Microfluidic Processors for Directed Evolution and Synthetic Biology

用于定向进化和合成生物学的微流体处理器

基本信息

批准号：
7994827
负责人：
Brian M Paegel
金额：
$ 24.4万
依托单位：
SCRIPPS FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-12-01 至 2011-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7994827
关键词：
Address Binding Biological Models Biology CCR5 gene Catalysis Cell membrane Cells Chemicals Chemistry Chemokine (C-C Motif) Receptor 5 Cholesterol Cloning Complex Devices Dextrans Emulsions Enzymes Evolution Exhibits Extravasation Fluorescein Fluorescence Galactosidase Generations Genetic Transcription Genotype Goals HIV Hemolysin Hepatitis C virus Hour Human In Vitro Individual Integral Membrane Protein Investigation Label LacZ Genes Libraries Ligands Lipids Liposomes Liquid substance Maps Membrane Membrane Proteins Mentors Methods Micelles Microfluidics Miniaturization Modeling Molecular Weight Morphology Mutation Oils Phase Phenotype Population Process Proteins Reaction Research Research Activity Scanning Screening procedure Signal Transduction Sorting - Cell Movement Speed Staining method Stains Structure System Technology Testing Therapeutic Translations Transmembrane Transport Variant Vesicle Viral Viral Proteins Water artificial vesicle dextran directed evolution fitness macromolecule novel novel therapeutics particle programs receptor research study single molecule small molecule synthetic biology tetramethylrhodamine tool

项目摘要

The chemical transformations of biology occur in a compartmentalized context inside the cell membrane. Systematically studying this compartmentalized chemistry and harnessing its benefits for therapeutic applications through directed enzyme evolution will require methods for controlled synthesis and functional screening of cell-like compartments. Mentored research activities significantly expanded on current efforts in microfluidic directed evolution by exploring circuitry for the controlled high-throughput synthesis of monodisperse water droplets in oil for in vitro compartmentalization (IVC). This strategy Is enabling new explorations of RNA's catalytic fitness landscape by prohibiting a single advantageous genotype from dominating in the selective amplification reaction, and exaggerating neutral drift ofthe population. A nozzle array microfluidic IVC (MIVC) circuit was developed for these experiments and enabled selections encompassing l e 8 individuals per hour. Directed evolution of proteins with complex phenotypes (transport, membrane display, catalysis) will form the theme for independent phase investigations. The pIVC system will be used to synthesize monodisperse lipid vesicles for compartmentalization and functional display of integral membrane proteins, P-galactosidase and hemolysin will serve as models for using the pIVC processor to evolve new catalytic and selective transport functions on cytosolic and transmembrane proteins, respectively. Long-tennn research program goals include evolving membrane receptors (CCR5 and CD4) in lipid vesicles, selecting for enhanced binding of viral protein-receptor complexes, evolutionary structure-function studies, and synthesizing membrane-bound evolvable ligands for applications In targeted and decoy therapeutics.

生物学的化学转化发生在细胞膜内部的分室环境中。系统地研究这种分室化的化学，并利用其对治疗的好处通过定向酶进化的应用将需要控制合成和功能的方法筛选细胞状隔室。指导的研究活动大大扩展了当前的努力微流体定向进化，通过探索控制电路的高通量合成的电路在油中的单分散水滴进行体外分室化（IVC）。该策略正在实现新通过禁止从在选择性扩增反应中占主导地位，并夸大了人口的中性漂移。喷嘴为这些实验开发了阵列微流体IVC（MIVC）电路，并启用了选择每小时8个人。具有复杂表型的蛋白质的定向演化（转运，膜显示，催化）将构成独立阶段调查的主题。 PIVC系统将用于合成单分散脂质囊泡以进行分隔和功能显示整体膜蛋白，p-半乳糖苷酶和hemolysin将用作使用PIVC的模型加工机在胞质和跨膜上发展新的催化和选择性传输功能蛋白质分别。 Long-Tennn研究计划的目标包括不断发展的膜受体（CCR5和 CD4）在脂质囊泡中，选择增强的病毒蛋白质受体复合物的结合，进化结构功能研究，并合成针对目标应用的膜结合的膜结合的配体和诱饵疗法。