Glycerol stocks of E. coli BL21(DE3)pLysS (NEB, Ipswich, MA) transformed with pET9c-ospA19 (link) were used to produce the vaccine. Untransformed E. coli BL21(DE3)pLysS were used as placebo controls. Multi-strain cultures of B. burgdorferi sensu stricto recovered from heart and bladder tissues from white-footed mice naturally infected with B. burgdorferi (Bb) in 2005, 2006, 2007 and 2008 are kept as glycerol stocks in the laboratory. We have sequenced DNA purified from ticks that fed on mice infected with this multi-strain culture and found it contains the following OspC variants: A, D, E, F, I, J, K, M, Q, T, X 25 (link). To generate a multi-strain culture for neutralization assays, 200ul of each stock (2005 to 2008) was combined and cultured in 7mL of Barbour-Stoenner-Kelly (BSK) media supplemented with 100x antibiotic mix for Borrelia at 34°C for 2–4 weeks until a cell density of 107 Bb/mL was reached. All bacterial glycerol stocks are stored at −80°C.
E coli bl21 de3 plyss
Manufactured by New England Biolabs
E. coli BL21(DE3)pLysS is a laboratory strain of Escherichia coli that is commonly used for the expression of recombinant proteins. It is designed to facilitate the efficient production of target proteins under the control of the T7 promoter system.
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2 protocols using e coli bl21 de3 plyss
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Multi-Strain Borrelia Neutralization Assay
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Bacterial Strain Cultivation Protocols
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All bacterial strains used in this study were provided by Goldberg-Klinik Kelheim GmbH, Germany, RWTH University Aachen, Germany, Bayerisches Landesamtf. Gesundheit u. Lebensmittelsicherheit (LGL), Germany, Universitätsklinikum Regensburg, Germany, DSMZ (German Collection of Microorganisms and Cell Cultures), KU Leuven (Belgium), University of Veterinary Medicine Hanover (TiHo, Germany), or Robert Koch Institute (Wernigerode, Germany), each of which has been assigned with a specific LiCC (Lisando Culture Collection) number. All strains were grown in brain heart infusion broth (BHI) (Oxoid Deutschland GmbH), except E. faecalis, S. aureus, E. coli and S. Typhimurium, which were grown in lysogeny broth (LB; Sambrook et al., 1989) with or without shaking at 37 °C. E. faecalis, S. suis, S. viridans group, S. sanguinis, and S. gordinii were grown under microaerophilic conditions (85% N2, 10% CO2, 5% O2). Likewise, different E. coli strains were used in this study: E. coli NEB Turbo (New England Biolabs GmbH, Country) for DNA cloning and cell stock storage, and E. coli BL21(DE3)pLysS (New England Biolabs) as host strain for protein expression. For proper selection, ampicillin (Roche Diagnostics, Mannheim, Germany) (100 μg/ml) was used.
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