Technical Mother Machine Core

技术母机核心

• 批准号: 10571888
• 负责人: Johan Paulsson
• 金额: $ 42.38万
• 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/10571888
• 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; Dedications; 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; Shapes; Staphylococcus aureus; Sum; Systems Analysis; Systems Biology; Technology; Time; Translating; Variant; Visualization software; Work; analysis pipeline; cell growth; cellular imaging; data handling; design; experience; experimental study; feeding; genetic variant; global health; innovation; interest; mutant; optic trap; 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.

摘要 母机是一种基于微流控技术的高通量成像和分析系统 测量大量(数千到数百万)单个细胞的属性和行为 细胞)。它能够识别种群中罕见的随机变量，如自发变量 以及随着时间的推移追踪这些稀有细胞的特性，或者对各种 治疗。它同样能够识别大量的表型改变的遗传变异 突变体库，例如通常用于高通量遗传的转座子插入文库 屏幕，如TN-SEQ。然而，不是依赖于基于计数的间接推断 几个循环的PCR后特定转座子插入连接的序列 通过放大，母机器可以随着时间的推移直接观察突变。此外，它还 允许与同一沟中约10个该遗传谱系的其他细胞进行比较，提供 极其严谨和精确的测量。与传统TN-SEQ相比的其他优势 方法包括识别不需要转换为变化的表型的能力 在染色体丰度(以及因此的序列计数)上，例如多核的形成 丝状细胞，或细胞生长或分裂的其他不规则。最后，直接成像单个 克隆谱系允许直接评估该特定的表型结果 在该特定方向上的转座子插入，而不是将所有插入的效果相加 跨越一个基因。这允许根据准确的插入位置进行表型差异 转座子的位置，以及它的取向有待评估，以便与其他不同的 同一基因中的插入。核心董事是这一领域的先驱，他已经做出了 这项技术的进步在其他任何地方都无法实现，包括能够 超过100万个战壕，以及从感兴趣的战壕中检索细胞的能力。这是专门的 先进技术现在不能在单独的实验室中重现，也不能使用 在商业上，为抗生素的重要研究提供了巨大的、独特的资源 葡萄球菌和肠球菌的耐药性和敏感性。此外，核心董事的 在细菌种群内随机变异方面的领域领先专业知识提供了重要的 研究抗生素永久剂特性的子项目智力资源 亚群-抗生素对它们的影响以及外排变异的贡献 对它们的起源的表达。这些能力与研究的能力是高度互补的 子项目，并显著提高了它们的吞吐量以及 以其他方式无法获得的数据。