Technical Mother Machine Core

总结 Mother Machine是一种基于微流体的高通量成像和分析系统， 测量大群体（数千到数百万）中单个细胞的特性和行为 细胞）。它能够识别群体中罕见的随机变异，例如自发性的 持续存在的细胞，并随着时间的推移跟踪这些罕见细胞的特性，或响应各种 治疗。它同样能够在大规模的基因组中识别表型改变的遗传变异。 突变体库，例如通常用于高通量遗传转化的转座子插入文库。 例如Tn-seq。然而，与其依赖于基于计数的间接推断， 在几个PCR循环后的特定转座子插入连接的序列 放大，母机允许随着时间的推移直接观察突变体。而且 允许与同一沟槽中该遗传谱系的大约10个其他细胞进行比较， 非常严格和精确的测量。与传统Tn-seq相比的其他优势 方法包括识别不一定转化为变化的表型的能力 在染色体丰度（因此序列计数），如形成多核 丝状细胞或细胞生长或分裂中的其他不规则性。最后，直接成像单个 克隆谱系允许直接评估特定的表型结果， 转座子插入在特定的方向，而不是所有插入的效果相加 一个基因。这允许依赖于精确插入位置的表型差异 转座子，其方向进行评估，个别比较，以其他不同的 在同一个基因中插入。核心总监是这一领域的先驱， 技术的进步在其他任何地方都无法获得，包括能够成像更多 超过100万个战壕，并且能够从感兴趣的战壕中检索细胞。这个专门 先进的技术现在不能在单个实验室中重新创造， 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，核心主任的 在细菌种群内随机变异方面的领域领先的专业知识提供了重要的 研究抗生素持续存在特性的子项目的知识资源 亚群-抗生素对它们的影响以及外排变化的贡献 表达他们的起源。这些能力与研究的能力高度互补 子项目，并显着提高了他们的吞吐量和严谨性和丰富性， 否则无法获得的数据。

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.