Experimentally-determined fitness landscape of beta-lactamase inhibitory protein

实验确定的 β-内酰胺酶抑制蛋白的适应性景观

• 批准号: 8656369
• 负责人: Courtney Elaine Gonzalez
• 金额: $ 4.27万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8656369
• 关键词: Address; Affect; Amino Acid Substitution; Amino Acids; Ampicillin; Antibiotic Resistance; Antibiotic Therapy; Antibiotics; Bacterial Infections; Base Pairing; Biological Process; Biology; Communicable Diseases; Coupled; DNA; DNA Sequence; Data; Data Set; Disease; Enzymes; Escherichia coli; Evolution; Gene Mutation; Generations; Genes; Genetic Code; Genetic Variation; Genomics; Hereditary Disease; High-Throughput Nucleotide Sequencing; Knowledge; Laboratories; Libraries; Medical; Methods; Minimum Inhibitory Concentration measurement; Modeling; Mutagenesis; Mutation; Nature; Organism; Outcomes Research; Prevention; Protein Engineering; Protein Inhibition; Proteins; Public Health; Relative (related person); Research; Shapes; Site-Directed Mutagenesis; Sorting - Cell Movement; System; Technology; Testing; Time; Variant; base; beta-Lactamase; common treatment; fitness; genetic selection; improved; infectious disease treatment; inhibitor/antagonist; member; microchip; three dimensional structure

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.

描述（由申请人提供）：拟议的项目旨在通过实验确定编码编码基因β-氨基氨基酰胺酶抑制性蛋白（BLIP）的每个最近邻居变体的适应性效应（与野生型序列不同的变体（与单个碱基对突变不同）的适应性效应（与WildType序列不同），以克服进化过程中的主要障碍。具体而言，这项研究旨在创建一个在DNA和氨基酸水平上的Blip的每个最近邻居变体的库，并量化这些变体的适应性。将使用Kunkle诱变（一种定点诱变方法）来实现库的创建，该方法可以产生具有所需突变的高百分比DNA。适合度将根据BLIP变体抑制TEM-1β-内酰胺酶的能力（一种水解β-酰胺抗生素（如氨苄青霉素）的抗生素耐药性酶）的能力。大肠杆菌将使用BLIP库进行转换，并将利用独特的带通路选择系统将库基于氨苄西林的最小抑制浓度（MIC）将库分开。由于最小抑制浓度与BLIP的功能呈竞相相关，因此该库将被分类为反映相对适应性的亚纤维。高通量测序技术将用于确定每个库成员的DNA序列。这样，可以确定整个基因的单基对突变引起的完整适应性分布。该结果将解决有关突变的许多问题，包括同义词与非同义突变的贡献，以及如何以线性序列和BLIP的三维结构分布适应性效应。这种对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。由于对抗生素耐药性抑制剂的了解增加以及发现改善的BLIP变体的可能性，这项研究的结果也将具有医学意义。