Design Principles of Molecular Computing Using Engineered Enzymes

每个细胞都可以被认为是一个计算设备，它接受各种物理和化学刺激作为输入，并使用生物分子相互作用的模拟电路将它们组合起来，以产生有助于其生存和执行其生理功能的输出。利用分子识别和化学动力学的基本生物物理原理，自下而上地构建这样的电路，提供了对生物系统潜在的物理化学设计原理的见解，并允许通过选择输入和输出来预测构建有用的电路，这些电路的输入和输出超出了自然界中发现的电路。该项目将开发一种新型快速生物电路，可以检测各种选择的输入，如光和小分子化学物质，并通过产生细胞存活和功能决定输出快速（和可控）响应。在这个项目中被询问的原则应该能够设计各种应用的人工电路，包括药物的可控生物制造和污染物的生物降解。该项目将为一些本科生提供培训和研究机会，特别是来自代表性不足的社区，通过参与由PI发起的罗格斯大学当地SACNAS分会。该项目还将支持大学生组成的团队参加三州地区的生物分子设计竞赛。在过去的十年里，已经建立了几个利用活细胞转录网络的合成生物电路，使活细胞的生物计算成为可能。然而，基于转录的电路性能的缓慢时间尺度（小时到天），异质性和缺乏对生物成分水平的精确控制，限制了转录电路的速度，适用性和稳健性。电路的速度对于需要快速传感和响应的应用尤为关键。本提案的目标是利用计算设计/工程蛋白酶开发基于蛋白酶的电路，该电路可以对选择的输入刺激（如小分子化学物质和光）做出响应，并且在底物选择性方面相互正交。表征良好、相互正交、种类多样的二聚化结构域化学/光诱导剂（作为传感器）、设计的分裂蛋白酶（作为致动器）和重新设计的细胞生存调节酶（作为输出）被用于构建、表征和优化基本逻辑门和电路元件。该项目涉及使用化学动力学建模方法优化电路元件的紧急特性，该方法将允许以复杂的方式组合多个输入信号。总体而言，本研究开发了一种计算和实验相结合的设计策略，以构建细胞中刺激响应的、基于蛋白酶的逻辑门，并在化学反应网络建模框架中使用对其紧急行为的数学处理来优化其性能。该项目由生物科学理事会分子和细胞生物科学部的分子生物物理学组和工程理事会化学、生物工程、环境和运输系统部的细胞和生物化学工程组共同支持。

Every cell can be thought of as a computing device that takes a variety of physical and chemical stimuli as inputs, and combines them using an analog circuit of biomolecular interactions to generate outputs that aid its survival and perform its physiological function. Building such circuits from the bottom up, using basic biophysical principles of molecular recognition and chemical dynamics, provides insights into the underlying physiochemical design principles of biological systems, and allows predictive construction of useful circuits with chosen inputs and outputs that go beyond the ones found in nature. This project will develop a new class of fast biological circuitry that can detect a variety of chosen inputs, such as light and small molecule chemicals, and rapidly (and controllably) respond by generating cell survival and function-determining outputs. The principles being interrogated in this project should enable the design of artificial circuits for a variety of applications including controllable biomanufacturing of pharmaceuticals and biodegradation of pollutants. The project will offer training and research opportunities for several undergraduate students especially from the underrepresented communities via involvement of the local SACNAS chapter at Rutgers University started by the PI. This project will also support teams built by undergraduates to participate in tri-state area biomolecular design competitions.Several synthetic biological circuits using transcriptional networks in living cells, to enable biological computation with living cells, have been built in the last decade. However, the slow timescales for transcription-based circuit performance (hours to days), heterogeneity and lack of precise control over the levels of biological components, limit the speed, applicability and robustness of transcriptional circuits. The speed of the circuit is especially critical for applications where rapid sensing-and-response is required. This proposal's goal is the development of protease enzyme-based circuits using computationally designed/engineered proteases that can be made responsive to chosen input stimuli such as small molecule chemicals and light, and mutually orthogonal with respect to their substrate selectivity. Well-characterized, mutually orthogonal and diverse class of chemical/light-inducers of dimerization domains (as sensors), designed split-protease enzymes (as actuators), and a redesigned cell survival-regulating enzyme (as output) are being used to construct, characterize and optimize elementary logic gates and circuit elements. This project involves optimization of the emergent properties of circuit elements using chemical dynamics modeling methods that will allow combining multiple input signals in complex ways. Overall, this research develops a combined computational and experimental design strategy to construct stimulus-responsive, protease-based logic gates in cells, and involves optimizing their performance using a mathematical treatment of their emergent behavior in a chemical reaction network modeling framework. This project is jointly supported by the Molecular Biophysics Cluster of the Molecular and Cellular Biosciences Division in the Directorate for Biological Sciences and Cellular and Biochemical Engineering Cluster in the Chemical, Bioengineering, Environmental and Transport System Division of Engineering Directorate.