Rapamycin-Mediated FRB-FKBP Dimerization Assay

To validate rapamycin-mediated FRB-FKBP dimerization in mycobacteria, Msm cells were grown to mid-log phase (OD₆₀₀ ≈ 0.5) and plated at a final OD₆₀₀ = 0.1 with 5-fold serial dilutions of rapamycin. The Nano-Glo® 5× reagent (furimazine substrate + buffer; Promega) was prepared and added to each sample. Luminescence was measured in opaque white plates after incubation with linear shaking at 37 °C for 5 min using a Spark 10 M plate reader (Tecan).

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Won H.I., Zinga S., Kandror O., Akopian T., Wolf I.D., Schweber J.T., Schmid E.W., Chao M.C., Waldor M., Rubin E.J, & Zhu J. (2024). Targeted protein degradation in mycobacteria uncovers antibacterial effects and potentiates antibiotic efficacy. Nature Communications, 15, 4065.

Publication 2024

Spark 10 M plate reader

Corresponding Organization :

Other organizations : Harvard University, Boston VA Research Institute, Chinese Academy of Sciences, Institute of Microbiology

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Variable analysis

independent variables

Rapamycin concentration (5-fold serial dilutions)

dependent variables

Luminescence

control variables

Msm cell density (OD600 = 0.1)
Incubation time (5 min)
Incubation temperature (37 °C)
Plate reader (Spark 10 M)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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