Team:Purdue/Contribution
For our 2021 Contribution, we focused on characterizing the parts we proposed to use for our molecular diagnostic assay. Each major part of our molecular diagnostic assay or the methods used to validate it is described as follows. We began Phase II with cloning codon-optimized tTago and h2ago inserts into a pWUR702 backbone. Using our resulting constructs from cloning, we tried to express tTago. We performed RPA on our target sequence (142 bp) and found it to be successful. Using tTago purchased from NEB, we tested our argonaute guide sequences designed in Phase I. We characterized the level of fluorescence produced from MB in response to target-sequence recognition versus random noise, allowing us to predict the limit of detection. We hoped to test and characterize different methods of chitosan elution to better understand resulting methods of elution efficiency and other variables which affect it. We present the results from testing chitosan-treated Standard 17 glass fiber pads using Yeast RNA. [1] DNA-guided DNA interference by a prokaryotic Argonaute. Swarts DC, Jore MM, Westra ER, Zhu Y, Janssen JH, Snijders AP, Wang Y, Patel DJ, Berenguer J, Brouns SJ, van der Oost J. Nature. 2014 Mar 13;507(7491):258-61. doi: 10.1038/nature12971. Epub 2014 Feb 16. 10.1038/nature12971 PubMed 24531762 [2] Swarts, D., Jore, M., Westra, E. et al. DNA-guided DNA interference by a prokaryotic Argonaute. Nature 507, 258–261 (2014). https://doi.org/10.1038/nature12971
[3] Hagan, K. A., Meier, W. L., Ferrance, J. P., & Landers, J. P. (2009). Chitosan-coated silica as a solid phase for RNA purification in a microfluidic device. Analytical Chemistry, 81(13), 5249–5256. https://doi.org/10.1021/ac900820z [4] Zhu, X., Zhao, J., Hu, A., Pan, J., Deng, G., Hua, C., Zhu, C., Liu, Y., Yang, K., & Zhu, L. (2020). A novel microfluidic device integrated with chitosan-modified capillaries for rapid ZIKV detection. Micromachines, 11(2), 186. https://doi.org/10.3390/mi11020186
Contribution
Molecular Cloning
Protein Expression
RPA
tTago Cleavage Assay
Molecular Beacon Design
Chitosan
References