Quantifying the Prevalence and Phenotypic Consequences of Transcriptional Irreversibility in Bacteria

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”。这些基因表达的变异可以在基因相同的细胞群体中导致不同的表型。例如，基因表达的变化可能导致基因相同的细菌群体中的一些细胞变得耐抗生素，而群体中的其他细胞仍然对抗生素产生怀疑。该项目将解决基因表达的变化如何导致细菌重要的表型变化。为了补充这项研究，将为高中学生开发一系列关于生物学数学的互动课程。这些课程将通过一系列教师讲习班分发。细胞状态的不可逆性、迟滞性和多稳定性已经在少数特定的细菌系统中得到了定量研究——枯草芽孢杆菌的产孢、lac抑制因子和裂解-溶生开关是现在的经典例子。在这里，研究人员试图将这些概念的理解扩展到基因组尺度：研究人员将检查可逆性的时间尺度，以及在大肠杆菌中所有已知功能基因的短暂抑制后不可逆性的流行。为了实现这一目标，研究人员将开发一种新的光诱导瞬时基因抑制试剂，称为LIT-CRISPRi。他们将结合实验数据和理论，在完全可逆的情况下（在没有迟滞的情况下）建立瞬时基因抑制后转录、翻译和生长速率恢复的时间尺度的期望，并描述一种被充分研究的不可逆情况（lac操纵子）的行为。然后，他们将使用这些工具来表征大肠杆菌在不同环境条件下的瞬时基因抑制之前，期间和之后的生长速度动态，以构建基因组规模的LIT-CRISPRi敲低文库。根据这些数据，研究人员将检查生长速率恢复时间的分布，确定不可逆性或异常长时间恢复（准不可逆性）的病例，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在短暂基因抑制之前，期间和之后对几种基因进行时间分辨转录组学，这些基因表现出我们的筛选确定的（准）不可逆动力学。总之，这项工作将建立对基因抑制后细胞适应和不可逆性的时间尺度的基本期望。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.