Team:Edinburgh OG/Experiments/Rhodococcus

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Introduction to Rhodococcus sp.

In the last three decades, there have been a number of patents and publications in the usage of several Rhodococcus strains as bioremediation and biotransformation agents. These gram-positive actinobacteria are commonly found in contaminated soils and toxic environments (Larkin et al., 2005). Along with the gram-negative Pseudomonas, they tolerate a range of organic solvents, exhibit unique enzymatic capabilities and have the ability to biodegrade several environmental pollutants. They have been used to bioremediate soil contaminated with hydrocarbons (Warhurst and Fewson, 1994), pesticides (Larkin et al., 2005), nitriles (Brandao and Bull, 2003) and xenobiotics (Martinkova et al., 2009). They have also been used to produce acrylamide (Hughes et al., 1998), triacylglycerols (Hernandez et al., 2008) and in fossil fuel desulfurization (Matsui et al., 2002).

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