In a sterile biosafety cabinet, stock vials of B. burgdorferi (1×107B. burgdorferi/tube), stored at −80°C, were thawed into BSK-H complete medium with 6% rabbit serum (50 mL, Sigma-Aldrich) or BSK-II complete medium with 6% rabbit serum (50 mL, generously made by the Chou lab or made in-house following protocol from Jenny Hyde’s Lab). Strain B. burgdorferi B31 A3 GFP was generously provided by George Chaconas. Strains B31 A3 p66 wildtype (WT) and B31 A3 p66 KO C3–14 (Δp66) were generated as described previously and plasmids profiled prior to use in experimnts18 (link),19 (link). ML23-pBBE22luc was generously provided by Jenny Hyde21 (link),22 (link). For the Δp66 B. burgdorferi cultures, we selected for the mutation in kanamycin (200 μg/mL, Sigma-Aldrich). Each culture was cultured in sealed tubes at 37°C unless otherwise stated.
Protocol detail
Culturing and Handling of B. burgdorferi
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Corresponding Organization : University of California, San Francisco
Other organizations : Massachusetts Institute of Technology, Stanford University, University of Tübingen, Medical College of Wisconsin
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Variable analysis
independent variables
- Strain of B. burgdorferi (B31 A3 GFP, B31 A3 p66 wildtype, B31 A3 Δp66)
- Growth medium (BSK-H complete medium with 6% rabbit serum, BSK-II complete medium with 6% rabbit serum)
- Growth of B. burgdorferi
- Storage temperature (-80°C)
- Incubation temperature (37°C)
- Presence of kanamycin (200 μg/mL) for Δp66 B. burgdorferi cultures
- B31 A3 GFP strain of B. burgdorferi
- B31 A3 p66 wildtype (WT) strain of B. burgdorferi
- B31 A3 Δp66 strain of B. burgdorferi
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