Difference between revisions of "Team:Newcastle/Results"

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<p class="legend"><strong>Table 3:</strong> Table of reactions performed according to the DoE salt supplement surface re-sponse design. CFPS reactions contained concentrations of magnesium glutamate, potassium glutamate, and sodium oxalate, according to the table above. The pattern column shows how much of each supplement was present in a reaction; very low concentration (a), low concentration (−), usual concentration (0), high concentra-tion (+), and very high concentration (A).</p>
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<p class="legend"><strong>Table 3:</strong> Table of reactions performed according to the DoE salt supplement surface response design. CFPS reactions contained concentrations of magnesium glutamate, potassium glutamate, and sodium oxalate, according to the table above. The pattern column shows how much of each supplement was present in a reaction; very low concentration (a), low concentration (−), usual concentration (0), high concentration (+), and very high concentration (A).</p>
 
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           <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> Experimental Procedure 2</h2>
 
           <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> Experimental Procedure 2</h2>
           <p>Cell extracts were prepared and CFPS reactions performed as before, except the magnesium glutamate, potassium glutamate, and sodium oxalate concentrations were according to the surface response experimental design. Ammonium acetate was kept at the default amount. CFPS activity was measured as fluorescence at each time point minus fluorescence at 15 mins (Figure below). Endpoint data was then used, along with the JMP software, to build a model predicting optimal concentrations for the three salts analysed (predictions visualised in the cube plot below). These predictions were then tested by preparing a supplement solution premix with amounts of magnesium glutamate, potassium glutamate, and sodium oxalate at concentrations of 6 mM, 195 mM, and 2 mM respectively. This supplement solution premix was used to supplement two batches of cell extract which were prepared identically. The first batch was the same extract used to collect data on which the predictions were made, whereas the second batch was newly prepared (Figure below). It was found that for the first extract, CFPS activity was enhanced when the premix containing ‘optimised’ concentrations of salts was used compared to the un-altered supplement solution premix. Additionally, CFPS activity was observed as being within the confidence intervals predicted by the DoE model.
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           <p>Cell extracts were prepared and CFPS reactions performed as before, except the magnesium glutamate, potassium glutamate, and sodium oxalate concentrations were according to the surface response experimental design. Ammonium acetate was kept at the default amount. CFPS activity was measured as fluorescence at each time point minus fluorescence at 15 mins (Figure 6). Endpoint data was then used, along with the JMP software, to build a model predicting optimal concentrations for the three salts analysed (predictions visualised in figure 7). These predictions were then tested by preparing a supplement solution premix with amounts of magnesium glutamate, potassium glutamate, and sodium oxalate at concentrations of 6 mM, 195 mM, and 2 mM respectively. This supplement solution premix was used to supplement two batches of cell extract which were prepared identically. The first batch was the same extract used to collect data on which the predictions were made, whereas the second batch was newly prepared. It was found that for the first extract, CFPS activity was enhanced when the premix containing ‘optimised’ concentrations of salts was used compared to the un-altered supplement solution premix (Figure 8a). Additionally, CFPS activity was observed as being within the confidence intervals predicted by the DoE model.
 
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           When CFPS reactions were performed using the second extract and the new premix, CFPS activity was lower compared to reactions using the second extract and the original premix. This supports the view that each extract requires separate optimisation of the supplement solution premix to reach optimal CFPS activity, and that a DoE approach is suitable for achieving this.
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           When CFPS reactions were performed using the second extract and the new premix, CFPS activity was lower compared to reactions using the second extract and the original premix (Figure 8b). This supports the view that each extract requires separate optimisation of the supplement solution premix to reach optimal CFPS activity, and that a DoE approach is suitable for achieving this.
 
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<p class="legend"><strong>Figure 6:</strong> CFPS activity of CFPS reactions with salt concentrations according to the surface response design (Table 3) was determined as fluorescence intensity at each time point minus fluorescence intensity at the 15 minute time point. Reactions 12 through 16 failed due to an error in set-up. These reactions were replicates of reactions 8-11 and their removal had little negative effect on the SRD according to the design diagnostics tool in JMP.</p>
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<img src="https://static.igem.org/mediawiki/2017/4/41/T--Newcastle--BB_CFPS_figure7.png" width="600px"/>
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<p class="legend"><strong>Figure 7:</strong> Cube plot generated by JMP using data collected for the SRD. The x-axis shows magnesium glutamate concentration, the y-axis shows potassium glutamate concentration, and the z-axis shows sodium oxalate concentration. The ovals shows predicted CFPS concentration. The maximal CFPS activity was found at 195 mM potassium glutamate, 6 mM magnesium glutamate, and 2 mM sodium oxalate. The minimal point was found at 65 mM potassium glutamate, 18 mM magnesium glutamate, and 2 mM sodium oxalate. This was verified as the maximum and minimum points using the surface profiler function in JMP.</p>
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<p class="legend"><strong>Figure 8:</strong> CFPS activity for two CFPS systems utilising two different cell extract batches prepared identically. A) Results for a system utilising the same extract batch used to test the SRD. B) Results for a system utilising a new batch of cell extract. The blue lines show systems using the normal CFPS supplement premix, and the purple lines show the systems with supplement premix with ‘optimised’ magnesium glutamate, potassium glutamate, and sodium oxalate as identified above. The system utilising extract from the same batch used in the SRD testing had a higher CFPS activity with the ‘optimised’ premix (purple) than with the original premix (blue), whereas the system utilising extract from a separate batch had higher activity with the original premix.</p>
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           <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> Design Stage 3</h2>
 
           <h2 style="font-family: Rubik; text-align: left; margin-top: 1%"> Design Stage 3</h2>

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