Microfluidic Processors for Directed Evolution and Synthetic Biology

描述（由申请人提供）：生物的化学转化不是在试管中发生的，而是在细胞膜内进行的。系统地研究这种区隔化化学，并通过定向酶进化利用其治疗应用的益处，将需要控制合成和细胞样区隔功能筛选的方法。指导的研究活动将通过探索用于体外区隔化（IVC）的单分散乳剂和脂质囊泡的受控高通量合成电路，大大扩展当前在微流体定向进化系统方面的努力。囊泡将连接酶核酶群中的单个分子分隔开来；通过禁止单一优势基因型主导反应，区隔化将允许对适应度景观进行彻底的探索，并允许选择反式作用的多周转率核酶。这将作为一个独立研究项目的基础。微流控IVC（¿lVC）处理器吞吐量将增加到每小时1x109阵列囊泡生成通道。对于需要进行液滴筛选的实验，通过多路膜阀阵列或电极阵列进行共聚焦荧光扫描和阵列分选；-半乳糖苷酶和-溶血素将作为使用- lVC处理器进化胞质和跨膜蛋白目标的模型。lVC处理器的长期研究计划目标包括脂质体中膜受体（例如CCR5和CD4）的进化，选择包膜蛋白受体复合物的增强结合，用于靶向递送治疗的脂质体的膜结合配体的进化，以及用于选择性包裹神经毒素的转运体的进化。

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.