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，它寻求建立新的生物系统(使用从自然界收集的积木)，这些系统可以定制，以满足可再生能源、特种化学品生产和生物技术各个领域的迫切需求。这一领域的一个重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。通过迭代的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往往彼此之间以及与生物体中先前存在的固有功能之间存在强烈的相互作用。例如，这种并发症给设计微生物和植物新陈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，例如从元基因组库中组装基因以合成新的小分子。在这些情况下，人们可能不知道在这样的合成组装中需要包括哪些遗传元件，更不用说为了最大限度地发挥特定功能而应该如何调节它们了。虽然这些特性使线性工程变得低效和困难，有时甚至令人望而却步，但大自然已经进化出处理这种复杂性的机制。这项研究项目将开发一种合成系统，利用这些自然机制的力量，使合成生物学家能够产生、多样化和提炼复杂的多基因功能。这项技术的核心将基于一种名为整合子的细菌创新，这是一种自然的克隆和表达系统，通过使用定点重组，并通过内部启动子的表达转化为功能基因，以基因盒的形式组装多个开放阅读框架。捕获不同的单个基因并将它们物理连接到适合共同表达的阵列中的能力是这些遗传元素所独有的特征。其结果是一组功能协调的基因在理论上促进了新表型的快速进化。该项目将产生一种基于合成整合子(同步整合子)的新技术平台，包括计算优化和分析工具，将能够通过持续定向进化来设计复杂的多基因功能(如紫杉醇等植物衍生小分子的生物合成)。广泛影响该项目将产生一种强大的技术，使包括微生物和植物在内的生物系统工程能够用于无数有用的目的。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改良。该项目还将为探索基因组组织和过程中的动力学提供科学工具，如出现微生物对小分子的抗药性和代谢途径进化。此外，这个项目将向研究生和本科生介绍合成生物学的潜力，包括通过一年一度的国际基因工程机器(IGEM)比赛。最后，该项目将通过科学、艺术和写作(SAW)倡议(SAW)吸引更广泛的社区(大学以外)--一个专门针对学龄儿童的跨课程科学教育计划(www.sawtrust.org)。这一倡议使用科学的主题和图像作为科学实验、艺术和创造性写作的起点，并通过这样做来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.