Department of Chemical Engineering, Institute of Biochemical Engineering, Tsinghua University; Key Laboratory of Industrial Biocatalysis, Ministry of Education, Tsinghua University.
Department of Chemical Engineering, Institute of Biochemical Engineering, Tsinghua University; Key Laboratory of Industrial Biocatalysis, Ministry of Education, Tsinghua University; Luoyang TMAXTREE Biotechnology Co., Ltd.
J Vis Exp. 2022 Feb 18(180). doi: 10.3791/62800.
Conventional microbial cultivation methods usually have cumbersome operations, low throughput, low efficiency, and large consumption of labor and reagents. Moreover, microplate-based high-throughput cultivation methods developed in recent years have poor microbial growth status and experiment parallelization because of their low dissolved oxygen, poor mixture, and severe evaporation and thermal effect. Due to many advantages of micro-droplets, such as small volume, high throughput, and strong controllability, the droplet-based microfluidic technology can overcome these problems, which has been used in many kinds of research of high-throughput microbial cultivation, screening, and evolution. However, most prior studies remain at the stage of laboratory construction and application. Some key issues, such as high operational requirements, high construction difficulty, and lack of automated integration technology, restrict the wide application of droplet microfluidic technology in microbial research. Here, an automated Microbial Microdroplet Culture system (MMC) was successfully developed based on droplet microfluidic technology, achieving the integration of functions such as inoculation, cultivation, online monitoring, sub-cultivation, sorting, and sampling required by the process of microbial droplet cultivation. In this protocol, wild-type Escherichia coli (E. coli) MG1655 and a methanol-essential E. coli strain (MeSV2.2) were taken as examples to introduce how to use the MMC to conduct automated and relatively high-throughput microbial cultivation and adaptive evolution in detail. This method is easy to operate, consumes less labor and reagents, and has high experimental throughput and good data parallelity, which has great advantages compared with conventional cultivation methods. It provides a low-cost, operation-friendly, and result-reliable experimental platform for scientific researchers to conduct related microbial research.
传统的微生物培养方法通常操作繁琐、通量低、效率低，耗费大量的劳动力和试剂。此外，近年来开发的基于微孔板的高通量培养方法由于溶解氧低、混合效果差以及严重的蒸发和热效应，微生物的生长状态和实验的并行化较差。由于微滴具有小体积、高通量和强可控性等诸多优点，基于液滴的微流控技术可以克服这些问题，已被用于高通量微生物培养、筛选和进化的多种研究中。然而，大多数先前的研究仍处于实验室构建和应用阶段。一些关键问题，如操作要求高、构建难度大以及缺乏自动化集成技术，限制了液滴微流控技术在微生物研究中的广泛应用。在这里，我们成功地基于液滴微流控技术开发了一种自动化微生物微滴培养系统（MMC），实现了微生物滴培养过程中所需的接种、培养、在线监测、亚培养、分选和采样等功能的集成。在本方案中，以野生型大肠杆菌（E. coli）MG1655和一株甲醇必需型大肠杆菌菌株（MeSV2.2）为例，详细介绍了如何使用 MMC 进行自动化和相对高通量的微生物培养和适应性进化。该方法操作简单，耗费的劳动力和试剂较少，具有较高的实验通量和良好的数据并行性，与传统培养方法相比具有很大的优势。它为科研人员进行相关的微生物研究提供了一个低成本、易于操作且结果可靠的实验平台。
