Optogenetic selection for dynamic phenotypes in bacteria

该项目的目标是开发基于动态、时变特征的筛选和选择单个细菌的方法，例如识别对环境变化表现出快速反应的细胞。筛选和筛选是有效鉴定和分离具有特定特性的细胞的有力工具，然而大多数现有的方法，如基于流式细胞术的方法，只能在单个时间点观察细胞。这种“快照”视图排除了基于动态特征的评估和选择，例如响应的速度、细胞的适应能力或细胞的生长速度。为了实现开发适合分离具有动态特征的细胞的选择方法的目标，研究人员将使用光诱导结构和空间图案化的光照明来激活目标细胞中抗生素耐药性基因的表达。研究人员将通过进行全面的实验来评估在营养胁迫下激活的启动子的时间反应，从而展示筛选动态表型的潜力。这些科学活动还得到了教育宣传倡议的补充。这些活动包括与STEM Path计划合作，为高中生举办活动，包括关于图像处理的编码黑客松和关于合成生物学设计的迷你大型聚会。此外，该项目还为本科生提供了培训机会。该项目的主要技术创新来源是开发用于细胞光遗传选择的工具。具体地说，研究人员将开发和优化基因结构，其中光可以用来触发抗生素耐药性基因的表达。将开发两种互补的遗传工具，基于蓝光诱导的Cre重组酶版本和红光/绿光诱导的CCAS-CCAR光遗传系统。研究人员将优化光照强度和曝光时间，以打开在微流控设备中生长的细胞的抗药性。使用一种基于荧光团表达的分析来选择细胞，研究人员将量化该方法的敏感性和特异性。这些工具的最终用途在于它们能够选择具有动态表型的细胞，以产生新的生物学见解。为了测试这一潜力，研究人员将专注于(P)ppGpp响应启动子，通过结合动态选择的池库方法有效地测量启动子的时间和随机属性。总体而言，该项目将引入利用光进行选择的光遗传工具，并将展示这些工具的力量，以揭示单个细菌如何应对环境压力的新见解。该奖项反映了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。

The goal of this project is to develop methods for screening and selecting individual bacteria based on dynamic, time-varying traits, such as identifying cells that exhibit a rapid response to an environmental change. Screening and selection are powerful tools that enable efficient identification and isolation of cells with specific properties, however most current approaches, such as those based on flow cytometry, only observe cells at a single time point. This "snapshot" view precludes assessment and selection based on dynamic features, such as the speed of a response, the ability of a cell to adapt, or a cell's growth rate. To achieve the goal of developing selection methods suited for isolating cells with dynamic traits, the researchers will use light-inducible constructs and spatially patterned light illumination to activate expression of antibiotic resistance genes in targeted cells. The researchers will demonstrate the potential of screening for dynamic phenotypes by conducting comprehensive experiments to assess temporal responses in promoters that are activated under nutrient stress. These scientific activities are complemented by educational outreach initiatives. These include partnering with the STEM Pathways program to host events for high school students including a coding hackathon on image processing and a mini-jamboree about synthetic biology design. In addition, the project provides training opportunities for undergraduate students.The primary source of technical innovation in this project is the development of tools for optogenetic selection of cells. Specifically, the researchers will develop and optimize genetic constructs where light can be used to trigger expression of an antibiotic resistance gene. Two complementary genetic tools will be developed, based on a blue-light inducible version of Cre recombinase and a red/green light inducible CcaS- CcaR optogenetic system. The researchers will optimize light intensities and exposure times to turn on drug resistance for cells growing within a microfluidic device. Using an assay to select cells based on expression of a fluorophore, the researchers will quantify the sensitivity and specificity of the approach. The ultimate utility of these tools is in their ability to select cells with dynamic phenotypes to generate novel biological insight. To test this potential, the researchers will focus on (p)ppGpp-responsive promoters, efficiently measuring temporal and stochastic properties of promoters with a pooled library approach coupled with dynamic selection. Overall, the project will introduce optogenetic tools for selection using light and will demonstrate the power of these tools to reveal new insights into how individual bacteria cope with environmental stress.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.