Stability and Robustness of Genetic Toggle Switches

因为一个生物体拥有的基因数量是有限的，每个基因能执行的功能数量也是有限的，所以一组基因的协调活动是细胞执行许多复杂任务所必需的。然而，对这种基因网络的组织和进化所知甚少。在这项研究中，一种结合体内/体外进化的方法将被用于“培育”各种特性的遗传回路。重点将放在最简单、最具特征的一类基因电路上，即双基因“拨动开关”。将进行分子和进化实验，以调查有关该系统的功能和进化的一些问题。具体来说，所提出的进化方法将用于生成相对于细胞噪声自发开关具有不同程度稳定性的开关。进化的调控序列将被分析，以确定与不同程度的电路稳定性相关的特征。这些开关对突变的鲁棒性也将被表征，并研究系统对噪声的稳定性与其对突变的鲁棒性之间的可能关系。最后，将通过对进化过程中各个中间阶段的进化区域进行测序来探索开关进化的动力学。这项研究的广泛影响包括在生物物理学、分子生物学和进化的界面和前沿培养新一代学生，以及为各种生物工程和生物医学应用开发设计遗传电路的实验协议的发展。

Because the number of genes an organism possesses is limited and the number of functions each gene can perform is also limited, coordinated activities of a group of genes are necessary for a cell to execute many complex tasks. However, very little is known about the organization and evolution of such gene networks. In this research, a combined in vivo/in vitro evolution approach will be used to "breed" genetic circuits of various properties. The focus will be on the simplest, and the best-characterized class of genetic circuits, the two-gene "toggle switch". Molecular and evolution experiments will be performed to investigate a number of issues concerning the function and evolution of this system. Specifically, the proposed evolutionary method will be used to generate switches with varying degrees of stability with respect to spontaneous switching by cellular noise. The evolved regulatory sequences will be analyzed to identify features that correlate with different degrees of circuit stability. The robustness of these switches to mutation will also be characterized, and possible relationship between the system's stability to noise and its robustness to mutation will be investigated. Finally, the dynamics of the switch evolution will be probed by sequencing the evolved regions at various intermediate stages of the evolution process. Broader impacts of this research include the training of a new generation of students at the interface and the forefront of biophysics, molecular biology, and evolution, and the development of experimental protocols to evolve designer genetic circuits for a variety of bioengineering and biomedical applications.