Multicopy plasmids play an important role in bacterial ecology and evolution by accelerating the rate of adaptation and providing a platform for rapid gene amplification and evolutionary rescue. Despite the relevance of plasmids in bacterial evolutionary dynamics, evaluating the population-level consequences of randomly segregating and replicating plasmids in individual cells remains a challenging problem, both in theory and experimentally. In recent years, technological advances in fluorescence microscopy and microfluidics have allowed studying temporal changes in gene expression by quantifying the fluorescent intensity of individual cells under controlled environmental conditions. In this paper, we will describe the manufacture, experimental setup, and data analysis pipeline of different microfluidic systems that can be used to study plasmid dynamics, both in single-cells and in populations. To illustrate the benefits and limitations of microfluidics to study multicopy plasmid dynamics, we will use an experimental model system consisting on Escherichia coli K12 carrying non-conjugative, multicopy plasmids (19 copies per cell, in average) encoding different fluorescent markers and β-lactam resistance genes. First, we will use an image-based flow cytometer to estimate changes in the allele distribution of a heterogeneous population under different selection regimes. Then we will use a mothermachine microfluidic device to obtain time-series of fluorescent intensity of individual cells to argue that plasmid segregation and replication dynamics are inherently stochastic processes. Finally, using a microchemostat, we track thousands of cells in time to reconstruct bacterial lineages and evaluate the allele frequency distributions that emerge in response to a range of selective pressures.

多拷贝质粒通过加快适应速度并为快速基因扩增和进化拯救提供平台，在细菌生态学和进化中发挥着重要作用。尽管质粒在细菌进化动力学中具有相关性，但评估在单个细胞中随机分离和复制质粒的群体水平后果仍然是一个具有挑战性的问题，无论是在理论上还是在实验上。近年来，荧光显微镜和微流体技术的进步已经允许通过在受控环境条件下量化单个细胞的荧光强度来研究基因表达的时间变化。在本文中，我们将描述制造、实验装置、和不同微流体系统的数据分析管道，可用于研究单细胞和群体中的质粒动力学。为了说明微流体在研究多拷贝质粒动力学方面的优势和局限性，我们将使用一个实验模型系统，该系统由大肠杆菌K12 携带非接合的多拷贝质粒（每个细胞平均 19 个拷贝），编码不同的荧光标记和β-内酰胺抗性基因。首先，我们将使用基于图像的流式细胞仪来估计不同选择机制下异质种群等位基因分布的变化。然后我们将使用一个 Mothermachine 微流体装置来获得单个细胞的荧光强度的时间序列，以论证质粒分离和复制动力学本质上是随机过程。最后，使用微恒化器，我们及时跟踪数千个细胞以重建细菌谱系并评估响应一系列选择压力而出现的等位基因频率分布。