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| − | <a name="Characterisation" id="Characterisation"></a><h2>Characterisation</h2>
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| − | <p>Strains made by transformation with required DNA. Overnight cultures with cells of the required strains in 2.5ml LB + 40mg/ml Cml were grown in the dark at 37C with shaking. ONs were as follows:</p>
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| − | <ul>
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| − | <li>JM109 – RLS.lacZ.YFP</li>
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| − | <li>envZ – RLS.lacZ.YFP</li>
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| − | <li>JM109 – YFP Control</li>
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| − | <li>envZ – YFP Control</li>
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| − | </ul>
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| − | <p>100μl of ONs were used to inoculate two sterile LB + 40mg/ml Cml making up a total volume of 4ml in 5ml flasks which were then incubated in both light and dark, at 37C with shaking, as follows:</p>
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| − | <ul>
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| − | <li>JM109 – RLS.lacZ.YFP (Light / Dark)</li>
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| − | <li>envZ – RLS.lacZ.YFP (Light / Dark)</li>
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| − | <li>JM109 – YFP Control (Light / Dark)</li>
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| − | <li>envZ – YFP Control (Light / Dark)</li>
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| − | </ul>
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| − | <p>At 50 minute time intervals 200μl of each sample was taken and mixed with 800μl of sterile water in plastic cuvettes. The optical density of each sample was taken after the spectrophotometer was set with a sample of 200μl of sterile LB and 800μl water as a control, as was the luminescence, with readings for the background luminescence being taken for the sample of LB and water.</p>
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| − | <p>Two readings for each sample at each time interval were taken and then an average was calculated for each time interval for both optical density and luminescence.</p>
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| − | <p>For each sample at each time interval the average luminescence (normalised by the background luminescence) was divided by the optical density and plotted on a graph, shown below as <a href="https://static.igem.org/mediawiki/2010/c/c9/Ed10-RedLightSensorCharData.png">Figure 2</a>.</p>
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| − | <center><br><br><p><img src="https://static.igem.org/mediawiki/2010/c/c9/Ed10-RedLightSensorCharData.png" width="600px"></p><br>
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| − | <p><b>Figure 2:</b> Characterisation data for the red light sensor.</p><br><br></center>
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| − | </div>
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| − | <div id="body" style="padding: 0px 60px 10px 60px; height: 1356px">
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| − | <p><a href="https://static.igem.org/mediawiki/2010/c/c9/Ed10-RedLightSensorCharData.png">Figure 3</a> shows further characterisation data for the red light sensor.</p>
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| − | <p>Analysis.</p><br>
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| − | YFP RLS Characterisation
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| − | Red light = less yellow colour fluorescence
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| − | No Red light = more yellow colour fluorescence
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| − | This assay shows the fluorescence per unit of estimated biomass for a number of different times of incubation. We can see that the controls for YFP in both the JM109 & envZ mutant strains show reasonable paired results for both Light and Dark. This means that the controls work as expected, however the large difference between the JM109 & the envZ control pairs implies that most of the result seen in JM109 is due to background envZ activity.
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| − | The rest of the results however are of limited usefulness beyond the testing of our assay procedure and the benchmark they represent of the RLS system which we can use to further improve our characterisation assays. The reasons for the affect seen are similar to those listed above in the lacZ RLS characterisation, listed below.
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| − | Though the data is not entirely useful we believe it may be due to a number of potential causes – such as the cofactor not being expressed properly, weak light intensity unable to activate the RLS system, or insufficient expression of the system as a whole - all of which we could test for given sufficient time, allowing us to improve our experimental technique to account for accordingly. We will endeavour to do so and bring further results to the Jamboree.
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