Alternative pathways in directed evolution

提案编号：1402101机构：约翰霍普金斯大学P.I.：作者声明：Marc A. 职务名称：进化的研究很重要，不仅因为它是生物学的核心，而且因为研究人员在实验室中应用进化来开发有益的蛋白质和细胞，用于许多领域，包括人类健康，化学和生物制剂的检测，以及环境中有毒化合物的补救。 该提案旨在评估一种在实验室中应用进化的全新方法。 巧合的是，目标是首先开发出在执行所需功能方面较差的蛋白质，然后重新设计这些蛋白质以改善功能。 这种方法是基于进化生物学对蛋白质复杂性的日益增长的认识，有时一组对蛋白质功能有负面影响的蛋白质变化，当结合起来时，可能会产生积极的影响。 该项目的成功将影响进化生物学和生物技术领域。 潜在地，这种新方法可以导致具有上级特性的蛋白质和细胞，而这些特性是使用现有方法无法容易地创建的。 该项目还将培养一名博士预科生和一名本科生从事研究工作，其目标是：（1）评估从根本上不同的定向进化选择策略，以使蛋白质从局部适应度峰值向其他远距离适应度峰值进化;（2）解决我们对波动环境中进化过程（即变量选择下的进化）理解的不足。TEM-15 β-内酰胺酶等位基因将在各种选择性压力方案下进行定向进化，这些方案旨在以不同的方式遍历适合度景观。这些方案包括（1）阳性选择，（2）中性漂移，（3）退行性选择，（4）振荡选择，和（5）波动抗生素环境。这种进化将利用我们的带通基因电路，允许在任何所需的适应性水平（例如远离适应性较低或较高的基因）选择β-内酰胺酶等位基因。进化的进展和结果将通过深度测序来监测，这将确定每个选择机制的突变的身份和分布。突变轨迹和结果的解释将通过全面确定TEM-15中单个突变的影响来帮助。由于该项目的跨学科性质，CBET部门的生物技术，生物化学和生物质工程计划的这一奖项由分子和细胞生物学部门的系统和合成生物学计划共同资助。

Proposal Number: 1402101Institution: Johns Hopkins UniversityP.I.: Ostermeier, Marc A. Title: Alternative pathways in directed evolutionThe study of evolution is important, not only because it is central to biology, but also because researchers apply evolution in the laboratory to develop beneficial proteins and cells for use in in many areas including human health, detection of chemical and biological agents, and remediation of toxic compounds in the environment. This proposal seeks to evaluate a fundamentally new approach for applying evolution in the laboratory. Paradoxically, the goal is to first develop proteins that are worse at performing the desired function and then re-engineering these to be improved in function. The approach is based on a growing appreciation in evolutionary biology of the complexity of proteins and that sometimes a set of changes to a protein that have a negative effect on protein function in isolation, can have a positive effect when combined. Success of this project would impact the fields of evolutionary biology and biotechnology. Potentially, this new approach could lead to proteins and cells with superior properties that could not be readily created using existing methods. The project will also train a pre-doctoral and undergraduate student in research.The objectives are (1) to evaluate fundamentally different directed evolution selection strategies for evolving proteins away from local fitness peaks towards other distant fitness peaks and (2) to address shortcomings in our understanding of evolutionary processes in fluctuating environments (i.e. evolution under variable selection). The TEM-15 beta-lactamase allele will be subjected to directed evolution under a variety of selective pressure schemes designed to traverse the fitness landscape in different ways. These schemes include (1) positive selection, (2) neutral drift, (3) retrogressive selection, (4) oscillating selection, and (5) fluctuating antibiotic environments. This evolution will exploit our band-pass gene circuit that allows the selection of beta-lactamase alleles at any desired fitness level (e.g. away from genes with lower or higher fitness). The progress and outcomes of evolution will be monitored by deep sequencing, which will determine the identity and distribution of mutations for each selection mechanism. The interpretation of mutational trajectories and outcomes will be aided by the comprehensive determination of the effect of individual mutations in TEM-15.Due to the interdisciplinary nature of the project, this award by the Biotechnology, Biochemical, and Biomass Engineering Program of the CBET Division is co-funded by the Systems and Synthetic Biology Program of the Division of Molecular and Cellular Biology.