Developing an ultra-high throughput droplet microfluidic workflow for genetic circuit characterization

开发用于遗传电路表征的超高通量液滴微流体工作流程

• 批准号: 10680017
• 负责人: Rohan Thakur
• 金额: $ 4.77万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10680017
• 关键词: Acceleration; Address; Antibodies; Attention; Back; Bar Codes; Behavior; Biological; Cancer Biology; Cell Separation; Cells; Cellular Morphology; Clinical; Communicable Diseases; Complex; Custom; DNA; Data; Data Set; Development; Developmental Biology; Disease; Elements; Engineering; Fluorescence; Fluorescent Antibody Technique; Gene Expression Profile; Genetic; Genome; Genomics; Goals; Hand; Individual; Libraries; Link; Maps; Membrane Proteins; Methods; Microfluidics; Microscopy; Optics; Pathology; Phenotype; Photobleaching; Plasmids; Proteome; RNA Sequences; Research; Resolution; Sampling; Scheme; Signal Transduction; System; Systems Biology; Technology; Time; Work; behavioral phenotyping; biological research; cell behavior; dosage; epigenome; frontier; interest; microscopic imaging; multiple datasets; multiple omics; novel; novel sequencing technology; sequencing platform; single cell sequencing; single cell technology; single-cell RNA sequencing; synthetic biology; targeted sequencing; technology platform; technology validation; tool; transcriptome; transcriptomics

PROJECT SUMMARY Existing single cell sequencing technologies provide an unprecedented understanding of the unique genomic and transcriptomic differences that underlie heterogeneous biological samples. These differences are key to understand disease pathologies in clinical samples and fundamental mechanisms in basic biological applications. Recent attention has been directed towards the development of multiomic technologies that better capture the differences in paired datasets such as genome and transcriptome or transcriptome and epigenome. Currently however, there is no single cell technology that can profile sequence information with the corresponding phenotypic behavior of the cell. The purpose of this project is to develop a novel sequencing platform to address this technology gap. To demonstrate the utility of this platform, I will then apply it to rapidly characterize a tunable genetic oscillator. To accomplish this goal, I propose the following 2 specific aims. In Aim 1, I will develop FAB-seq (Fluorescence Annotated Barcoding and Sequencing). I will first demonstrate a novel dual barcoding approach that co-delivers optical and DNA barcodes to create single cell maps between microscopy image data and sequence data. I will also show that FAB-seq can be used to perform targeted sequencing according to an arbitrary phenotype. Then, I will leverage additional injected DNA barcodes to enhance the total barcode space of FAB-seq. In Aim 2, I will then demonstrate that FAB-seq can be used to rapidly characterize a tunable genetic oscillator. First, I will demonstrate that FAB-seq can detect oscillatory phenotypes from a circuit library containing oscillatory and non-oscillatory genetic circuits. Then I will show that when the oscillatory behavior of the circuit is perturbed, FAB-seq can map individual oscillation dynamics to the corresponding single cell transcriptome. The long-term goal of this project is to develop a platform technology that can map single cell microscopy data to single cell sequencing data at ultra-high throughput. I envision FAB-seq to be a transformative tool in addressing questions at the frontier of the genomics field.

项目概要 现有的单细胞测序技术提供了对独特基因组的前所未有的了解 以及异质生物样品背后的转录组差异。这些差异是关键 了解临床样本中的疾病病理和基础生物学的基本机制 应用程序。最近的注意力集中在多组学技术的开发上，这些技术可以更好地 捕获配对数据集（例如基因组和转录组或转录组）中的差异 表观基因组。然而，目前还没有单细胞技术可以分析序列信息 细胞相应的表型行为。 该项目的目的是开发一种新颖的测序平台来解决这一技术差距。到 为了演示该平台的实用性，我将应用它来快速表征可调谐遗传振荡器。 为了实现这一目标，我提出以下两个具体目标。在目标 1 中，我将开发 FAB-seq （荧光注释条形码和测序）。我将首先演示一种新颖的双条形码 共同提供光学和 DNA 条形码以在显微镜图像之间创建单细胞图谱的方法 数据和序列数据。我还将展示 FAB-seq 可用于根据 到任意表型。然后，我将利用额外注入的 DNA 条形码来增强总条形码 FAB-seq 的空间。在目标 2 中，我将演示 FAB-seq 可用于快速表征可调参数 遗传振荡器。首先，我将演示 FAB-seq 可以从电路库中检测振荡表型 包含振荡和非振荡遗传电路。然后我将证明当振荡行为 当电路受到扰动时，FAB-seq 可以将个体振荡动态映射到相应的单细胞 转录组。 该项目的长期目标是开发一种可以绘制单细胞显微镜数据图谱的平台技术 以超高通量获得单细胞测序数据。我设想 FAB-seq 成为一个变革性工具 解决基因组学领域的前沿问题。