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

合成生物学中快速、精确 DNA 模块重排的平台

• 批准号: BB/K003356/1
• 负责人: Marshall Stark
• 金额: $ 416.07万
• 依托单位: University of Glasgow
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2013
• 资助国家: 英国
• 起止时间: 2013 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FK003356%2F1
• 关键词: platform; rapid; precise; DNA; module

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.

最近，出现了一个新的科学领域，称为合成生物学，该领域旨在应用工程原理（例如，使用模块化组件的使用以及“设计建筑测试模型”改进方法）来开发生物系统出于有用的目的。合成生物学的一个主要目标是创建转基因的微生物，以经济上的高产和低环境影响产生有价值的化学物质，或者进行有益的化学转化，例如对废水中污染物的中和。为了创建这些生物，通常有必要引入一组新基因（以DNA序列编码），并在生物体长固有的DNA序列（“基因组”）内的指定位置组装它们。目前可用于此“组装”任务的遗传技术仍然非常原始且不足，而基因组装被认为是导致有用微生物发展的工作中的严重瓶颈。我们提出的研究计划的第一个主要目的是为此基因组装过程建立复杂的新方法，该方法将在创造新的微生物菌株的速度和效率方面实现逐步变化。为此，我们将适应一种称为丝氨酸积分酶的非凡细菌酶，其自然任务是进行这种遗传重排，但迄今为止，它们是合成生物学的工具。我们将设计快速，健壮和有效的方法来制作基因组DNA中许多不同指定位置（“着陆垫”）中的任何一个可以插入（使用丝氨酸积分）中的基因盒。通过这样做，我们可以组装基因集合以在特定的微生物中排序。此外，我们可以选择将基因放在基因组和以什么顺序中的位置，并用不同版本代替任何单个部分。这允许对复杂遗传系统的优化比目前可能容易得多。使用我们的新方法，我们打算设计微生物细胞以制造下一代生物燃料，通过微生物发酵而不是使用化石燃料为塑料工业制造化学物质，并合成新的抗生素。可以以巧妙的方式响应外部信号的“智能细胞”（例如，在其环境中的光，高温或化学物质），或者如果它们暴露于特定信号以及多少次，则可以“记住”。因此，这些智能单元只能在需要时才能打开以执行有用的功能，或者可以编程以执行有序的一系列任务，就像一台洗衣机的洗衣服旋转循环一样。我们将为基因组装创建的基于丝氨酸积分酶的工具，可以构建简单但高效的细胞内设备来检测和计数信号。因此，我们程序的第二部分是展示这些内存设备的设计和构建的方式，并证明它们可以像我们设想的方式一样工作。

项目成果

Budding yeast centromeric DNA and A+T rich bacterial DNA can function as centromeres in the fission yeast Schizosaccharomyces pombe

芽殖酵母着丝粒 DNA 和富含 A T 的细菌 DNA 可以充当裂殖酵母裂殖酵母中的着丝粒

• DOI: 10.1101/513150
• 发表时间: 2019
• 作者: Barbosa A

Site-specific recombinases: Methods and protocols

位点特异性重组酶：方法和方案

• 发表时间: 2017
• 作者: Femi J Olorunniji

A novel Streptomyces spp. integration vector derived from the S. venezuelae phage, SV1.

• DOI: 10.1186/1472-6750-14-51
• 发表时间: 2014-05-30
• 期刊: BMC biotechnology
• 影响因子: 3.5
• 作者: Fayed B;Younger E;Taylor G;Smith MC
• 通讯作者: Smith MC

Rapid metabolic pathway assembly and modification using serine integrase site-specific recombination.

• DOI: 10.1093/nar/gkt1101
• 发表时间: 2014-02
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Colloms SD;Merrick CA;Olorunniji FJ;Stark WM;Smith MC;Osbourn A;Keasling JD;Rosser SJ
• 通讯作者: Rosser SJ

Multiplexed integrating plasmids for engineering of the erythromycin gene cluster for expression in Streptomyces spp. and combinatorial biosynthesis.

• DOI: 10.1128/aem.02403-15
• 发表时间: 2015-12
• 期刊: Applied and environmental microbiology
• 影响因子: 4.4
• 作者: Fayed B;Ashford DA;Hashem AM;Amin MA;El Gazayerly ON;Gregory MA;Smith MC
• 通讯作者: Smith MC