For a full list of detailed protocols, please visit our protocols page. This page will just show an informative pipeline of the order of our experiments, making them easy to exactly replicate. See the main results of our experiments here. The specific activities that were performed by each team member are shown on a day to day basis here, where all development (successes and failures) are documented.
Figure 1: Pipeline diagram of the methods we used. We used GoldenGate cloning to assemble our parts, using E. coli and A. tumefaciens as our intermediate chassis organisms. We finally transformed N. benthamiana with the constructs and tested for their presence.
During our experiments we found better protocols that helped us improve our project. We also found some that we wanted to use, but couldn't due to either lack of time or resources. The only major change that we made during our project was heat shocking our plants at 37 degrees Celsius, either 1 or 2 DPI, as it has been recently reported that this can increase transgene expression by 4-5 fold(1). As you will see from our results page, the detection and testing side of our siRNAs was the main shortcoming of our project. This is because we lacked the resources for dedicated siRNA detection kits, such as the one found here. Then, we lacked the lab time to perform the aphid toxicity experiments, due to aphid generation time and needing multiple generations for reliable results. Had we had the time, we would have performed an experiment analogous to that of Rodriguez et al. in 2014(2).
(1) - Norkunas, K., Harding, R., Dale, J. and Dugdale, B. (2018). Improving agroinfiltration-based transient gene expression in Nicotiana benthamiana. Plant Methods 14.
(2) - Rodriguez, P., Hogenhout, S. and Bos, J. (2014). Leaf-Disc Assay Based on Transient Over-Expression in Nicotiana benthamiana to Allow Functional Screening of Candidate Effectors from Aphids. Methods in Molecular Biology:137-143.