A platform for rapid and precise DNA module rearrangements in Synthetic Biology

最近，一个新的科学领域出现了，称为合成生物学，其目标是将工程原理(例如，使用模块化组件，以及“设计-建造-测试-修改”的改进方法)应用到生物系统的开发中，以达到有用的目的。合成生物学的一个主要目标是创造转基因微生物，以经济、高产和对环境影响小的方式生产有价值的化学物质，或进行有益的化学转化，如中和废水中的污染物。为了创造这些生物体，通常需要引入一组新的基因(以DNA序列编码)，并将它们组装在生物体内在的长DNA序列(基因组)中的特定位置。目前可用于这一“组装”任务的基因技术仍然相当原始和不足，基因组装被认为是导致开发有用微生物的工作中的一个严重瓶颈。我们建议的研究计划的第一个主要目标是为这种基因组装过程建立一种复杂的新方法，这将实现创造新微生物菌株的速度和效率的阶段性变化。为此，我们将改造一组引人注目的细菌酶，称为丝氨酸整合酶，它的自然任务是进行这种基因重排，但到目前为止，它一直没有被用作合成生物学的工具。我们将设计快速、坚固和高效的方法来制造基因盒，这些基因盒可以(使用丝氨酸整合酶)插入基因组DNA中许多不同的指定位置中的任何一个。通过这样做，我们可以在特定的微生物中组装成有序的基因集合。此外，我们可以选择基因在基因组中的位置和顺序，并将任何单独的部分替换为不同的版本。这使得复杂基因系统的优化比目前可能的要容易得多。使用我们的新方法，我们打算设计微生物细胞来制造下一代生物燃料，通过微生物发酵而不是化石燃料来制造塑料工业的化学品，并合成新的抗生素。合成生物学的第二个主要目标是制造能够以聪明的方式对外部信号(例如，光、高温或环境中的化学物质)做出反应的“智能细胞”，或者能够“记住”它们是否暴露在特定信号下以及暴露了多少次。因此，只有当我们需要时，这些智能电池才能启动以执行有用的功能，或者可以被编程为执行一系列有序的任务，就像洗衣机的洗涤-漂洗-旋转-干燥循环一样。我们将创建的基于丝氨酸整合酶的基因组装工具有助于构建简单但高效的细胞内设备，用于检测和计数信号。因此，我们计划的第二部分是展示这些存储设备的设计和建造方法，并证明它们可以以我们设想的方式工作。

Recently, a new field of science has emerged called Synthetic Biology, which aims to apply engineering principles (for example, the use of modular components, and a "design-build-test-modify" approach to improvement) to the development of biological systems for useful purposes. One major target in Synthetic Biology is the creation of genetically modified microorganisms, to produce valuable chemical substances economically, in high yield and with low environmental impact, or to carry out beneficial chemical transformations such as neutralization of pollutants in waste water. To create these organisms, it is often necessary to introduce a set of new genes (encoded in DNA sequence) and assemble them in specified positions within the organism's long intrinsic DNA sequence ('genome'). The genetic techniques currently available for this 'assembly' task are still quite primitive and inadequate, and gene assembly is considered to be a serious bottleneck in the work leading to the development of useful microorganisms. The first main aim of our proposed research programme is to establish a sophisticated new methodology for this gene assembly process which will achieve a step-change in the speed and efficiency of creating new microorganism strains. For this purpose we will adapt a remarkable group of bacterial enzymes called the serine integrases, whose natural task is to carry out this kind of genetic rearrangement but which have hitherto been underused as tools for Synthetic Biology. We will design rapid, robust and efficient ways of making gene cassettes that can be slotted in (using serine integrases) to any one of a number of different specified positions ('landing pads') in genome DNA. By doing this we can assemble collections of genes to order within a particular microorganism. Furthermore we can choose where to place the genes in the genome and in what order, and replace any individual parts with different versions. This permits much easier optimization of complex genetic systems than is currently possible. Using our new methods we intend to engineer microbial cells to make next-generation biofuels, to make chemicals for the plastics industry by microbial fermentation instead of by using fossil fuel, and to synthesise new antibiotics.A second major target in Synthetic Biology is to make 'smart cells' that can respond in clever ways to external signals (for example, light, high temperature, or a chemical in their environment), or that can 'remember' if they have been exposed to a particular signal and how many times. These smart cells could thus be switched on to perform a useful function only when we need it, or could be programmed to carry out an ordered series of tasks, rather like the wash-rinse-spin-dry cycles of a washing machine. The serine integrase-based tools that we will create for gene assembly lend themselves to the construction of simple yet highly effective intracellular devices for detecting and counting signals. So a second part of our programme is to show the way to the design and construction of these memory devices, and prove that they can work in the way we envisage.