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<figcaption>Figure 3: Bitis gabonica AMC experiment, with different concentrations of venom</figcaption> | <figcaption>Figure 3: Bitis gabonica AMC experiment, with different concentrations of venom</figcaption> | ||
</figure> | </figure> | ||
+ | |||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/2/2d/T--DTU-Denmark--NN_AMC_experiment.png" alt="NN AMC experiment" width="500"> | ||
+ | <figcaption>Figure 4: Naja nigricolis AMC experiment, with different concentrations of venom</figcaption> | ||
+ | </figure> | ||
+ | |||
+ | <p>We repeated the experiment with two different substrate concentrations, along with three different venom concentrations, similar results were obtained. The bigger substrate concentration produced much higher fluorescence intensity. You can find the raw data here (excel link of second AMC-substrate experiment). | ||
+ | </p><br /> | ||
+ | |||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/2/26/T--DTU-Denmark--venom100_AMC_experiment2.png" alt="AMC experiment 2 data" width="300"> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/2/26/T--DTU-Denmark--venom100_AMC_experiment2.png" alt="AMC experiment 2 data" width="300"> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/2/26/T--DTU-Denmark--venom100_AMC_experiment2.png" alt="AMC experiment 2 data" width="300"> | ||
+ | <figcaption>Figure 5: Difference in cleavage at different venom concentrations</figcaption> | ||
+ | </figure> | ||
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</div> | </div> |
Revision as of 14:23, 1 November 2017
Results
Visit experimental design for the theory and design behind our experiments, and protocols for the protocols of our experiments.
AMC Experiment
In this experiment, we used the AMC fluorescent molecule, coupled with a peptide sequence (A-L-K) known to be cleaved by serine proteases from literature. The molecule emits fluorescence when it is released from the peptide it is coupled with. For that reason, we expected to see fluorescence when the peptide was cleaved by proteases in the snake venom. We first generated a standard curve of the AMC molecule without the peptide sequence as seen in figure 1.
![AMC standard curve](https://static.igem.org/mediawiki/2017/e/ec/T--DTU-Denmark--AMC_standardcurve.png)
We tested the AMC-substrate peptide against the venom from our three snakes of interest, Bitis arietans, Bitis gabonica and Naja nigricolis. We made measurements in five different timepoints, with five different venom concentrations. The background noise was deducted from all measurements. The experiment showed that this particular substrate is cleaved significantly by the two venoms from Bitis arietans and Bitis gabonica, but not by the venom of Naja nigricolis as seen in figure 2-4. Different concentrations of venom had great effect in the fluorescence intensity. You can find the raw data here (excel link of 1st AMC-substrate experiment).
![BA AMC experiment](https://static.igem.org/mediawiki/2017/b/bc/T--DTU-Denmark--BA_AMC_experiment.png)
![BG AMC experiment](https://static.igem.org/mediawiki/2017/a/a9/T--DTU-Denmark--BG_AMC_experiment.png)
![NN AMC experiment](https://static.igem.org/mediawiki/2017/2/2d/T--DTU-Denmark--NN_AMC_experiment.png)
We repeated the experiment with two different substrate concentrations, along with three different venom concentrations, similar results were obtained. The bigger substrate concentration produced much higher fluorescence intensity. You can find the raw data here (excel link of second AMC-substrate experiment).
![AMC experiment 2 data](https://static.igem.org/mediawiki/2017/2/26/T--DTU-Denmark--venom100_AMC_experiment2.png)
![AMC experiment 2 data](https://static.igem.org/mediawiki/2017/2/26/T--DTU-Denmark--venom100_AMC_experiment2.png)
![AMC experiment 2 data](https://static.igem.org/mediawiki/2017/2/26/T--DTU-Denmark--venom100_AMC_experiment2.png)