Team:Edinburgh OG/Experiments/Rhodococcus

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Introduction to Rhodococcus sp.

Our second organism is the Gram positive bacterium Rhodococcus jostii. Such bacteria have applications in bioremediation as they are able to degrade polychlorinated biphenyls (PCBs), which are a variety of chlorinated compounds that, even though banned in the United States, they have been found in 500 of the 1,598 National Priorities List sites already identified by the Environmental Protection Agency (EPA) [13] and affect animal and human health by causing skin conditions, liver damage and even cancer [14].

Experiments and Protocols

This is a record of protocol and optimization that we did


1. Competent cells

Competent cells were obtained following the iGEM protocol Help:Protocols/Competent Cells

2. Transformation

E. coli DH5α was transformed using the protocol Help:Protocols/Transformation

3. Fluorence Intensity Measurement

The Fluorescence Intensity was measured using the standardized protocol from iGEM Plate_Reader_Protocol_Update .
The Plate Reader (Fluostar omega, BMG LABTECH) was calibrated using the solutions included in the Interlab Measurement Kit.

4. Flow Cytometry

Fluorescence Intensity was measured using the Flow Cytometer (Attune NxT, Thermo Fisher Scientific) in cells grown in LB following guidelines from iGEM. The Flow Cytometer was calibrated using Sphero®Rainbow Calibration Particles (BD Bioscience), 8 peaks, calibrated for MEFL (Molecules of Equivalent Fluorescein). Four drops of calibration particles were dissolved in sheath fluid (1ml). Samples were prepared for measurement in the Flow Cytometer washing the culture media in filtered 1X PBS. Cells cultures were diluted 1:100, adding PSB in a 96-well microtiter plate (Thermofisher Scientific). The instrument was configured with a channel for GFP measurement with 488 nm laser and 530/30 filter