Team:WashU StLouis/Future Directions
Due to safety concerns, we were unable to use Pgt in the lab and engineered S. cerevisiae EBY100 to also produce AvrSr35 which would normally come from the fungus to be detected and the Sr35 and AvrSr35 interaction occurs within one cell. When our engineered yeast are used in the field, AvrSr35 will be unable to enter the inside of the cell, so Sr35 will need to be expressed on the cell wall. To do this, we propose a construct that combines our Aga2 and Sr35 parts. With this design, the AvrSr35 can interact with Sr35 on the cell surface and produce a fluorescent signal.
Wild-type avirulence factors found in Pgt will not contain the nYFP tag, introducing limitations to this step of our project. In order to be used in the field, the system must be able to produce a fluorescent signal without a component on the avirulence factor and hopefully, with increased research, Sr35 could be modified differently to produce a signal when activated without tagging AvrSr35.
Currently, our project is only capable of determining the virulence of the race of Pgt present to Sr35. We hope that as more resistance protein’s genes are sequenced and the interactions with their corresponding avirulence factors are studied, the concepts of our project and our device can be expanded to other common resistance genes.
Though our human practices work, we have found that many small-scale farmers in developing nations are unaware of the specific resistance proteins that are present in their line of wheat. They continue to use the same stock of seeds that lack newly developed resistance genes, making their crops susceptible to almost every race of Pgt, given the recently high mutation rate. Therefore, these farmers will mainly utilize the first component of our project.
However, some farmers are aware of the line of wheat they are planting and the resistance genes present in their crops. Our mechanism would be tailored to their specific strain of wheat by having our S. cerevisiae EBY100 express the same resistance genes as their wheat. Therefore, if Pgt is detected, we will also be able to distinguish if the race of Pgt present is virulent or not to the specific wheat crops present and if fungicide application is necessary.
Unfortunately, due to cloning difficulties with our yeast expression plasmid, pRS424, we were unable to complete experiments with our AvrSr35 and Sr35 construct as well as our surface display construct. Both constructs were successfully cloned and transformed into S. cerevisiae, but the plates had not grown by the time of Wikifreeze. However, we will wait for the plates to grow and attempt to run our experiments by the Jamboree.
To expand upon our hardware, we would like to connect our device to the internet using a Raspberry Pi as suggested by Microsoft so that researchers and government officials can more easily collect data on the presence of rust fungus. We would also like to expand into a greater variety of construction materials, using more durable plastics and testing the durability when exposed to common sanitizing agents. In addition to the physical improvements, we would like to conduct further testing of our hardware and software to determine how well our model could be expanded to other proteins. We would also like to model spore travel and test the quantity of spores collected and germinated using polyethylene disks compared to the polystyrene tested previously. In addition, we would also like to test how well our polyethylene disks break down in the environment.