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

0302618诺盖拉的学术价值：这项研究的主要成果将是建立一种数学方法，以促进鱼类探针的设计。在研究人员确定了特定的16S rRNA序列后，我们的模型将有助于(1)使用在任务2中构建的无偏向图检查特定目标位置的可及性，(2)微调探针以获得高强度信号(例如，调整探针长度以达到G0 Ov-15 kcal/mol)，(3)使用特定于位置的m值(在任务1中获得)来生成目标和非目标微生物的理论变性曲线，以及(4)设计具有稳定的茎环结构的探针，以增加目标和非目标的分化，目前，遵循任务3中制定的指导方针。这些重要的评估都是不可能的。此外，由于FISH和类似杂交方法的广泛适用性，这项研究的结果可能会跨越多学科的边界。广泛的影响：该项目将有助于两名研究生的教育。其中一位是土木工程与环境工程专业三年级博士生萨法克·耶尔马兹。第二名研究生将从土木工程与环境工程专业的申请者中招聘，并将特别考虑多样性申请者。国际工程学院将利用工程学院和华盛顿大学现有的基础设施来促进教育公平，例如研究生教育研究学者计划和工程学院工程机会会议。此外，在这个项目期间，PI将通过现有的生物和工程暑期项目(SRP-Bio和Sure/REU)为不同类别的本科生提供暑期实习机会。最后，我们计划建立一个网站，用户可以访问在这个项目中产生的大型数据库，并使用该模型进行FISH协议的机械设计。模型本身、生成的16S rRNA图谱(即m值和不可获得性)以及模型应用程序将在同行评议出版物和现有的最佳电子方法中分发给科学界。

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.