Engineering Bacteriophage T7 RNA Polymerase/Promoter System for Mammalian Synthet

描述(申请人提供)：基因组学技术和基因工程的进步使合成生物学这一新领域的发展成为可能，合成生物学寻求通过引入新的遗传元素并将这些元素整合到电路中来产生更复杂的行为来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，合成生物学的许多研究一直局限于细菌和酵母等更简单的生物。然而，由于哺乳动物合成生物学在治疗学和诊断学方面的巨大潜力，迫切需要更多的努力来关注这一领域。合成生物学的一个基本需求是可以用来精细控制基因表达的正交转录机器。我建议使用先进的基因组工程技术，如可跟踪多重重组工程(TRMR)和蛋白质序列活性关系(ProSAR)，来设计正交的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，是本项目研究的良好起点。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突变。组合突变也将通过多重自动化基因组工程技术进行探索。我将对工程RNA聚合酶/启动子对进行表征，以确定启动子的强度和正交性。最终，这些完全具有特征的RNA聚合酶/启动子系统可以用作哺乳动物细胞中遗传电路设计和整合的部分。

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.