Development of a Mechanistic Model of Fluorescent In Situ Hybridization (FISH) based on Equilibrium Thermodynamics

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0302618 Noguera Intellectual Merit: The main outcome of this research will be the establishment of a mathematical methodology to facilitate the design of probes for FISH. After a researcher has identified specific 16S rRNA sequences, our model will assist with (1) checking the specific target sites for accessibility using the unbiased map constructed in Task 2, (2) fine-tuning the probes to achieve high intensity signals (e.g., adjusting probe length to achieve .G 0 ov-15 kcal/mol), (3) using site-specific m values (obtained during Task 1) to generate theoretical denaturation curves for the target and non-target microorganisms, and (4) designing probes with stable stem-loop structures to increase target and non-target differentiation, following guidelines developed during Task 3. Currently, none of these important evaluations are possible. Moreover, due to the wide applicability of FISH and similar hybridization methods, the outcome of this study is likely to cross multidisciplinary boundaries.Broader Impacts: This project will contribute to the education of two graduate students. One of them is Mr. Safak Yilmaz, a third-year PhD student in Civil and Environmental Engineering. The second graduate student will be recruited from the pool of applicants to the Civil and Environmental Engineering program and special consideration will be given to diversity applicants. The PI will use the existing infrastructure in the College of Engineering and the UW to foster educational equity, such as the Graduate Education Research Scholars Program, and the College of Engineering Opportunities in Engineering Conference. In addition, during the duration of this project, the PI will offer summer internships to diversity undergraduate students through existing summer programs in biology and engineering (SRP-Bio and SURE/REU). Finally, we plan to establish a website where users can access the large databases generated in this project and use the model for the mechanistic design of FISH protocols. The model itself, the generated 16S rRNA maps (i.e., m values and inaccessibility), and the model applications will be distributed to the scientific community in peer-review publications and the best available electronic approach.