldrich, St. Louis, MO, United States) supplemented with 0.5% glucose (w/v) (Labsynth, São Paulo, Brazil) (G-M17). The Erythromycin antibiotic (Sigma-Aldrich) was added at the indicated concentration as necessary; 500 μg/mL for E. coli and 125 μg/mL for L. lactis. Pure cultures of bacteria were kept as stock cultures in 40% glycerol (v/v) (Sigma-Aldrich) for E. coli and 25% glycerol (v/v) for L. lactis at −80°C.

Coelho-Rocha N.D., de Castro C.P., de Jesus L.C., Leclercq S.Y., de Cicco Sandes S.H., Nunes A.C., Azevedo V., Drumond M.M, & Mancha-Agresti P. (2018). Microencapsulation of Lactic Acid Bacteria Improves the Gastrointestinal Delivery and in situ Expression of Recombinant Fluorescent Protein. Frontiers in Microbiology, 9, 2398.

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Oral Gavage of Engineered Lactobacillus

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C57BL/6 mice received 100 μL of bacterial suspension (L. lactis MG1363 FnBPA⁺ (pValac::dts::IL-4) and L. lactis MG1363 FnBPA⁺ (pValac::IL-10)) at a final dose of 1 × 10⁹ CFU in 0.9% saline by oral gavage. The spleen, liver, and mesenteric lymph nodes (MLNs) were collected under strict aseptic conditions 2 h, 4 h, 6 h, 12 h, and 24 h after bacterial administration. The tissues were macerated in 500 μL of 0.9% saline, and 100 μL of this homogenate was plated in M17 medium (Sigma-Aldrich) supplemented with agar, 0.5% glucose, chloramphenicol (10 μg/mL) (Sigma-Aldrich), and erythromycin (5 μg/mL) (Sigma-Aldrich). The plates were incubated at 30°C for 24 h.

Preisser T.M., da Cunha V.P., Santana M.P., Pereira V.B., Cara D.C., Souza B.M, & Miyoshi A. (2021). Recombinant Lactococcus lactis Carrying IL-4 and IL-10 Coding Vectors Protects against Type 1 Diabetes in NOD Mice and Attenuates Insulitis in the STZ-Induced Model. Journal of Diabetes Research, 2021, 6697319.

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Bacterial Strain and Growth Conditions

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The bacterial strains and plasmids used in this study are listed in Table S1 and the primers used for this study are listed in Table S2. E. coli cells were incubated at 37°C in a Luria‐Bertani medium (tryptone, 10 g/L; yeast extract, 5 g/L; NaCl, 10 g/L) whereas E. faecalis cells were cultured at 37°C in M17 medium (Sigma). In a few cases, we used AC medium (tryptone, 10 g/L; yeast extract, 10 g/L; glucose 1 g/L; K₂HPO₄ 5 g/L) which contains low levels of fatty acids. Antibiotics were added in the following concentrations (in mg/L): kanamycin sulfate 30 for E. coli; erythromycin at 250 for E. coli and 10 for E. faecalis; chloramphenicol 30 for E. coli and 10 for E. faecalis; fatty acids were added at 0.1 mM unless otherwise stipulated.

Dong H, & Cronan J.E. (2022). Unsaturated fatty acid synthesis in Enterococcus faecalis requires a specific enoyl‐ACP reductase. Molecular Microbiology, 118(5), 541-551.

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Heterologous Production of Fatty Acids

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Malonyl-CoA, NADPH, fatty acids, antibiotics, M17 medium and agmatine sulfate salt were purchased from Sigma-Aldrich. DNA polymerases, Gibson assembly kit, restriction endonucleases and T4 ligase were from NEB. Sodium [1-¹⁴C]acetate (specific activity, 58.6 mCi/mmol) was provided by Moravek. Ni-NTA resin and the DNA purification kits were from Qiagen, and the silver nitrate silica gel thin layer plates were from Analtech. All the other reagents were of the highest available quality. Oligonucleotide primers were synthesized by Integrated DNA Technologies and DNA sequencing was provided by ACGT, Inc.

Dong H, & Cronan J.E. (2022). Unsaturated fatty acid synthesis in Enterococcus faecalis requires a specific enoyl‐ACP reductase. Molecular Microbiology, 118(5), 541-551.

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Culturing E. coli and E. faecalis with Antibiotics

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The bacterial strains and plasmids used in this study are listed in Table S1 and the primers used for this study are listed in Table S2. E. coli cells were incubated at 37°C in Luria-Bertani medium (tryptone, 10 g/L; yeast extract, 5 g/L; NaCl, 10 g/L) whereas E. faecalis cells were cultured at 37°C in M17 medium (Sigma). In a few cases we used AC medium (tryptone, 10 g/L; yeast extract, 10 g/L; glucose 1 g/L; K₂HPO₄ 5 g/L) which contains low levels of fatty acids. Antibiotics were added at the following concentrations (in mg/L): kanamycin sulfate 30 for E. coli; erythromycin at 250 for E. coli and 10 for E. faecalis; chloramphenicol 30 for E. coli and 10 for E. faecalis; Fatty acids were added at 0.1 mM unless otherwise stipulated.

Dong H, & Cronan J.E. (2022). Unsaturated fatty acid synthesis in Enterococcus faecalis requires a specific enoyl‐ACP reductase. Molecular Microbiology, 118(5), 541-551.

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L. lactis Strain Cultivation Protocols

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The strains used in this work are listed in Table 1. L. lactis MG1363 FnBPA⁺, L. lactis MG1363 FnBPA⁺ (pValac::dts::IL-4), and L. lactis MG1363 FnBPA⁺ (pValac::IL-10) were grown in M17 medium (Sigma-Aldrich) supplemented with 0.5% glucose with or without chloramphenicol (10 μg/mL) (Sigma-Aldrich) and erythromycin (5 μg/mL) (Sigma-Aldrich) at 30°C.

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Biosynthesis of Fatty Acids

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Malonyl‐CoA, NADPH, fatty acids, antibiotics, M17 medium, and agmatine sulfate salt were purchased from Sigma‐Aldrich. DNA polymerases, Gibson assembly kit, restriction endonucleases and T4 ligase were from NEB. Sodium [1‐¹⁴C]acetate (specific activity, 58.6 mCi/mmol) was provided by Moravek. Ni‐NTA resin and the DNA purification kits were from Qiagen, and the silver nitrate silica gel thin layer plates were from Analtech. All the other reagents were of the highest available quality. Oligonucleotide primers were synthesized by Integrated DNA Technologies and DNA sequencing was provided by ACGT, Inc.

Dong H, & Cronan J.E. (2022). Unsaturated fatty acid synthesis in Enterococcus faecalis requires a specific enoyl‐ACP reductase. Molecular Microbiology, 118(5), 541-551.

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Inducible Expression of M. leprae Hsp65 in L. lactis

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The invasive L. lactis FnBPA+ (pXYCYT:Hsp65) strain^{20 (link)} was grown at 30 °C without agitation in M17 medium (Sigma-Aldrich, São Paulo, Brazil) supplemented with 0.5% glucose, chloramphenicol (10 µg/mL) (Sigma-Aldrich, São Paulo, Brazil) and erythromycin (5 µg/mL) (Sigma-Aldrich, São Paulo, Brazil). To induce expression of the M. leprae Hsp65 ORF, the recombinant L. lactis NCDO2118 FnBPA+ (pXYCYT:Hsp65) strain was grown with 2% xylose (Dinâmica, Indaiatuba, Brazil) and 0.5% galactose (Vetec, Duque de Caxias, Brazil) for approximately 8 h (until the cells reached an OD_{600 nm} of ~ 2)^{17 (link),20 (link)}.

da Cunha V.P., Preisser T.M., Santana M.P., Machado D.C., Pereira V.B, & Miyoshi A. (2020). Mycobacterial Hsp65 antigen delivered by invasive Lactococcus lactis reduces intestinal inflammation and fibrosis in TNBS-induced chronic colitis model. Scientific Reports, 10, 20123.

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