Engineered Selective Pressure: Challenges and Opportunities

在活细胞中复杂遗传电路的正向工程方面的快速进展证明了合成生物学这门年轻的学科具有改变现代生物技术的潜力。对生命系统的预测性理解是生物工程设计操作和医学知情干预的先决条件。这样的理解需要定量测量、数学分析和理论抽象。强大的测量技术和计算能力的出现，使生物学能够推动下一次科学革命。合成生物学为利用工程基因电路的定量数据开发和测试模型和一般网络原理提供了一个天然的平台。合成生物学的主要基本问题之一是合成电路进化和消除其预期功能的倾向。因此，重要的是发展一种定量的方法，使遗传网络更加健壮和稳定，这也将使它们在治疗应用中更有用。该项目的总体目标是通过结合尖端实验和计算工具，探索合成生物学中不可避免的选择压力带来的挑战和利用机遇。该项目的成功完成将在理解合成电路与宿主基因组在进化背景下的相互作用方面取得重大进展。该项目将为新一代数量生物学家的跨学科培养提供充足的机会。此外，为了将这个项目的影响扩大到学术界以外，其参与者将扩展一个非常成功的小学科学项目，促进合作小学的研究人员和教师之间的合作，以改善实践科学教育。为了达到项目的总体目标，将使用最近开发的“同步裂解电路”作为主要重点。这种合成基因回路的功能是，在一个临界值的种群密度下，触发大约90%的细菌死亡，让剩下的10%的细胞重新生长到临界值，重新开始这个循环。这产生了一种独特的表型，其特征是种群循环，可以使用微型和台式化学调节器在大范围的长度尺度上进行监测。它还引入了一种强大的选择压力，可以导致快速进化。研究人员将开发定量测量技术和计算模型，这将导致电路宿主进化过程的定量统计特征。沿着实验的时间进程对宿主基因组的电路和突变部分进行测序将用于重建系统所采取的进化路径。数学建模将指导实验，并帮助提取关键参数表征同步裂解电路的进化动力学。利用所获得的知识，研究人员将设计一个“裂解电路稳定器”模块，杀死失去裂解效率的突变细菌，并探索如何将裂解电路作为合成电路定向进化的工具。该奖项由生物科学理事会分子和细胞生物科学（MCB）部门的系统和合成生物学（SSB）项目以及工程理事会化学、生物工程、环境和运输系统（CBET）部门的生物技术和生化工程（BBE）项目共同资助。

Rapid advances in the forward engineering of complex genetic circuits in living cells proved that the young discipline of Synthetic Biology has the potential to transform modern biotechnology. A predictive understanding of living systems is a prerequisite for designed manipulation in bioengineering and informed intervention in medicine. Such an understanding requires quantitative measurements, mathematical analysis, and theoretical abstraction. The advent of powerful measurement technologies and computing capacity has positioned biology to drive the next scientific revolution. Synthetic Biology provides a natural platform for the development and testing of models and general network principles using quantitative data from engineered genetic circuits. One of the major fundamental problems of Synthetic Biology is the propensity of synthetic circuits to evolve and obliterate their intended functionality. Thus, it is important to develop a quantitative approach to make genetic networks more robust and stable which also will make them more useful in therapeutic applications. The overall goal of this project to explore the challenges and exploit the opportunities that accompany the inevitable selective pressure that arises in synthetic biology through a combination of cutting-edge experimental and computational tools. Successful accomplishment of this project will lead to significant advances in understanding the interaction between synthetic circuits and host genome in the evolutionary context. This project will provide ample opportunities for cross-disciplinary training of the new generation of quantitative biologists. Furthermore, to broaden the impact of this project beyond academia, its participants will expand a highly successful elementary school science program that that foster collaboration between researchers and teachers at partner elementary schools to improve hands-on science education.To reach the overall goal of the project, the recently developed "synchronized lysis circuit" will be used as the major focus. This synthetic gene circuit functions by triggering death of around 90% of the bacteria at a threshold population density, leaving the remaining 10% of the cells to grow back to threshold and restart the cycle. This generates a distinct phenotype characterized by population cycling that can be monitored across a wide range of length scales using micro- and bench-top chemostats. It also introduces a strong selective pressure that can lead to rapid evolution. The investigators will develop quantitative measurement technology and computational modeling that will lead to a quantitative statistical characterization of the circuit-host evolution process. Sequencing of the circuit and mutated sections of the host genome along the time course of the experiments will be used to reconstruct the evolutionary path the systems have taken. Mathematical modeling will guide experiments and help extract key parameters characterizing evolutionary dynamics of the synchronized lysis circuit. Using the gained knowledge, the researchers will engineer a "lysis circuit stabilizer" module that kills mutant bacteria losing lysis efficiency, and explore ways to use lysis circuit as a tool in directed evolution of synthetic circuits.This award was co-funded by the Systems and Synthetic Biology (SSB) program in the Molecular and Cellular Biosciences (MCB) Division in the Biological Sciences Directorate and the Biotechnology and Biochemical Engineering (BBE) program of the Division of Chemical, Bioengineering, Environmental and Transport Systems (CBET) in the Engineering Directorate.