Development of an open source platform for synthetic genomics

在过去的15年里，合成基因组的创建取得了长足的进步。除了推进分子微生物学的基础知识外，由设计基因组控制的细菌有望在帮助社会应对21世纪的重大挑战方面发挥非常重要的作用。工程微生物的一些最明显的应用包括生物修复、纳米制造以及疾病治疗。然而，只有少数显著修改的基因组结构被探索，主要是因为大多数模式生物的相对较高的复杂性，这显著增加了这类项目的成本和工作量。因此，我们对合成基因组的设计规则的理解仍然有限。我的实验室正在使用近乎最小的细菌花中浆体作为快速生长的非致病细胞模型，用于基础研究和细胞工程努力。花分支杆菌是最简单的自由生物之一，其基因组只有800kb左右，编码不到700个基因。我的研究小组已经开发了一些遗传工具，如质粒、抗生素耐药性标记和有效的转化方案。此外，我们已经在酵母中克隆了整个花分支杆菌基因组作为人造染色体，在接受细菌进行全基因组移植之前，可以对其进行遗传操作。我的团队还制作了对M.florum物理和生理特性的深入表征，这是一种几乎完全基于实验数据的功能注释，以及一个强大的全细胞模型，它忠实地预测了这种细菌的许多表型。我的研究计划的长期目标是了解塑造细菌基因组结构的规则，我们将利用这些规则来生产合成有机体。在接下来的五年里，我们的主要目标是将这种生物发展成为科学界免费获得的开源基因组原型平台，并启动对选定的合成基因组变体的研究。更准确地说，我们建议生成并表征标准化DNA部分的目录，这将有助于准确控制花生菌中的基因表达。我们还对为大规模细菌基因组工程开发一种新的模块化和简单的策略感兴趣。这种新的方法利用丝氨酸重组酶，通过用合成DNA组装的变体取代40kb的染色体片段，极大地促进了工程基因组的创建。我们将进行两种不同的设计，一种是最小的花分支杆菌基因组，另一种是基因聚集在功能模块中的重构菌株。总而言之，这些努力将有助于解决有关基因组设计和细胞限制的问题，否则这些问题将非常复杂，很难用常见的模式生物来解决，并将有助于合成基因组学的发展。

The creation of synthetic genomes has made considerable progress during the past 15 years. In addition to advancing fundamental knowledge in molecular microbiology, bacteria controlled by designer genomes promise to play a very important role in helping society address major challenges of the 21st century. Some of the most obvious applications of engineered microbes include bioremediation, nanofabrication as well as disease treatment. However, only a few significantly modified genome architectures have been explored, mainly because of the relatively high complexity of most model organisms, which significantly increase the cost and workload for this type of project. As a consequence, our understanding of design rules for synthetic genomes remains limited. My laboratory is using the near-minimal bacterium Mesoplasma florum as a fast-growing, non-pathogenic cell model for fundamental research and cell engineering efforts. M. florum is one of the simplest free-living organisms with a genome of only ~800kb encoding fewer than 700 genes. Genetic tools, such as plasmids, antibiotic resistance markers, and efficient transformation protocols have been developed by my research group. In addition, we have cloned the entire M. florum genome as an artificial chromosome in yeast, where it can be genetically manipulated prior to whole-genome transplantation in a recipient bacterium. A deep characterization of the physical and physiological properties of M. florum, a functional annotation based almost exclusively on experimental data as well as a robust in silico whole-cell model that faithfully predicts many phenotypes for this bacterium were also produced by my team. The long-term objective of my research program is to understand the rules that shape genome architecture in bacteria, which we will leverage for the production of synthetic organisms. During the next five years, our main goal is to develop this organism as a freely available open-source genome prototyping platform for the scientific community, and to initiate the investigation of selected synthetic genome variants. More precisely, we propose to generate and characterize a catalog of standardized DNA parts that will be useful to accurately control gene expression in M. florum. We are also interested in developing a new modular and straightforward strategy for large-scale bacterial genome engineering. This novel approach takes advantage of serine recombinases to considerably facilitate the creation of engineered genomes by replacing 40kb chromosome sections by variants assembled using synthetic DNA. We will work on two different designs, a minimal M. florum genome as well as a refactored strain in which genes are clustered in functional modules. Taken together these efforts will help address questions about genome design and cell constraints that would otherwise be very complex and difficult to address with common model organisms, and will contribute to the development of synthetic genomics.