Designing and analyzing multi-generational switching in gene circuits for single cell biology

设计和分析单细胞生物学基因电路的多代转换

• 批准号: 1615487
• 负责人: Philippe Cluzel
• 金额: $ 61.61万
• 依托单位: Harvard University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1615487&HistoricalAwards=false
• 关键词: Designing; analyzing; multi; generational; switching

Memory is central to all living organisms from single-celled bacteria to humans and yet very little is known about how memory is controlled in biology. This project proposes to uncover this long-standing mystery by building circuits that are capable of memory in bacteria. Bacterial systems are ideal for this study because they are more accessible than multi-cellular organisms, and they allow for ultra-high precision measurements. The project relies on innovative biologically friendly chips to collect streaming data from live bacteria. Specific questions address in this study are (1) How is memory inherited through hundreds of generations in bacteria? (2) How might memory be robust enough to withstand large changes at the molecular level such as synthesis and degradation of proteins? The project will have a large impact on the teaching of science; many courses have already adopted preliminary examples from this work. More broadly, since the goal of this project is to demonstrate that engineered circuits can accurately perform predefined tasks in living cells, this effort will fundamentally change the way we develop new tools for medicine and other uses.One of the main goals of synthetic biology is to construct genetic circuits whose properties are not only robust on average as conditions change, but which can operate accurately in single cells without being corrupted by stochastic fluctuations. In many cases the dynamics also need to play out on a multi-generational time scale, which is particularly challenging because the states must be remembered even though the molecular hardware is replaced. Natural systems often face similar challenges, and though epigenetic memory is typically associated with higher organisms, bacteria must also decide between alternative states that can be maintained for tens or even hundreds of generations. Based on insights from stochastic theory for chemical reactions, and encouraged by substantial preliminary successes, this project will build synthetic oscillators and switches with multigenerational dynamics and highly precise timing in single cells. It will also characterize the on- and off-states of natural genetic circuits that exhibit slow switching, with a particular focus on DNA looping. Finally, a combination of classic bacterial genetics, microfluidic engineering, synthetic genetic oscillators, pulse generators based on DNA looping, and mathematical analyses will be used to probe the slow dynamics of flagellar promoters, which again reveal intriguing multi-generational effects. Thus, the project takes a broad systems and synthetic approach to quantify multigenerational switching at the level of individual bacterial cells.

记忆是从单细菌到人类的所有生物体的核心，但对于生物学中的记忆是如何控制的，知之甚少。该项目建议通过建立能够在细菌中记忆的电路来揭示这种长期存在的谜团。 细菌系统是本研究的理想选择，因为它们比多细胞生物更容易获得，并且可以进行超高的精确测量。该项目依靠创新的生物友好芯片来从实时细菌中收集流媒体数据。本研究中的特定问题要解决的是（1）细菌中如何通过数百代遗传记忆？ （2）记忆如何足够稳健以承受分子水平的大变化，例如蛋白质的合成和降解？ 该项目将对科学教学产生重大影响；许多课程已经从这项工作中采用了初步示例。更广泛地说，由于该项目的目标是证明工程电路可以准确执行活细胞中的预定任务，因此这项工作将从根本上改变我们为医学和其他用途开发新工具的方式。合成生物学的主要目标之一是构建遗传电路的主要目标，其遗传电路的属性不仅可以随着条件而无需在单个细胞中而不受单个细胞而不受单个细胞的损害。在许多情况下，动态还需要在多代时尺度上发挥作用，这尤其具有挑战性，因为即使更换了分子硬件，也必须记住各州。天然系统通常面临类似的挑战，尽管表观遗传记忆通常与较高的生物体有关，但细菌还必须在替代状态之间做出决定，这些状态可以维持数十甚至几代。基于从随机理论的化学反应的见解，并受到实质性成功的鼓励，该项目将构建具有多代动力学的合成振荡器和开关，并在单个细胞中建立高度精确的时机。它还将表征表现出缓慢开关的自然遗传回路的现行和外种状态，特别关注DNA循环。最后，将使用经典细菌遗传学，微流体工程，合成遗传振荡器，基于DNA循环的脉冲发生器以及数学分析的组合来探测鞭毛启动子的慢速动力学，这再次揭示了吸引人的多代作用。因此，该项目采用广泛的系统和合成方法来量化单个细菌细胞水平的多代转换。