An Integrated Approach for Genome-Engineering

本提案的总体目标是开发一种基于合成生物学和基因组学方法应用的基因组工程综合方法，这些方法在研究小组的实验室中率先应用。提出的方法汇集了DNA合成、基因组学和重组的进展，使数十亿合理设计的基因改变同时和组合的定量评估成为可能。具体来说，10000个独特的DNA低聚物被平行合成，高频重组到大肠杆菌基因组中，并使用深度测序或分子条形码策略进行定量追踪。这个过程可以递归地自动进行，以便迅速积累有益的修改，形成优化的菌株。在这里，我们建议将这些进展应用于一个广泛重要的工程模型系统：在大肠杆菌中生产生物柴油。作为更大努力的一部分，大肠杆菌产生的平台分子将被分泌、提取，并提供给无机催化，以生产C12/C14柴油分子混合物和C4下一代生物燃料（即丁醇）。与替代生物燃料方法相比，这种方法有可能改善经济效益和碳平衡，这应该会扩大这些努力的总体影响。

The overall objective of this proposal is to develop an integrated approach to genome engineering based on the application of synthetic biology and genomics methods pioneered in the laboratories of the investigatory team. The proposed approach brings together advances in DNA synthesis, genomics, and recombineering to enable the quantitative evaluation of billions of rationally designed genetic alterations simultaneously and combinatorially. Specifically, 10,000 unique DNA oligomers are synthesized in parallel, recombined at high-frequency into the Escherichia coli genome, and quantitatively tracked using deep sequencing or molecular barcoding strategies. This process can be performed recursively and automatically in order to rapidly accumulate beneficial modifications into an optimized strain. Here, we propose to apply these advances within the context of engineering a broadly significant model system: the production of biodiesel in E. coli. As part of a larger effort, the platform molecule produced by E. coli will be secreted, extracted, and provided for inorganic catalysis to produce a C12/C14 diesel molecule mixture and a C4 next generation biofuel (i.e.butanol). This approach has the potential to improve economics and carbon-balances compared to alternative biofuels approaches, which should broaden the overall impact of these efforts.