Collaborative Research: Synthetic integrons for continuous directed evolution of complex genetic ensembles

合成生物学是一门新兴的科学和工程学科，旨在建立新的生物系统（使用从自然界收集的构建块），这些生物系统可以定制，以满足可再生能源、特种化学品生产和各种生物技术领域的迫切需求。这一领域的一个巨大挑战是需要能够在生物系统中构建新的复杂功能的技术。当这些功能涉及多个基因的表达和协调时，构建它们变得越来越困难。通过迭代方法在生物体中组装多基因功能既费力又困难，因为工程基因及其产物经常相互作用，并与生物体中已有的天然功能相互作用。例如，这样的复杂情况给设计微生物和植物代谢的努力带来了重大挑战。此外，合成生物学的许多理想应用包括复杂的新功能和巨大的遗传多样性，例如组装来自宏基因组文库的基因以合成新的小分子。在这些情况下，人们可能无法先验地知道哪些遗传元素需要包含在这样的合成组装中，更不用说如何调节它们以最大限度地发挥特定功能。虽然这些特性使得线性工程效率低下且困难重重，有时甚至令人望而却步，但大自然已经进化出了处理这种复杂性的机制。该研究项目将开发一个合成系统，利用这些自然机制的力量，使合成生物学家能够产生、多样化和完善复杂的多基因功能。这项技术的核心将基于一种被称为整合子的细菌创新，整合子是一种天然的克隆和表达系统，它通过使用特定位点的重组和内部启动子表达转化为功能基因，以基因盒式的形式组装多个开放阅读框。捕获不同的个体基因并将它们物理连接到适合共表达的阵列的能力是这些遗传元件所特有的特性。结果是功能协调基因的组装理论上促进了新表型的快速进化。该项目将产生一个基于合成整合子（syntegrons）的新技术平台，包括计算优化和分析工具，这将使复杂的多基因功能的工程（如植物源小分子的生物合成，如紫杉醇）通过持续的定向进化。更广泛的影响该项目将产生一种强大的技术，使生物系统的工程，包括微生物和植物，为无数有用的目的。值得注意的例子包括可再生生物燃料和生物材料的生产，用于特种化学品的小生物分子的合成，生物修复和农业作物的改良。该项目还将为探索微生物对小分子耐药性的出现和代谢途径进化等过程中的基因组组织和动力学提供科学工具。此外，该项目将向研究生和本科生介绍合成生物学的潜力，包括通过年度国际基因工程机器（iGEM）竞赛。最后，该项目将通过科学、艺术和写作（SAW）计划（一个专门针对学龄儿童的跨学科科学教育计划）吸引更广泛的社区（在大学环境之外）参与（www.sawtrust.org）。这一倡议以科学主题和图像作为科学实验、艺术和创意写作的起点，以此激发创造力和科学好奇心。

Synthetic biology is emerging scientific and engineering discipline that seeks to make it possible to build new biological systems (using building blocks gleaned from the natural world) that can be customized to meet pressing needs in areas such as renewable energy, specialty chemical production, and various areas of biotechnology. A grand challenge in this field is the need for technologies that enable the construction of novel complex functions in biological systems. When these functions involve the expression and coordination of multiple genes, building them becomes increasingly difficult. Assembling multigenic functions in an organism by an iterative approach is both laborious and difficult, since the engineered genes and their products often interact strongly with both one another and with the pre-existing native functions in the organism. For example, such complications have presented major challenges to efforts to engineer metabolism in microbes and plants. Moreover, many desirable applications of synthetic biology comprise complex novel functions and great genetic diversity, such as the assembly of genes from a metagenomic library in order to synthesize novel small molecules. In these cases, one might not know a priori which genetic elements need to be included in such a synthetic assembly, much less how they should be regulated in order to maximize the performance of a particular function. While these properties make linear engineering inefficient and difficult, sometimes prohibitively so, Nature has evolved mechanisms to deal with such complexity. This research project will develop a synthetic system that harnesses the power of these natural mechanisms to enable synthetic biologists to generate, diversify, and refine complex multigenic functions. The core of this technology will be based on a bacterial innovation called integrons, which are natural cloning and expression systems that assemble multiple open reading frames, in the form of gene cassettes, by using site-specific recombination and conversion to functional genes by expression from an internal promoter. The ability to capture disparate individual genes and physically link them in arrays suitable for co-expression is a trait unique to these genetic elements. The result is an assembly of functionally coordinated genes theoretically facilitating the rapid evolution of new phenotypes. This project will generate a novel technology platform based on synthetic integrons (syntegrons), including computational optimization and analysis tools, that will enable the engineering of complex multigenic functions (such as the biosynthesis of plant-derived small molecules like taxol) through continuous directed evolution.Broader impactsThis project will generate a robust technology enabling the engineering of biological systems, including both microbes and plants, for myriad useful purposes. Notable examples include the production of renewable bio-fuels and biomaterials, the synthesis of small biomolecules for applications in specialty chemicals, bioremediation, and improvement of crops for agriculture. This project will also provide a scientific tool for probing genome organization and dynamics in processes such as the emergence of microbial resistance to small-molecules and metabolic pathway evolution. In addition, this project will introduce students at both graduate and undergraduate levels to the potential of synthetic biology, including exposure through the annual International Genetically Engineered Machine (iGEM) competition. Finally, this project will engage the broader community (outside the university setting) through the Science, Art and Writing (SAW) initiative - a cross-curricular science education program that is particularly targeted towards school-age children (www.sawtrust.org). This initiative uses themes and images from science as the starting point for scientific experimentation, art and creative writing, and in doing so stimulates creativity and scientific curiosity.