Exploring the potential of synthetic cell-free systems for low-cost bioproduction

探索合成无细胞系统用于低成本生物生产的潜力

• 批准号: 2273129
• 金额: --
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2273129
• 关键词: Exploring; potential; synthetic; cell; free

PhD project strategic theme: Bioscience for renewable resources and clean growthFor the purposes of bioengineering, the complexity of the cell poses a great number of challenges. The major issue encountered when attempting to introduce synthetic gene circuits into a host cell is unpredictability. There are numerous ways in which a synthetic circuit can fail inside a host cell, with some common examples being; issues with initiation and termination of transcription, cross-talk between circuit and host, and overburdening of the host cell through competition for a shared resource. An alternative route for the application of synthetic gene circuits is found in cell-free systems, which can be used to harness cellular biochemistry without the need or constraints of anintact cell.Cell-free systems involve the use of cell-lysates, prepared from whole-cells through a variety of means including sonication, purification, and bead lysis. These lysates contain the transcription and translation machinery which can be combined with a DNA template to perform protein synthesis in vitro.Cell-free systems offer an unparalleled tool for the rapid and facile prototyping of synthetic genetic circuits. This is a feature which is invaluable for the development of bioprocesses to be used in bioproduction. It has already seen applications in the production of therapeutic proteins, along with renewable fuels and commodity platform chemicals.There is the potential of cell-free systems to achieve higher levels of productivity than whole-cell based systems, at a lower cost. These systems can also facilitate the production of products which would either be toxic to cells or impossible for them to produce, while also alleviating some of the biosecurity concerns associated with engineered organisms. The ability to freeze dry cell-free systems for long term storage and transport opens the door for applications in point of usediagnostics and in education.A plant-based system would also be an ideal target for low-cost bioproduction, particularly if synthetic circuits could be targeted to the chloroplast. Plant systems offer access to agricultural-level scaling of production, with the infrastructure already in place for growth and harvest. However, until recently the transformation of chloroplast genomes has been a difficult task that represents a bottleneck in the application of synthetic biology to plant systems. Recent work has shown that pentatricopeptide repeat proteins (PPR proteins) can be engineered to promote transgene expression in chloroplasts.There clearly offers great potential for the application of synthetic gene circuits within the chloroplast. I propose to explore and expand upon the cell-free platform as a tool for gene circuit prototyping, and also to investigate its utility as a testbed for circuits that could be introduced into the chloroplast.

博士项目战略主题：可再生资源和清洁增长的生物科学对于生物工程的目的，细胞的复杂性提出了许多挑战。当试图将合成基因电路引入宿主细胞时，遇到的主要问题是不可预测性。合成电路在宿主细胞内发生故障的方式有很多种，一些常见的例子是；转录的起始和终止，电路和宿主之间的串扰，以及通过竞争共享资源而使宿主细胞负担过重的问题。另一种应用合成基因电路的途径是在无细胞系统中发现的，它可以在不需要完整细胞的情况下利用细胞生物化学。无细胞系统包括使用细胞裂解物，从全细胞通过各种手段制备，包括超声，纯化和头裂解。这些裂解物含有转录和翻译机制，可以与DNA模板结合，在体外进行蛋白质合成。无细胞系统提供了一个无与伦比的工具，快速和方便的原型合成遗传电路。这一特性对于开发用于生物生产的生物工艺是非常宝贵的。它已经应用于生产治疗性蛋白质，以及可再生燃料和商品平台化学品。与基于全细胞的系统相比，无细胞系统有可能以更低的成本实现更高的生产力水平。这些系统还可以促进对细胞有毒或无法生产的产品的生产，同时还可以减轻与工程生物体相关的一些生物安全问题。冷冻无干细胞系统用于长期储存和运输的能力为在使用点诊断和教育中的应用打开了大门。基于植物的系统也将是低成本生物生产的理想目标，特别是如果合成电路可以针对叶绿体。植物系统提供了农业级生产规模的途径，并且已经具备了用于生长和收获的基础设施。然而，直到最近，叶绿体基因组的转化一直是一项艰巨的任务，是合成生物学在植物系统中应用的瓶颈。最近的研究表明，可以设计五肽重复蛋白（PPR蛋白）来促进叶绿体中的转基因表达。这显然为叶绿体内合成基因回路的应用提供了巨大的潜力。我建议探索和扩展无细胞平台作为基因电路原型的工具，并研究其作为可以引入叶绿体的电路测试平台的效用。