The Procedure of Wet Lab Experiments
- Fungal experiments: Botrytis cinerea
(Because of their high infectiousness to many kinds of hosts (around 200 types of species) and also the short lifespan)
- DNA cloning: E. coli DH5α
- Peptide cloning: E. coli BL21 Rosetta-Gami
- Peptide expression: E. coli BL21 Rosetta-Gami
1.Experimental Validation for Parabase
We did the experimental validation for our prediction system using the predicted peptides.
- Inhibition Zone: the measurement of inhibiting mycelia
- Spore Germination: the measurement of inhibiting spores
- Botany Experiment: the observation of the disease occurring in reality
2.Further Application in Agriculture
For the ultimate goal of realizing the system in farmlands, we first attempted to cost down by producing peptides from E. coli to prepare for the mass production.
We added several peptides already known to be antifungal to do the following experiments.
After filtering samples that were not suitable to apply to farms, we tried to transform samples into E. coli. We have submitted Parts to iGEM.
We then tried to express them by SDS-PAGE.
3. Future Work
For the proteins expressed, we will do the three fungal experiments to do double check, then attempt to apply them into farmlands by the use of IoT and the disease occurrence model.
For the proteins that aren’t suitable to be expressed by SDS-PAGE, we will try other expression methods.
4. How to select the predicted antifungal peptides to do the validation?
We collected unknown peptides from UniProt database and calculated their scores through the scoring card. After calculating the scores, we selected hundreds of peptides which scores were higher than the threshold.
Then the peptides would pass through some filters, and then chose our final peptides for wet lab experiments:
First , we deleted the peptides which amino acid lengths were longer than one hundred amino acids or lower than ten amino acids because the lengths of most antifungal peptides are between this range. Second, we checked the UniProt to confirm the sequence correctness of peptides and ensure they were not signal peptides or propeptides. Last, searching the original function of the peptide. If the group of peptides had the same original function, we would only choose the one which possessed the highest score. After filtering the peptides, we have chosen six peptides for following wet lab experiments.