Alternative pathways in directed evolution

定向进化的替代途径

基本信息

批准号：
1402101
负责人：
Marc Ostermeier
金额：
$ 35万
依托单位：
Johns Hopkins University
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
2014
资助国家：
美国
起止时间：
2014-06-01 至 2018-05-31
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1402101&HistoricalAwards=false
关键词：
Alternative pathways directed evolution

项目摘要

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.

提案编号：1402101institution：Johns Hopkins Universityp.I。：Ostermeier，MarcA。该建议旨在评估一种从根本上应用实验室进化的新方法。矛盾的是，目标是首先开发蛋白质，这些蛋白质在执行所需功能然后重新设计这些蛋白质方面会改善功能。该方法是基于对蛋白质复杂性进化生物学的越来越多的欣赏，有时对蛋白质的一组变化对蛋白质的作用有负面影响，对蛋白质功能隔离，在合并时可能会产生积极的效果。该项目的成功将影响进化生物学和生物技术领域。这种新方法可能会导致具有出色特性的蛋白质和细胞，这些特性无法轻易使用现有方法创建。该项目还将培训研究学前和研究生的研究。（1）旨在评估从根本上不同的定向进化选择策略，以使蛋白质从局部健身峰变成其他遥远的健身峰以及（2）以解决我们对流动环境中进化过程中的理解中的缺点。 TEM-15β-内酰胺酶等位基因将在各种旨在以不同方式穿越健身景观的选择性压力方案下进行定向进化。这些方案包括（1）阳性选择，（2）中性漂移，（3）倒出选择，（4）振荡选择，以及（5）波动的抗生素环境。这种进化将利用我们的带通基因电路，该基因允许在任何所需的健身水平（例如，远离较低或更高适应度的基因）中选择β-内酰胺酶等位基因。进化的进步和结果将通过深层测序来监测，这将确定每个选择机制的突变的身份和分布。通过全面确定TEM-15中个体突变对项目的跨学科性质的影响，CBET部门的生物技术和生物量工程计划的奖项由Systems and Synthetic Biologal of Molecormoliolar of Merecular of Merecular of Molecorlul and Cillular of Molecormult and Cillular and Cillular和Cylologular collogular和Cylologular collogular conso，这将有助于突变轨迹和结果的解释。