Reversible long-term memory devices in bacteria inspired by mammalian chromatin modification circuits

使细菌细胞能够记住长时间的环境线索对于许多过程至关重要，包括感知水或土壤中的污染物，记录人体肠道压力的生物标志物，以及触发细胞死亡以避免在环境中逃逸。与此同时，如果科学家能够建立这种能力，他们也将了解其原理，从而有可能揭示人类细胞如何在生物体的整个生命周期内记住自己的身份，这是在某些生物过程中被破坏的一个关键特性。这个项目的独特创新是在细菌中设计一个电路基序，模仿哺乳动物细胞中染色质状态的长期记忆过程。这种设计有望实现细菌基因状态的长期记忆。这个项目将丰富麻省理工学院每年开设的生物分子反馈系统课程。这项研究将培养麻省理工学院机械、生物和电气工程方面的研究生，并为每年通过麻省理工学院夏季研究计划和本科生机会研究计划来到麻省理工学院校园的麻省理工学院和非麻省理工学院本科生提供本科研究经验。麻省理工学院的本科生将通过“新工程转型教育计划”进一步获得该项目下的培训机会。细菌中的可逆记忆装置，可以在瞬态输入刺激下在两种稳定状态之间切换，以前就有人设计过。然而，在输入刺激被移除后，稳定状态的记忆通常在几天后就消失了。这限制了细菌记忆装置的适用性。更广泛地说，人们对控制记忆时间持续时间的分子机制缺乏了解。在这里，研究人员试图建立这种理解，在细菌中设计长期记忆装置，并展示这些装置在生物控制试验台的应用。为此，研究人员建议在细菌过程中实现与哺乳动物细胞中染色质状态的长期维持有关的电路基元。研究人员将利用转化酶进行DNA转化作为设计的核心过程，因为与染色质修饰一样，DNA转化也是一种酶促反应。关键的创新，反映了哺乳动物染色质修饰回路，是引入DNA逆转录的自催化作用，通过让逆转录的DNA表达一种逆转录酶来催化逆转录本身。研究人员提出，首先证明DNA倒置需要自催化来实现倒置DNA状态的稳定性。然后，他们提出，两种对立的自催化DNA反转，共享相同的底物，创造了一个双稳态的记忆开关，并且两种状态的记忆持续时间取决于自催化的强度。他们最终提出证明，他们可以将记忆延长到几周，他们可以通过小分子快速逆转记忆，并且这种设计可以应用于生物防护。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Enabling bacterial cells to remember long time environmental cues is essential for numerous processes including, sensing pollutants in water or soil, for recording biomarkers of stress in the human gut, and for triggering cell death to avoid escape in the environment. At the same time, if scientists can build this capability, they will also understand its principles, and thereby potentially shed light on how human cells can remember their identity for the life-time of an organism, a critical property that is broken in some biological processes. The unique innovation of this project is the engineering of a circuit motif in bacteria that mimics processes associated with long-term memory of chromatin states in mammalian cells. This design is expected to enable long-term memory of gene states in bacteria. This project will enrich the curriculum of the Biomolecular Feedback Systems Course, which is taught every year at MIT. This research will train graduate students in Mechanical, Biological, and Electrical Engineering at MIT, as well as provide undergraduate research experience to both MIT and non-MIT undergraduate students who come to the MIT campus yearly through the MIT Summer Research Program and Undergraduate Opportunity Research Program. MIT undergraduate students will further obtain a training opportunity under this project through the New Engineering Transformation Education Program. Reversible memory devices in bacteria, which can be switched between two stable states with a transient input stimulus, have been engineered before. However, after the input stimulus is removed, memory of the stable state usually vanishes after only a few days. This limits the applicability of bacterial memory devices. More broadly, there is a lack of understanding of the molecular mechanisms that control the temporal duration of memory. Here, the investigators seek to establish this understanding, engineer long-term memory devices in bacteria, and demonstrate these devices’ application to a biocontainment test-bed. To this end, the investigators propose to implement with bacterial processes the circuit motifs that are implicated in the long-term maintenance of chromatin states in mammalian cells. The investigators will use DNA inversion through invertase enzymes as the core process of the design since, just like chromatin modification, DNA inversion is an enzymatic reaction. The key innovation, which mirrors mammalian chromatin modification circuits, is to introduce autocatalysis of DNA inversions by having the inverted DNA express an invertase enzyme that catalyzes the inversion itself. The investigators propose to first demonstrate that autocatalysis of DNA inversion is required to achieve stability of the inverted DNA state. They then propose to show that two antagonistic autocatalytic DNA inversions, sharing the same substrate, create a bistable memory switch, and that the duration of memory of each of the two states hinges on the strength of autocatalysis. They finally propose to demonstrate that they can extend memory to last several weeks, that they can quickly reverse it by small molecules, and that the design can be applied for biocontainment.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.