Microfluidic Processors for Directed Evolution and Synthetic Biology

描述（由申请人提供）：生物学的化学转化不是发生在试管中，而是发生在细胞膜内。系统地研究这种区室化化学并通过定向酶进化将其益处用于治疗应用将需要用于细胞样区室的受控合成和功能筛选的方法。指导的研究活动将显着扩大目前在微流体定向进化系统的努力，探索电路的控制高通量合成的单分散乳液和脂质囊泡在体外区室化（IVC）。囊泡将从连接酶核酶群体中划分单个分子;划分将允许通过禁止单一有利基因型主导反应来彻底探索适应性景观，并允许选择反式作用的多营业额核酶。这将作为一个独立的研究计划的基础。通过排列囊泡生成通道，微流体IVC（IVC）处理器的吞吐量将增加到每小时1 × 109。对于需要液滴筛选的实验，2D囊泡阵列的共聚焦荧光扫描和阵列的分选将通过多路复用膜阀阵列或使用电极阵列的介电电泳来完成;半乳糖苷酶和α-溶血素将用作使用IVC处理器来进化胞质和跨膜蛋白靶的模型。lVC处理器的长期研究计划目标包括进化膜受体（例如，CCR 5和CD 4），选择包膜蛋白-受体复合物的增强结合，进化用于靶向递送治疗剂的脂质体的膜结合配体，以及进化用于选择性包封神经毒素的转运蛋白。

DESCRIPTION (provided by applicant): The chemical transformations of biology occur not in the test tube, but compartmentalized 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 activity will significantly expand on current efforts in microfluidic directed evolution systems by exploring circuitry for the controlled high-throughput synthesis of monodisperse emulsions and lipid vesicles for in vitro compartmentalization (IVC). Vesicles will compartmentalize individual molecules from a population of ligase ribozymes; compartmentalization will permit a thorough exploration of the fitness landscape by prohibiting a single advantageous genotype from dominating the reaction and will permit selection for trans-acting, multiple-turnover ribozymes. This will serve as the foundation for an independent research program. The microfluidic IVC (¿lVC) processor throughput will be increased to 1x109 per hour by arraying vesicle generation channels. For experiments requiring droplet screening, confocal fluorescence scanning of 2D vesicle arrays and sorting of the array will be accomplished either by multiplexed membrane valve arrays or dielectrophoresis using electrode arrays; ¿-galactosidase and a-hemolysin will serve as models for using the ¿lVC processor to evolve cytosolic and transmembrane protein targets. Long-term research program goals for the ¿lVC processor include evolving membrane receptors (e.g., CCR5 and CD4) in liposomes, selecting for enhanced binding of envelope protein-receptor complexes, evolving membrane-bound ligands for liposomes used in the targeted delivery of therapeutics, and evolving transporters for selective encapsulation of neurotoxins.