Engineered gene circuits for basic science and biotechnology

描述(申请人提供)：在拟议的项目中，我们将继续设计、建造和表征基因电路。我们将使用微流控工具在精确控制的环境条件下培养和观察单个细胞。单细胞数据将提供一组数学模型，这些模型将用于确定关键的设计特征，然后将使用先前建立的分子生物学技术进行严格的测试。这种多学科的方法将增加我们对基因调控的理解，并为合成生物学社区带来新的工具。我们的第一个目标是探索“嵌套时钟”之间的相互作用。我们之前构建了一个健壮的细胞内时钟和一个细胞间同步的时钟群体。对这些系统的表征表明，天然的酶机制诱导了等待“降解”的不稳定蛋白质之间的偶联。在目标1中，我们将探索这种细胞内耦合如何导致在多个(细胞内和细胞间)尺度上同步的时钟。在下一个目标中，我们将探索两种细胞间耦合机制的使用，以开发一个新的合成生物学平台。我们以前已经展示了如何使用群体感应和氧化还原通信来设计宏观(1厘米)生物传感器。在目标2中，我们将展示这些耦合机制如何导致在单细胞水平上具有高度一致的开关跃迁的极其稳定的触发开关。在下一个目标中，我们将设计产生复杂时空动力学的光敏电路。最近，其他几个小组开发了光遗传电路，我们计划将光敏元件耦合到我们的电路中，以探索光引导信号在空间上扩展的细胞群体中的传播。在目标4中，我们将继续研究哺乳动物振荡器。在这里，我们将设计一个新的合成哺乳动物电路，它依赖于一个负反馈机制，该负反馈机制是由一个转录抑制因子介导的，该转录抑制因子在结合杂交启动子时通过诱导局部染色质重塑来发挥作用。我们将把我们的合成电路集成到细胞基因组中，以研究分子动力学如何在染色体调控的背景下发挥作用。最后，在目标5中，我们将开发细菌微细胞作为向哺乳动物细胞输送合成电路的平台。为了提高微细胞的功能，我们将构建并向微细胞转移一个额外的合成网络，提供补充的RNA聚合酶，使微细胞从亲本细菌分离后很长一段时间内能够独立表达基因。我们将定制用于容纳和跟踪微型细胞的微芯片设备，并将使用时间推移荧光显微镜来表征电路行为。这个项目的成功完成将促进我们对基因调控的理解，并最终导致可编程逻辑在基因传递方面的应用。1

DESCRIPTION (provided by applicant): In the proposed project, we will continue to design, construct and characterize genetic circuits. We will use microfluidic tools to grow and observe single cells in precisely controlled environmental conditions. Single cell data will inform a set o mathematical models that will be used to identify key design characteristics, which will then be rigorously, tested using previously established molecular biology techniques. This multi- disciplinary approach will increase our understanding of gene regulation and lead to new tools for the synthetic biology community. Our first aim will be to explore the interaction of "nested clocks". We previously constructed a robust intracellular clock and an intercellularly synchronized colony of clocks. Characterization of these systems revealed that the native enzymatic machinery induces a coupling between destabilized proteins that are \waiting" to be degraded. In Aim 1, we will explore how such intracellular coupling can lead to clocks that are synchronized at multiple (intra- and intercellular) scales. In the next aim, we will explore the us of two intercellular coupling mechanisms to develop a new platform for synthetic biology. We have previously shown how quorum sensing and redox communication can be used to design a macroscopic (1cm) biosensor. In Aim 2, we will show how these coupling mechanisms can lead to an extremely stable toggle switch with switching transitions that are highly uniform at the single cell level. In the next aim, we will engineer light-sensitive circuits that produce complex spatiotemporal dynamics. Optogenetic circuits have recently been developed by several other groups and we plan to couple light-sensitive elements to our circuits to explore the light-guided propagation of signals throughout a spatially extended population of cells. In Aim 4, we will continue our work on a mammalian oscillator. Here, we will engineer a novel synthetic mammalian circuit that relies on a negative feedback mechanism that is mediated by a transrepressor that acts by inducing local chromatin remodeling upon binding the hybrid promoter. We will integrate our synthetic circuits into the cell genome in order to study how the molecular dynamics function within the chromosomal regulatory context. Finally, in Aim 5 we will develop bacterial minicells as a platform for delivering synthetic circuits to mammalian cells. To improve the functionality of minicells, we will construct and transfer to minicells an additional synthetic network that provides supplemental RNA polymerase, enabling independent gene expression long after minicell separation from parental bacteria. We will tailor our microuidic devices for housing and tracking minicells and will characterize circuit behavior using time-lapse uorescence microscopy. The successful completion of this project will lead to advances in our understanding of gene regulation and could ultimately result in the utilization of programmable logic in a gene-delivery context. 1