Wet Lab Results When we initially hydrated our lyophilized genes of interest, a mistake was made with the concentration of the DNA for ADT2 scrambled. When no results were observed on the agarose gels for ADT2 scrambled only, we tested the concentration of the reverse samples, and got a concentration of only 3.906 µg/mL of ADT2 scrambled. Another sample was then hydrated with the proper amounts, and lab work was repeated, with a final concentration resulting in 435.7 µg/mL. From this point forward, all samples contained adequate proportions of DNA.
Once we had the appropriate DNA ratios we performed PCRs to amplify the gene sequences while adding specific prefix and suffix sequences. Once the PCR’s were analyzed through agarose gels and concluded as successful, gel extraction, cleanup, and IVT’s were conducted. tRNA touchup was undergone to lead to an IVT which transformed tRNA to siRNA. Once we had the RNA-DNA hybrid to increase the stability of the construct we proceeded with an RNase H digestion to hydrolyze the phosphodiester bond and gain our end product.
Testing On August 19th members from our team gathered at the university and met with Elizabeth Shultz to discuss the use of Arabidopsis plants to aid with our project. We planted approximately 400 seeds into 40 pots, and the plants have now matured to the stage where experimental application processes can be conducted. We plan to use these plants to test our siRNA construct, hopefully, to see the target genes silenced resulting in the death of the plant. We also plan to use various application methods such as spraying, application through a surfactant, as well as adding lyophilized construct into the soil. These tests will act as our proof of concept. Taking into consideration that RNA is unstable, we intend to store our construct in BioClay. This will allow our construct to maintain stability while simultaneously reducing the risks to environmental and human health.
Future Plans If the target genes in Arabidopsis are successfully silenced and result in the death of the plant, comparing kill rates between the different trials would allow our team to decide which construct to use in the final herbicide. Further development of our mathematical models would also assist in predicting the amount of herbicide needed to apply to regions. After thorough testing of the construct and application methods, our team would continue to develop the security and safety of the project through integrated human practices, ensuring the project is not used for the benefit of a group of individuals. After this, our project can then be implemented in wildfire-affected regions of Waterton, where Spotted Knapweed is problematic.
Public Support
Realized the struggles of controlling knapweed with Helen Schuler Center’s volunteer spotted knapweed removals
Changed our robot approach to a spray application as suggested by MindFuel in their Tech Futures Challenge
Used feedback from Melanie Triganne on how to handle herbicides
Implemented the “one-pot” method for producing siRNA using Karolinska Institute’s Petzold lab’s 2020 paper
Will market using Megan Coverdale’s feedback on selling herbicides
Experimental Results
Successfully located target gene, the chloroplast Clp protease using BLAST
Also located potential target gene, Adt2 using BLAST
Configured applications consisting of the Clpp gene alone, the Adt2 gene alone, and the gene sequences combined
