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).

Potential model industrial organism

There are currently 58 different Rhodococcus species (reference list periodically updated at http://www.bacterio.net/rhodococcus.html) and based on 16S rRNA data (McMinn et al., 2000) they are classified into three main subclades: R. erythropolis, R. equi and R. rhodochrous. Of the R. erythropolis subclade, the three most characterised industrial species are R. erythropolis, R. opacus, and R. jostii (Goodfellow et al., 2012). R. erythropolis was the first characterised bacteria in this subclade while R. jostii was the first to have its genome sequenced (McLeod et al., 2006). R. opacus has the unique ability of accumulating up to 40% of its dry cell mass as triacylglycerols (Kurosawa et al., 2010). All three species catabolise a wide range of oligosaccharides and organic compounds, but R. erythropolis has two industrial advantages of tolerating a wider range of temperatures and possessing a significantly smaller genome, corresponding to faster growth rate. However, the larger genomes of R. jostii and R. opacus gives them the ability to metabolise a wider range of organic substances.

ErmE Promoter Phytobrick

A widely used constitutive promoter, ErmE (promoter region of erythromycin-resistance gene) was isolated from Streptomycin (Bibb et al., 1985). It works with related gram positive actinobacteria, such as Rhodococcus.

P-45 Promoter Phytobrick

A strong constitutive promoter from Corynebacterium (Patek et al., 1996) that works with related gram positive actinobacteria, such as Rhodococcus. We have this part in pUPD2, a MoClo-compatible level 0 vector with no illegal sites for MoClo assembly standard, GoldenBraid assembly standard and iGEM’s pairwise assembly standard.

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