Engineering Bacteriophage T7 RNA Polymerase/Promoter System for Mammalian Synthet

描述（由申请人提供）：基因组技术和基因工程的进步使合成生物学这一新领域得以发展，合成生物学旨在通过引入新的遗传元件并将这些元件集成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控、电路构建和电路集成的复杂性增加，合成生物学的许多研究仅限于细菌和酵母等更简单的生物体。然而，由于哺乳动物合成生物学在治疗和诊断方面的巨大潜力，迫切需要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，它可以用来精细地控制基因表达。我建议使用先进的基因组工程技术，如可追踪多重重组（TRMR）和蛋白质序列活性关系（ProSAR），来设计正交RNA聚合酶/启动子对。噬菌体T7 RNA聚合酶/启动子系统转录速度快，终止率低，启动子序列特异性高，是本项目研究的一个很好的起点。我将合理设计寡核苷酸，搜索蛋白质空间，确定影响启动子结合的“热点”，然后对这些区域进行饱和诱变。组合突变也将通过多重自动化基因组工程技术进行探索。我将表征工程RNA聚合酶/启动子对，以确定启动子的强度和正交性。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遗传电路设计和整合的一部分。

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.