Technical Mother Machine Core

技术母机核心

• 批准号: 10327901
• 负责人: Johan Paulsson
• 金额: $ 44.04万
• 依托单位: MASSACHUSETTS EYE AND EAR INFIRMARY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10327901
• 关键词: Ablation; Address; Algorithms; Antibiotic Resistance; Antibiotic susceptibility; Antibiotics; Bacillus subtilis; Bacteria; Behavior; Cell Size; Cell division; Cells; Cellular Assay; Cellular Morphology; Chromosomes; Complex; Data; Data Analyses; Data Scientist; Deoxyribonucleases; Devices; Dimensions; Engineering; Ensure; Enterococcus; Enterococcus faecalis; Environment; Environmental Risk Factor; Escherichia coli; Generations; Genes; Genetic; Genetic Screening; Genotype; Genus staphylococcus; Growth; Health; Image; Individual; Infrastructure; Investigation; Laboratories; Lasers; Libraries; Location; Mammalian Cell; Measurement; Measures; Methods; Microbial Biofilms; Microfluidics; Microscopy; Morphology; Mothers; Noise; Output; Phenotype; Physiologic pulse; Physiological; Population; Predisposition; Property; Publishing; Reporter; Research; Research Project Grants; Resources; Retrieval; Running; Science; Series; Staphylococcus aureus; Sum; Systems Analysis; Systems Biology; Technology; Time; Translating; Ursidae Family; Variant; Visualization software; Work; analysis pipeline; base; cell growth; cellular imaging; data handling; design; experience; experimental study; feeding; genetic variant; global health; innovation; interest; mutant; optical traps; programs; response; statistics; technology development; tool; transposon sequencing; virtual

SUMMARY The Mother Machine is a microfluidics-based high throughput imaging and analysis system for measuring the properties and behaviors of single cells in a large population (thousands to millions of cells). It is capable of identifying rare stochastic variants in a population, such as spontaneous persister cells, and tracking the properties of those rare cells over time, or in response to various treatments. It is equally capable of identifying phenotypically-altered genetic variants in a large pool of mutants, such as a transposon insertion library typically used for high throughput genetic screens such as Tn-seq. However, rather than relying on indirect inferences based on counts of sequences of particular transposon insertion junctions following several cycles of PCR amplification, the Mother Machine allows direct observation of the mutant over time. Moreover, it allows comparison to about 10 other cells of that genetic lineage in the same trench, providing extremely rigorous and precise measurements. Additional advantages over conventional Tn-seq approaches include the ability to identify phenotypes that do not necessary translate to changes in chromosome abundance (and hence sequence counts), such as the formation of multinucleate filamentous cells, or other irregularities in cell growth or division. Finally, direct imaging of single clonal lineages allows direct assessment of the phenotypic consequence for that particular transposon insertion in that particular orientation, rather than summing the effects of all insertions across a gene. This allows phenotypic differences that depend on the precise insertion location of a transposon, and its orientation to be assessed, for individual comparison to other different insertions within the same gene. The Core Director is a pioneer in this field who has made advancements in the technology not available anywhere else, including the ability to image more than 1 million trenches, and the ability to retrieve cells from trenches of interest. This specialized advanced technology cannot now be recreated in individual laboratories, and is not available commercially, providing a tremendous, unique resource for the important investigation of antibiotic resistance and susceptibility of staphylococci and enterococci. Additionally, the Core Director’s field-leading expertise in stochastic variation within bacterial populations provides an important intellectual resource for subprojects investigating the properties of antibiotic persister subpopulations – the effects of antibiotics on them as well as the contribution of variation in efflux expression to their genesis. These capabilities are highly complementary to those of the research subprojects, and significantly enhances both their throughput and the rigor and richness of the data that cannot otherwise be obtained.

概括 母机是一种基于微流体的高吞吐量成像和分析系统 测量大量人群中单细胞的特性和行为（数千至数百万人 细胞）。它能够识别人群中的罕见随机变体，例如赞助 迫害细胞，并随着时间的推移跟踪这些稀有细胞的特性，或者响应各种 治疗。它同样能够识别大型遗传变异的大型遗传变异 突变体库，例如通常用于高通量遗传的转座子插入库 屏幕，例如TN-Seq。但是，与其基于计数依靠间接推论 在几个循环的PCR循环之后，特定转座子插入连接的序列 放大，母机可以随着时间的推移直接观察突变体。而且，它 允许在同一沟渠中与该遗传谱系的其他大约10个细胞进行比较，提供 极其严格和精确的测量。与常规TN-Seq相比 方法包括识别不一定转化为变化的表型的能力 在染色体抽象（因此序列计数）中，例如多核形成 丝状细胞或细胞生长或分裂中的其他不规则性。最后，直接成像 克隆谱系可以直接评估该特定的表型后果 在该特定方向上的跨座椅插入，而不是总结所有插入的效果 跨基因。这允许表型差异取决于精度插入位置 转座子及其要评估的方向，以与其他不同的不同 同一基因中的插入。核心主任是该领域的先驱 该技术在其他任何地方都无法获得的进步，包括能够形象更多的能力 超过100万的沟渠，以及从感兴趣的战es中检索细胞的能力。这个专业 先进的技术现在不能在单个实验室中重新创建，并且不可用 商业上，为抗生素的重要投资提供了巨大的独特资源 葡萄球菌和肠球菌的抗性和敏感性。此外，核心导演的 细菌种群中随机变化方面的现场领先专业知识提供了重要的 用于调查抗生素持久性能的智力资源 亚群 - 抗生素对它们的影响以及外排的贡献 表达其起源。这些功能对于研究的功能非常完整 副标理，并显着增强了它们的吞吐量和严格性和丰富性 无法获得的数据。