CAREER: Synthetic approaches to unravel heterogeneous decision-making in individual microbes and populations

职业：揭示个体微生物和群体异质决策的综合方法

• 批准号: 2045493
• 负责人: Megan McClean
• 金额: $ 62.99万
• 依托单位: University of Wisconsin-Madison
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-15 至 2026-01-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2045493&HistoricalAwards=false
• 关键词: CAREER; Synthetic; approaches; unravel; heterogeneous

Microbes that are identical at the level of their genetic material can still behave differently (this is referred to as ‘phenotypic heterogeneity’) and one of the fundamental challenges in understanding and controlling the behavior of microbes is identifying the factors that control this heterogeneity. The goals of this project are to understand which specific factors control phenotypic heterogeneity and what the consequences of this heterogeneity are for populations of microbes. We will use new tools that allow us to control cellular processes with light to determine the effect of different factors (specifically transcription factors) on phenotypic heterogeneity in a model microbe: yeast. To determine the consequences of phenotypic heterogeneity on outcomes for microbial communities we will measure how populations of microbes grow and survive under different stress and drug treatments. In addition to this research, the project will focus on developing human resources at the interface of engineering and life sciences research starting at the middle-school level and reaching through graduate levels. This project will integrate research methods and findings from the research to develop new curricula and mentor trainees. Development of outreach and educational materials aimed specifically at middle-school students, including an innovative interactive modeling game, will enhance science literacy and maintain students in the STEM pipeline.This project will utilize new approaches from synthetic and quantitative biology to understand (1) how the dynamics and heterogeneity of effector activity interact with additional cellular components to contribute to cell-to-cell variability in gene expression and (2) how heterogeneity in individual microbial decisions determines population-level phenotypes such as growth and stress resistance. The project will pursue these objectives using the yeast Saccharomyces cerevisiae and combine new optogenetic tools to control effector (specifically transcription factor, TF) activity with measurement of gene expression, single-cell decisions, and population-level outcomes. Optogenetic control of transcription factor activity in combination with a computational model will allow us to (1) identify transcriptional targets that propagate or suppress fluctuations in their cognate TFs activity and (2) characterize how TF activity dynamics control transcriptional bursting. Furthermore, to characterize how heterogeneous decision-making determines population-level phenotypes in isogenic microbial populations we will quantify growth and stress survival while using optogenetic control of TF activity to modulate cell-to-cell variability in gene expression. The results of this research will increase understanding of how effector activity contributes to observed heterogeneity in gene expression and phenotype between individual microbes and the effect of this heterogeneity on population-level outcomes.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

在遗传物质水平上相同的微生物仍然可以表现出不同的行为（这被称为“表型异质性”），理解和控制微生物行为的基本挑战之一是确定控制这种异质性的因素。该项目的目标是了解哪些特定因素控制表型异质性，以及这种异质性对微生物种群的影响。我们将使用新的工具，使我们能够用光来控制细胞过程，以确定不同因子（特别是转录因子）对模型微生物（酵母）中表型异质性的影响。为了确定表型异质性对微生物群落结果的影响，我们将测量微生物种群如何在不同的压力和药物治疗下生长和生存。除了这项研究外，该项目还将侧重于开发工程和生命科学研究界面的人力资源，从中学开始，一直到研究生阶段。该项目将整合研究方法和研究结果，以制定新的课程和指导受训人员。开发专门针对中学生的宣传和教育材料，包括创新的互动建模游戏，本项目将利用合成和定量生物学的新方法来理解（1）效应物活性的动态和异质性如何与其他细胞组分相互作用，以促进细胞对细胞的作用，基因表达的细胞变异性，以及（2）个体微生物决策的异质性如何决定群体水平的表型，如生长和抗逆性。该项目将使用酵母酿酒酵母实现这些目标，并将联合收割机与新的光遗传学工具相结合，通过测量基因表达、单细胞决策和群体水平结果来控制效应子（特别是转录因子，TF）活性。转录因子活性的光遗传学控制与计算模型相结合将使我们能够（1）鉴定传播或抑制其同源TF活性波动的转录靶点，以及（2）表征TF活性动力学如何控制转录爆发。此外，为了表征异质决策如何决定同基因微生物种群中的种群水平表型，我们将量化生长和应激生存，同时使用TF活性的光遗传学控制来调节基因表达的细胞间变异性。这项研究的结果将增加对效应子活性如何影响个体微生物之间观察到的基因表达和表型异质性以及这种异质性对群体水平结果的影响的理解。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估来支持。

项目成果

Lustro: High-Throughput Optogenetic Experiments Enabled by Automation and a Yeast Optogenetic Toolkit

• DOI: 10.1021/acssynbio.3c00215
• 发表时间: 2023-07-11
• 期刊: ACS SYNTHETIC BIOLOGY
• 影响因子: 4.7
• 作者: Harmer, Zachary P. P.;McClean, Megan N. N.
• 通讯作者: McClean, Megan N. N.

Transcription factor localization dynamics and DNA binding drive distinct promoter interpretations.

• DOI: 10.1016/j.celrep.2023.112426
• 发表时间: 2023-05-30
• 期刊: Cell reports
• 影响因子: 8.8

Optogenetic Tools for Control of Public Goods in Saccharomyces cerevisiae.

• DOI: 10.1128/msphere.00581-21
• 发表时间: 2021-08-25
• 期刊: mSphere
• 影响因子: 4.8
• 作者: Moreno Morales N;Patel MT;Stewart CJ;Sweeney K;McClean MN
• 通讯作者: McClean MN