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，我们利用工程原理将不同的生物部分组合起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细菌，其中包含用于检测和预防疾病的遗传电路。自然界中的许多细菌都具有感知环境微小变化的能力，并能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”（用于检测疾病症状）和“效应器”（用于产生感兴趣的分子）部分结合起来，我们就能设计出可以作为“智能”疗法的菌株。这种治疗菌株可以留在我们体内，自主检测和治疗致病菌感染，或预防癌症或糖尿病等遗传或代谢紊乱。具有合成遗传电路的工程细菌作为我们身体的最终诊断和治疗的潜力是深刻而令人兴奋的。过去十年的合成生物学研究已经非常成功地扩展了这种合成遗传电路的“传感器”和“效应器”模块的遗传部分的曲目。然而，当组合到电路中时，性能一直是次优的，难以预测。此外，这种电路的功能寿命非常短，因为有害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。合成遗传电路的性能差和功能寿命短，使得工程细菌在“细菌治疗”中的巨大潜力难以实现。我们建议建立一个定向进化管道来设计适合治疗应用的合成基因电路。该管道涉及一种用于创建电路序列变异的新方法，一种用于比较每种变体的电路功能及其对携带细胞健康（功能寿命的主要决定因素）的影响的变革性测试平台，以及一种用于分离选定变体的新技术。该系统是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而开发的，包括最近开发的微流体技术，使我们能够在多个小时内并行监测100万个细菌细胞，时间分辨成像技术以高帧率记录每个细胞的生长和荧光，以及机器学习算法，使我们能够准确分析这种高通量数据。该管道将优化电路，以改善其功能和功能寿命，使其能够在很长一段时间内以稳健的方式工作。由于我们需要将含有这种回路的治疗性细菌应用于我们的身体，我们需要确保这些回路不会丢失，也不会转移到我们体内的其他细菌细胞中。因此，为了设计出安全的治疗性细菌，我们将把合成电路集成到细菌的染色体上，这样它们就不会轻易丢失或转移。目前，还没有优化集成到染色体上的电路的管道，也没有同时量化合成电路的功能和功能寿命的平台。在这项工作中开发的定向进化管道将是同类中第一个能够对合成电路进行原位优化，同时提高功能和功能寿命的管道。我们计划利用这种定向进化管道来设计和优化一种“智能”治疗细菌，这种细菌携带一个传感器模块，用于检测病原体引起的炎症，并激活一种抗菌肽的振荡生产，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.