Functional Selection of High Performance Enzymes

高性能酶的功能选择

基本信息

批准号：
8057644
负责人：
Christopher J Easley
金额：
$ 19.92万
依托单位：
LUCIGEN CORPORATION
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-08-12 至 2012-12-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8057644
关键词：
Bacterial Genome Bacteriophages Biochemical Biological Biology Bioreactors Caring Cells Chemistry Complex DNA DNA Repair DNA Sequence DNA amplification DNA-Directed DNA Polymerase Data Development Devices Diagnostic Disease Emulsions Enzymes Equipment Evolution Expression Library Gene Expression Gene Proteins Generations Genes Genomics Genotype Goals Hand Hour In Vitro Individual Libraries Lighting Major Histocompatibility Complex Measures Metagenomics Methods Microfluidic Microchips Microfluidics Molds Nucleic Acids Oils Operon Performance Peripheral Phase Polymerase Polymerase Gene Process Property Proteins Proteomics RNA Reaction Reading Refractory Research Research Personnel Scientist Screening procedure Sequence Analysis Small Business Innovation Research Grant Solutions Source Speed System Taq Polymerase Technology Time Translating UV sensitive Universities Variant Viral Virus Work aqueous base catalyst cost design directed evolution enzyme activity genetic analysis genetic selection high throughput screening human disease improved in vivo instrument instrumentation interest micro-total analysis system microbial microorganism nanolitre next generation novel nucleotide analog repair enzyme replicase tool viral DNA

项目摘要

DESCRIPTION (provided by applicant): Microfluidic systems are emerging as critically important tools for high throughput cell based analysis for applications including genotyping, sequencing, gene expression analysis, nucleic acid amplification, and diagnostics. Lab-on-a-chip devices that are conventionally designed to perform microchemical reactions in a continuous flow require external components, such as microvalves, and micropumps for handling solution, and complicated instruments to control everything. Most of these systems are large, their fabrication is expensive and they require proprietary chemistry and disposables, which increases their costs. One goal of this proposal is to develop an affordable, simple tool for correlating genomic and proteomic data from individual cells using droplet based microfluidic technology. This simple handheld device requires no electrical connections or instrumentation, but will allow for the formation of precisely controlled droplets, enabling massively parallel arrays of bioreactors suitable for enzyme screening, directed evolution, emulsion PCR of large DNA fragments, single-cell high throughput screening and numerous other applications. This will provide an alternative to conventional chemistries that are inadequate for these tasks and more complicated and expensive newer-generation instruments. The second goal of this proposal is to utilize this device to discovery novel thermostable viral replisomes de novo by their ability to auto amplify large operons containing their genes. We will optimize these multi-subunit enzymes through directed evolution to perform 100 kb PCR with cellular replication-like fidelity, enabling straightforward genetic analysis of highly complex disease loci. PUBLIC HEALTH RELEVANCE: Many complex problems in biology are essentially intractable without large-scale manipulation and processing. The genetic analysis of large complex disease loci is prohibitively expensive and time consuming. We propose to develop a simple system to readily produce millions of micro bioreactors to study cells, proteins and genes. We will use this device to develop powerful new enzymes for amplifying very large DNAs. This work will enable researchers to better understand cells, proteins and genes in normal and disease situations.

描述（由申请人提供）：微流体系统正在成为基于高通量细胞的分析的极其重要的工具，其应用包括基因分型、测序、基因表达分析、核酸扩增和诊断。通常设计用于在连续流中进行微量化学反应的芯片实验室设备需要外部组件，例如用于处理溶液的微型阀和微型泵，以及用于控制一切的复杂仪器。这些系统大多数都很大，制造成本昂贵，并且需要专有的化学物质和一次性用品，这增加了成本。该提案的一个目标是开发一种经济实惠、简单的工具，使用基于液滴的微流体技术关联单个细胞的基因组和蛋白质组数据。这种简单的手持设备不需要电气连接或仪器，但可以形成精确控制的液滴，从而实现适合酶筛选、定向进化、大 DNA 片段的乳液 PCR、单细胞高通量筛选和生物反应器的大规模并行阵列。许多其他应用程序。这将为无法胜任这些任务的传统化学方法和更复杂、更昂贵的新一代仪器提供替代方案。该提案的第二个目标是利用该装置通过其自动扩增包含其基因的大型操纵子的能力来从头发现新型热稳定病毒复制体。我们将通过定向进化来优化这些多亚基酶，以执行具有细胞复制保真度的 100 kb PCR，从而能够对高度复杂的疾病位点进行直接的遗传分析。公共卫生相关性：如果不进行大规模操作和处理，生物学中的许多复杂问题本质上都是难以解决的。大型复杂疾病位点的遗传分析非常昂贵且耗时。我们建议开发一种简单的系统，可以轻松生产数百万个微生物反应器来研究细胞、蛋白质和基因。我们将使用这个装置来开发强大的新酶来扩增非常大的 DNA。这项工作将使研究人员能够更好地了解正常和疾病情况下的细胞、蛋白质和基因。