Difference between revisions of "Team:Edinburgh OG/Experiments/Rhodococcus"

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                     <h2 class="section-heading">Introduction to <i>Synechocystis sp.</i></h2>
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                     <h2 class="section-heading">Introduction to <i>Rhodococcus sp.</i></h2>
 
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                     <p class="text-faded" style="font-size: 15px">The first host organism we will be working on is the cyanobacterium Synechocystis sp. strain PCC 6803sp. PCC 6803. Cyanobacteria constitute a unique class of microorganisms that are able to fix and metabolize carbon dioxide using the energy derived from sunlight. Cyanobacteria also present some of the highest solar energy conversion efficiencies found in nature (Dismukes et al., 2008). As cyanobacteria require nutrients like phosphorus and nitrogen for growth, they can also theoretically be used to sequester these pollutants from both industrial and agricultural streams, thereby further reducing production costs and environmental impact. Synechocystis exhibits robust and predictable growth in laboratory settings and the effect of different parameters including salt concentration, pH, temperature, UV light and CO2 level on its growth has been investigated (Lopo et al., 2012). Despite being one of the most extensively studied cyanobacterial species, the use of Synechocystis as a synthetic biology chassis awaits further investigation.</p>
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                     <p class="text-faded" style="font-size: 15px">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].</p>
 
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Revision as of 16:43, 17 October 2016

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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