Engineering Bacteriophage T7 RNA Polymerase/Promoter System for Mammalian Synthet

用于哺乳动物合成的工程噬菌体 T7 RNA 聚合酶/启动子系统

• 批准号: 8521522
• 负责人: Nanette Regina Boyle
• 金额: $ 2.13万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2013-08-16
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8521522
• 关键词: Adverse effects; Amino Acid Sequence; Antibiotic Resistance; Area; Bacteria; Bacteriophage T7; Behavior; Binding; Biological Markers; Cell Death; Cells; Characteristics; Chromosomes; Complex; Conditioned Culture Media; DNA-Directed RNA Polymerase; Data; Detection; Development; Diagnostic; Disease; Elements; Engineering; Ensure; Escherichia coli; Fluorescence; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Engineering; Genetic Transcription; Genome engineering; Genomics; Growth; Hereditary Disease; Hot Spot; Libraries; Life; Logic; Malignant Neoplasms; Mammalian Cell; Methods; Microbe; Mutagenesis; Mutation; Nature; Oligonucleotides; Organism; Peptide Sequence Determination; Physiologic pulse; Protein Engineering; Protein Region; Proteins; Radiation; Regulatory Element; Research; Research Project Grants; Resolution; Source; Specificity; Speed; Structure; System; T7 RNA polymerase; Techniques; Technology; Temperature; Testing; Therapeutic; Variant; Work; Yeasts; biological systems; cancer cell; cancer therapy; cellular engineering; chemotherapy; chromosome replication; combat; combinatorial; conventional therapy; design; genetic element; human disease; improved; novel; promoter; public health relevance; response; sensor; synthetic biology; therapeutic target; tool

DESCRIPTION (provided by applicant): Advances in genomics technology and genetic engineering enabled the development of a new field, synthetic biology, which seeks to create novel behaviors in cells by introducing new genetic elements and integrating such elements into circuits to produce more complex behaviors. Unfortunately, due to the added complexity of mammalian gene regulation, circuit construction, and circuit integration, much research in synthetic biology has been limited to simpler organisms like bacteria and yeast. However, due to the great potential of mammalian synthetic biology for therapeutics and diagnostics, it is imperative that more effort be focused towards this area. One basic need for synthetic biology is orthogonal transcription machinery which can be used to finely control gene expression. I propose using advanced genome engineering techniques, such as Trackable Multiplex Recombineering (TRMR) and Protein Sequence Activity Relationship (ProSAR), to engineer orthogonal RNA polymerase/promoter pairs. The bacteriophage T7 RNA polymerase/promoter system is an excellent starting point for this project research due to its rapid transcription spee, low termination rate and high specificity for its promoter sequence. I will rationally design oligo to search the protein space to determine 'hot spots' that effect promoter binding and then subject these areas to saturation mutagenesis. Combinatorial mutations will also be explored via multiplex automated genome engineering techniques. I will characterize the engineered RNA polymerase/promoter pairs to determine strength of promoter and orthogonality. Ultimately, these fully characterized RNA polymerase/promoter systems can then be used as parts for genetic circuit design and integration in mammalian cells.

描述（由申请人提供）：基因组技术和遗传工程的进步使新领域，合成生物学的发展，旨在通过引入新的遗传元素并将此类元素整合到电路中以产生更复杂的行为，从而在细胞中创造新的行为。不幸的是，由于哺乳动物基因调节，电路结构和电路整合的复杂性增加，因此在合成生物学方面进行了许多研究，仅限于细菌和酵母等简单生物。但是，由于哺乳动物合成生物学对治疗和诊断的巨大潜力，因此必须将更多的努力集中在这一领域。合成生物学的一个基本需求是正交转录机制，可用于精心控制基因表达。我建议使用先进的基因组工程技术，例如可跟踪的多重重组（TRMR）和蛋白质序列活性关系（PROSAR），以设计正交RNA聚合酶/启动子对。噬菌体T7 RNA聚合酶/启动子系统由于其快速转录SPEE，低终止率和启动子序列的高特异性而成为该项目研究的绝佳起点。我将在理性上设计寡核，以搜索蛋白质空间，以确定影响启动子结合的“热点”，然后使这些区域受到饱和诱变。组合突变还将通过多重自动基因组工程技术探索。我将表征工程的RNA聚合酶/启动子对，以确定启动子和正交性的强度。最终，这些完全表征的RNA聚合酶/启动子系统可以用作哺乳动物细胞中遗传回路设计和整合的零件。