Engineering robust, stable, and safe synthetic genetic circuits for smart therapeutics

合成生物学是一个新兴的研究领域，我们使用工程原理来结合不同的生物部分来构建工程生物系统。合成生物学中最活跃的研究领域之一是工程治疗性细菌，这种细菌包含用于检测和预防疾病的基因电路。自然界中的许多细菌都有能力感知环境中的微小变化，并产生具有治疗潜力的生物分子。如果我们能有效地将来自不同细菌的“传感器”(用来检测疾病症状)和“效应器”(用来产生感兴趣的分子)部分结合起来，我们就可以设计出可以作为“智能”疗法的菌株。这种治疗菌株可以留在我们的身体里，自主地检测和治疗病原菌的感染，或者预防癌症或糖尿病等遗传或代谢疾病。具有合成遗传电路的工程细菌作为我们身体的终极护理诊断和治疗的潜力是深远的和令人兴奋的。过去十年的合成生物学研究在扩大这种合成遗传电路的“传感器”和“效应器”模块的遗传部分方面非常成功。然而，当结合到电路中时，性能一直处于次优状态，很难预测。此外，这种电路的功能寿命非常短，因为受损的突变体出现并接管了种群，或者电路本身随着时间的推移而丢失。合成基因电路的糟糕性能和短暂的功能寿命使其难以实现工程菌在“细菌疗法”中的巨大潜力。我们建议建立一条定向进化管道，以设计适合于治疗应用的合成遗传电路。这条流水线包括一种在电路序列中产生变异的新方法，一种用于比较电路功能及其对每个变体的承载单元健康状况(功能寿命的主要决定因素)的影响的变革性试验台，以及一种分离所选变体的新技术，两者都有所改进。该系统的开发基于我们在分析细菌生长、生理和基因表达方面的主要研究成果，包括最近开发的微流体技术，使我们能够在数小时内并行监测10万个细菌细胞，以高帧速率记录每个细胞中的生长和荧光的时间分辨率成像技术，以及使我们能够准确分析这些高通量数据的机器学习算法。这条管道将优化电路，以改善它们的功能和功能寿命，使它们能够在长时间内以强大的方式发挥作用。由于我们需要含有这些电路的治疗性细菌应用于我们的身体，我们需要确保这些电路不会丢失，也不会转移到我们体内的其他细菌细胞。因此，为了设计安全的治疗性细菌，我们将把合成电路集成到细菌的染色体上，在那里它们不会轻易丢失或转移。目前，没有优化集成到染色体上的电路的流水线，也没有同时量化合成电路的功能和功能寿命的平台。这项工作中开发的定向进化管道将是此类管道中的第一个，能够实现合成电路的现场优化，同时提高功能和功能寿命。我们计划利用这一定向进化管道来设计和优化一种“智能”治疗细菌，它带有一个传感器模块，用于检测病原体引起的炎症，并激活抗菌肽的振荡生产，以消除感染病原体。

Synthetic biology is an emerging field of research where we use engineering principles for combining different biological parts to construct engineered biological systems. One of the most active areas of research in synthetic biology is engineering therapeutic bacteria that contain genetic circuits for detection and prevention of diseases. Many bacteria in nature possess the capabilities to sense minute changes in the environment and to produce biomolecules with therapeutic potential. If we can effectively combine the 'sensor' (to detect disease symptoms) and 'effector' (to produce molecules of interest) parts from different bacteria we can engineer strains that can act as 'smart' therapeutics. Such a therapeutic strain could stay in our body and autonomously detect and treat infections from pathogenic bacteria or prevent genetic or metabolic disorders such as cancer or diabetes. The potential for engineered bacteria with synthetic genetic circuits to act as the ultimate point-of-care diagnostic and therapeutic for our body is profound and exciting.The past decade of research in synthetic biology has been very successful in expanding the repertoire of genetic parts for 'sensor' and 'effector' modules of such synthetic genetic circuits. However, when combined into the circuit, the performance has been suboptimal and difficult to predict. Furthermore, the functional lifetime of such circuits has been very short, as impairing mutants appear and take over the population or the circuit itself gets lost over time. The poor performance and short functional lifetime of synthetic genetic circuits have made it difficult to realise the enormous potential of engineered bacteria in 'bacterial therapy'. We are proposing to build a directed evolution pipeline to engineer synthetic genetic circuits that are suitable for therapeutic applications. This pipeline involves a novel method for creating variation in circuit sequence, a transformative testbed for comparing the circuit function and its impact on the health of the carrying cell (the main determinant of functional lifetime) for each variant, and a new technology to isolate the selected variants with improvements in both. This system is developed based on key achievements in our research on analysing growth, physiology, and gene-expression of bacteria, including the recently developed microfluidic technology that enables us to monitor >100k individual bacterial cells in parallel over many hours, time-resolved imaging techniques to record the growth and fluorescence in each cell with a high framerate, and machine-learning algorithms that enable us to accurately analyse this high-throughput data. This pipeline will optimise circuits to improve their function and functional lifetime such that they can act in robust manner over a long period of time.Since we need the therapeutic bacteria containing such circuits to be applied to our body, we need to make sure that the circuits do not get lost and are not transferred to other bacterial cells in our body. Therefore, to engineer safe therapeutic bacteria, we will integrate the synthetic circuit to the chromosome of the bacteria, where they can't be easily lost or transferred. Currently, there is no pipeline for optimising circuits integrated to the chromosome and no platform for simultaneously quantifying function and functional lifetime of a synthetic circuit. The directed evolution pipeline developed in this work will be the first of its kind to enable in situ optimisation of synthetic circuits to simultaneously improve the function and functional lifetime. We plan to use this directed evolution pipeline to engineer and optimise a 'smart' therapeutic bacterium that carries a sensor module for detecting inflammation caused by pathogens and activates oscillatory production of an antimicrobial peptide to eliminate the infecting pathogen.