EAGER: Functional Integration of Synthetic Kinases into Endogenous Signaling Networks

细胞可以通过自然机制(如水平基因转移)或人工手段(如基因工程)获得新的基因。我们开始了解新获得的基因是如何整合到宿主的基因组中的，但这些基因产生的蛋白质如何在功能上整合到细胞的调控网络中的进化机制在很大程度上仍不清楚。在这个项目中，将使用合成生物学和定向进化方法在实验室中重新创建和分析这种网络重新布线过程。利用从哺乳动物丝裂原活化蛋白激酶级联中提取的信号模块设计的酵母，将在实验室应用进化策略来奖励获得突变的单个酵母细胞，这些突变导致这些引入的哺乳动物蛋白质的功能利用。在这个选择过程中，将进行基因组测序，以确定发生的突变的特征，并重建导致宿主信号网络功能重新布线的进化过程。更广泛的影响：这项研究背后的科学原理将用于培训研究生和本科生，包括那些来自代表性不足的少数族裔的学生。合成生物学中的进化论模块将被添加到由首席研究员教授的本科课程中。本科生还将有机会通过国际基因工程机器(IGEM)比赛学习合成生物学。通过暑期培训计划，将针对高中教师和高中生进行更多的外联活动。从这个项目中获得的见解将对理解微生物中出现的抗生素耐药性、转基因作物的栽培和特性以及新型合成生物学设备的设计具有重要意义。

Cells can acquire new genes by natural mechanisms (e.g. horizontal gene transfer) or by artificial means (e.g. genetic engineering). We are beginning to understand how newly-acquired genes are integrated into the host's genome, but the evolutionary mechanisms by which the proteins produced by these genes become functionally incorporated into the cell's regulatory networks remain largely unknown. In this project, synthetic biology and directed evolution approaches will be used to recreate and analyze in the laboratory this network rewiring process. Using yeast that have been engineered with signaling modules taken from the mammalian mitogen-activated protein kinase cascade, evolutionary strategies will be applied in the laboratory to reward individual yeast cells that acquire mutations resulting in functional utilization of these introduced mammalian proteins. During this selection process, genome sequencing will be performed to characterize the mutations that occur and to reconstruct the evolutionary processes that result in functional rewiring of the host's signaling networks. Broader Impacts: The scientific principles underlying this research will be used to train graduate and undergraduate students, including those from underrepresented minorities. Modules on evolution in synthetic biology will be added to an undergraduate course taught by the principal investigator. Undergraduates will also have the opportunity to learn about synthetic biology through the International Genetically Engineered Machine (iGEM) competition. Additional outreach will be targeted to high school teachers and high school students through summer training programs. The insights gained from this project will have relevance for understanding the emergence of antibiotic resistance in microbes, the cultivation and properties of genetically modified crops, and the design of novel synthetic biology devices.