Experimentally-determined fitness landscape of beta-lactamase inhibitory protein

描述（由申请人提供）：拟议项目旨在通过实验确定编码β-内酰胺酶抑制蛋白（BLIP）的基因的每个最近邻变体（与野生型序列相差一个碱基对突变的变体）的适应性效应分布，克服进化中突变研究的主要障碍。具体来说，这项研究旨在创建一个BLIP在DNA和氨基酸水平上的每个最近邻变体的库，并量化这些变体的适应性。文库创建将使用Kunkle诱变完成，Kunkle诱变是一种定点诱变方法，可以产生高百分比的具有所需突变的DNA。适应性将基于BLIP变体抑制TEM-1 β-内酰胺酶（一种水解β-内酰胺抗生素，如氨苄青霉素的抗生素抗性酶）的能力来表征。E.将用BLIP文库转化大肠杆菌，并且将利用独特的带通选择系统基于氨苄青霉素的最小抑制浓度（MIC）将文库分离到箱中。因为最小抑制浓度与BLIP的功能负相关，所以文库将被分选成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。以这种方式，将确定由于整个基因的单碱基对突变引起的完整适应度分布。这一结果将解决一些关于突变的问题，包括同义突变与非同义突变的贡献，以及健身效果如何分布在线性序列和BLIP的三维结构。这种突变效应的知识有可能显着提高进化生物学，蛋白质工程和医学基因组学领域的理解。这项研究的结果也将具有医学意义，因为人们对抗生素耐药性抑制剂的了解有所增加，并且有可能找到一种改进的BLIP变体。

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.