Experimentally-determined fitness landscape of beta-lactamase inhibitory protein

描述（由申请人提供）：拟议的项目旨在通过实验确定编码β -内酰胺酶抑制蛋白（BLIP）基因的每个最近邻变体（与野生型序列不同的单个碱基对突变的变体）的适合度效应分布，从而克服进化突变研究中的一个主要障碍。具体来说，本研究的目的是在DNA和氨基酸水平上建立一个BLIP的每个最近邻居变体的文库，并量化这些变体的适应度。文库的创建将使用Kunkle诱变来完成，这是一种定点诱变方法，可以产生高比例的具有所需突变的DNA。适应度将基于BLIP变异抑制TEM-1 β -内酰胺酶（一种抗生素抗性酶，可水解β -内酰胺类抗生素，如氨苄西林）的能力来表征。BLIP文库将转化大肠杆菌，并利用独特的带通选择系统根据氨苄西林的最小抑制浓度（MIC）将文库分成箱。由于最小抑制浓度与BLIP的功能呈负相关，因此将文库分类为反映相对适合度的子文库。高通量测序技术将用于确定每个文库成员的DNA序列。这样，就可以确定整个基因由于单碱基对突变而产生的完整适应度分布。这一结果将解决一些关于突变的问题，包括同义突变和非同义突变的贡献，以及适应度效应如何在线性序列和BLIP的三维结构中分布。这种突变效应的知识有可能显著提高对进化生物学、蛋白质工程和医学基因组学领域的理解。由于对抗生素耐药性抑制剂的了解增加以及发现改进的BLIP变体的可能性，这项研究的结果也将具有医学意义。

DESCRIPTION (provided by applicant): The proposed project seeks to overcome a major barrier in the study of mutations in evolution by experimentally determining the distribution of fitness effects of every nearest neighbor variant (variants that differ from the wildtype sequence by a single base pair mutation) of the gene encoding beta- lactamase inhibitory protein (BLIP). Specifically, this research aims to create a library of every nearest neighbor variant of BLIP on the DNA and on the amino acid level and to quantify the fitness of these variants. Library creation will be accomplished using Kunkle mutagenesis, a site-directed mutagenesis method that can produce a high percentage of DNA with the desired mutations. Fitness will be characterized based on the ability of BLIP variants to inhibit TEM-1 beta-lactamase (an antibiotic resistance enzyme that hydrolyzes beta-lacam antibiotics, such as ampicillin). E. coli will be transformed with the BLIP library and a unique band-pass selection system will be utilized to separate the library into bins based on the minimal inhibitory concentration (MIC) for ampicillin. Because the minimum inhibitory concentration inversely correlates to the function of BLIP, the library will be sorted into sub-libraries that reflect relative fitness. High-throughput sequencing technology will be used to determine the DNA sequence of every library member. In this way, a complete fitness distribution due to single base pair mutations for the entire gene wil be determined. This result will address a number of questions about mutations, including the contributions of synonymous versus non-synonymous mutations, and how fitness effects are distributed in the linear sequence and in the three-dimensional structure of BLIP. This knowledge of mutational effects has the potential to significantly improve understanding in the fields of evolutionary biology, protein engineering, and medical genomics. The outcomes of this research will also be of medical significance owing to the increased understanding of an antibiotic resistance inhibitor and the possibility of finding an improved BLIP variant. PUBLIC HEALTH RELEVANCE: This project focuses on the study of mutations, which are the cause of genetic diseases and disorders. Understanding the global picture of how these mutations affect proteins would be beneficial to the prevention and treatment of these diseases and disorders. Furthermore, because the model gene for this research encodes a protein that inhibits antibiotic resistance, the proposed project will also be beneficial in developing improved antibiotic resistance inhibitors for the treatment of bacterial infection.