Quantifying the Prevalence and Phenotypic Consequences of Transcriptional Irreversibility in Bacteria

量化细菌转录不可逆性的普遍性和表型后果

• 批准号: 2206974
• 负责人: Kimberly Reynolds
• 金额: $ 109.38万
• 依托单位: University of Texas Southwestern Medical Center
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-15 至 2025-07-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2206974&HistoricalAwards=false
• 关键词: Quantifying; Prevalence; Phenotypic; Consequences; Transcriptional

A key question in biology is how genetically identical cells achieve varied appearances and behaviors. These distinct cell states are realized by variations in gene expression within certain cells in the population; different genes are turned “on” or “off” in these cells. These variations in gene expression can lead to different phenotypes within a population of genetically identical cells. For example, variations in gene expression can lead some cells in a population of genetically identical bacteria to become antibiotic tolerant while other cells in the population remain suspectable to antibiotics. This project will address how variations in gene expression lead to important phenotypic changes in bacteria. To complement the research, an interactive series of lessons on mathematics in biology will be developed for high school students. These lessons will be distributed through a series of teacher workshops.Irreversibility, hysteresis, and multistability in cell state have been quantitatively studied in a handful of specific bacterial systems — B. subtilis sporulation, the lac repressor, and the lysis-lysogeny switch are now classic examples. Here, the investigators seek to expand the understanding of these concepts to the genomic scale: the investigators will examine the time scales of reversibility, and the prevalence of irreversibility, following transient repression of all genes with known function in E. coli. To accomplish this, the investigators will develop a new reagent for light-inducible transient gene repression called LIT-CRISPRi. They will use a combination of experimental data and theory to establish expectations for the time scale of transcriptional, translational, and growth rate recovery following transient gene repression in the fully reversible case (in the absence of hysteresis) and characterize behavior for one well-studied irreversible case (the lac operon). They will then use these tools to characterize E. coli’s growth rate dynamics before, during, and after transient gene repression for a genome scale library of LIT-CRISPRi knockdowns under different environmental conditions. From these data, the investigators will examine the distribution of growth rate recovery times, identify cases of irreversibility or exceptionally long timescale recovery (quasi-irreversibility), and further validate these phenotypes through secondary experiments. Finally, they will use RNAtag-Seq to perform time-resolved transcriptomics before, during, and after transient gene repression for several genes exhibiting (quasi-)irreversible dynamics identified by our screen. Taken together, the work will establish fundamental expectations for the time scales of cellular adaptation and irreversibility following gene repression.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.

生物学中的一个关键问题是，基因相同的细胞如何实现不同的外观和行为。这些不同的细胞状态是通过群体中某些细胞内基因表达的变化来实现的；在这些细胞中，不同的基因被打开或关闭。基因表达的这些差异可能会导致遗传相同的细胞群体中出现不同的表型。例如，基因表达的变化可能会导致基因相同细菌种群中的一些细胞对抗生素产生耐受性，而种群中的其他细胞仍然对抗生素持怀疑态度。这个项目将解决基因表达的变化如何导致细菌的重要表型变化。为了补充这项研究，将为高中生开发一系列关于生物数学的互动课程。这些课程将通过一系列的教师工作坊分发。在一些特定的细菌系统中已经定量地研究了细胞状态下的不可逆性、滞后和多稳定性-枯草杆菌芽胞形成、乳胶抑制物和裂解-溶源开关现在是经典的例子。在这里，研究人员试图将对这些概念的理解扩展到基因组规模：研究人员将检查在大肠杆菌中所有已知功能的基因被瞬时抑制后，可逆性和不可逆性的流行程度。为了实现这一点，研究人员将开发一种新的试剂，用于光诱导的瞬时基因抑制，称为LIT-CRISPRi。他们将使用实验数据和理论的组合来建立对完全可逆情况下(在没有滞后的情况下)瞬时基因抑制后转录、翻译和生长速度恢复的时间尺度的预期，并描述一个研究得很好的不可逆情况(乳胶操纵子)的行为。然后，他们将使用这些工具来表征在不同环境条件下对LIT-CRISPRi基因敲除的基因组规模文库进行瞬时基因抑制之前、期间和之后大肠杆菌的生长速度动态。根据这些数据，研究人员将检查生长速度恢复时间的分布，识别不可逆性或超长时间尺度恢复(准不可逆性)的情况，并通过二次实验进一步验证这些表型。最后，他们将使用RNAtag-Seq在瞬时基因抑制之前、期间和之后对我们的屏幕确定的表现(准)不可逆动态的几个基因进行时间分辨转录。综上所述，这项工作将建立对细胞适应和基因抑制后的不可逆性时间尺度的基本预期。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。