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Bioingegneria

使用微生物滴培养系统（MMC）的自动微生物培养和适应性进化

Published: February 18, 2022
doi:
10.3791/62800
, , , , , ,
1Department of Chemical Engineering, Institute of Biochemical Engineering,Tsinghua University, 2Key Laboratory of Industrial Biocatalysis, Ministry of Education,Tsinghua University, 3Luoyang TMAXTREE Biotechnology Co., Ltd., 4Biochemical Engineering Research Group, School of Chemical Engineering and Technology,Xi’an Jiaotong University, 5Center for Synthetic & Systems Biology,Tsinghua University, 6School of Life Science and Technology,Tokyo Institute of Technology

Summary

该协议描述了如何使用微生物滴培养系统（MMC）进行自动微生物培养和适应性进化。MMC可以自动连续地培养和分培养微生物，并在线监测其生长，具有相对较高的通量和良好的并行化性，减少了劳动力和试剂消耗。

Abstract

传统的微生物培养方法通常操作繁琐，通量低，效率低，消耗大量劳动力和试剂。此外，近年来开发的基于微孔板的高通量培养方法由于其溶解氧低，混合物不良以及严重的蒸发和热效应，微生物生长状况较差，实验平行化程度低。由于微液滴的诸多优点，如体积小、通量高、可控性强等，液滴基微流控技术可以克服这些问题，已应用于多种高通量微生物培养、筛选、进化的研究。然而，大多数先前的研究仍处于实验室建设和应用阶段。一些关键问题，如操作要求高，施工难度高，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，基于液滴微流技术成功开发了一种自动化微生物微滴培养系统（MMC），实现了微生物液滴培养过程中所需的接种、培养、在线监测、亚培养、分选、取样等功能的集成。以野生型大肠杆菌MG1655和甲醇必需大肠杆菌菌株（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。该方法操作简便，消耗的人工和试剂少，实验通量高，数据并行度好，与常规培养方法相比具有很大的优势。它为科研人员开展相关微生物研究提供了一个低成本、操作友好、结果可靠的实验平台。

Introduction

微生物培养是微生物科学研究和工业应用的重要基础，广泛应用于微生物的分离、鉴定、重建、筛选和进化1，2，3。传统的微生物培养方法主要采用试管、摇瓶、实心板作为培养容器，结合振荡培养箱、分光光度计、酶标仪等设备进行微生物培养、检测和筛选。但是，这些方法存在许多问题，例如操作繁琐，通量低，效率低，消耗大量劳动力和试剂。近年来开发的高通量培养方法主要基于微孔板。但微孔板溶解氧水平低，混合性能差，蒸发和热效应严重，往往导致微生物生长状态和实验平行化不良4，5，6，7;另一方面，它需要配备昂贵的设备，如液体处理工作站和酶标仪，以实现自动化培养和过程检测8，9。

作为微流控技术的一个重要分支，液滴微流体近年来在传统的连续流微流体系统的基础上发展起来。它是一种离散流动微流体技术，它使用两个不混溶的液相（通常是油 – 水）来产生分散的微液滴并在其上运行10。由于微液滴具有体积小、比表面积大、内部传质率高、区室化不造成交叉污染等特点，以及液滴可控性强、通量高的优点，因此应用液滴微流控技术应用于微生物高通量培养、筛选和进化等已有多种研究11.然而，仍有一系列关键问题需要使液滴微流控技术普及和广泛应用。首先，液滴微流体的操作繁琐复杂，对操作人员的技术要求很高。其次，液滴微流控技术结合了光学、机械和电气元件，需要与生物技术应用场景相关联。如果没有多学科协作，单个实验室或团队就很难建立高效的液滴微流体控制系统。第三，由于微液滴体积小（从皮升（pL）到微升（μL）），对于一些基本的微生物操作，如亚培养、分选、取样，实现液滴的精确自动化控制和实时在线检测需要很大的难度，并且难以构建一体化设备系统12。

为了解决上述问题，基于液滴微流控技术成功开发了一种全自动微生物微滴培养系统（MMC）。MMC由四个功能模块组成：液滴识别模块、液滴光谱检测模块、微流控芯片模块和采样模块。通过所有模块的系统集成和控制，准确建立包括液滴生成、培养、测量（光密度（OD）和荧光）、分裂、融合、分选在内的自动化操作系统，实现微生物液滴培养过程中所需的接种、培养、监测、分培养、分选、采样等功能的集成。MMC可容纳多达200个2-3μL体积的重复液滴培养单元，相当于200个摇瓶培养单元。微滴培养系统可以满足微生物生长过程中无污染、溶解氧、混合、质能交换的要求，通过生长曲线测量、自适应进化、单因子多级分析、代谢物研究与分析（基于荧光检测）等多种集成功能，满足微生物研究的各种需求13，14。

在这里，该协议详细介绍了如何使用MMC进行自动化和微生物培养以及适应性进化（图1）。以野生型大肠杆菌MG1655为例，以生长曲线测量和甲醇必需的大肠杆菌菌株MesV2.215为例，证明了MMC的适应性进化。开发了MMC的操作软件，使操作非常简单明了。在整个过程中，用户需要准备初始细菌溶液，设置MMC的条件，然后将细菌溶液和相关试剂注入MMC。随后，MMC将自动执行液滴生成，识别和编号，培养和自适应进化等操作。它还将以高时间分辨率对液滴进行在线检测（OD和荧光），并在软件中显示相关数据（可以导出）。操作员可以根据结果随时停止培养过程，并提取目标液滴以进行后续实验。MMC操作简便，消耗的劳动力和试剂少，实验通量相对较高，数据并行度好，与常规培养方法相比具有显著优势。它为研究人员进行相关的微生物研究提供了一个低成本，操作友好且强大的实验平台。

Protocol

1. 仪器和软件安装 选择清洁无菌环境（如洁净工作台）作为MMC的专用永久空间。在空间中稳定地安装MMC。注意：使MMC远离强电场，磁场和强热辐射源的干扰。避免剧烈振动影响光学检测元件。向MMC提供AC220 V，50 HZ的电源。有关 MMC 的详细信息，请参阅 材料表 和 MMC16 的网站。 从 MMC.zip 文件安装操作软件注意：请与作者联系以获取 MMC…

Representative Results

该协议使用 大肠杆菌 MG1655和MesV2.2菌株作为示例，以展示MMC中具有自动化和相对高通量策略的微生物培养和甲醇必需的适应性进化。生长曲线测量主要用于表征微生物培养。通过自动连续亚培养和在每个亚培养过程中加入高浓度甲醇作为选择压力进行适应性进化。通过每个次培养期间液滴最大OD值的变化趋势来估计是否实现了适应性进化。MMC的可调参数和精度参数如 表 2<…

Discussion

该协议介绍了如何使用微生物滴培养系统（MMC）进行自动微生物培养和长期适应性进化。MMC是一种小型化，自动化和高通量微生物培养系统。与传统的微生物高通量培养方法和仪器相比，MMC具有劳动力和试剂消耗少、操作简单、在线检测（OD和荧光）、高时间分辨率数据采集、超并行化等诸多优点。MMC还具有一些与常规液滴微流体技术不同的特殊优势，后者通常使用pL和nL液滴。以前报道的大多数?…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

本研究得到了国家重点研发计划（2018YFA0901500）、国家自然科学基金国家重点仪器设备专项（21627812）和清华大学自主研发计划（20161080108）的支持。我们还感谢Julia A. Vorholt教授（苏黎世联邦理工学院生物学系微生物研究所，苏黎世联邦理工学院，苏黎世8093，瑞士）提供甲醇必需 的大肠杆菌 菌株2.2版（MeSV2.2）。

Materials

0.22 μm PVDF filter membrane Merck Millipore Ltd. SLGPR33RB Sterilize the MMC oil
4 °C refrigerator Haier BCD-289BSW For reagent storage
Agar Becton, Dickinson and Company 214010 For solid plate preparation
CaCl2·2H2O Sinopharm Chemical Reagent Beijing Co., Ltd. 20011160 Component of the special medium for MeSV2.2.
Clean bench Beijing Donglian Har Instrument Manufacture Co., Ltd. DL-CJ-INDII For aseptic operation and UV sterilization
CoCl2·6H2O Sinopharm Chemical Reagent Beijing Co., Ltd. 10007216 Component of the special medium for MeSV2.2.
Computer Lenovo E450 Software installation and MMC control
Constant temperature incubator Shanghai qixin scientific instrument co., LTD LRH 250 For the microbial cultivation using solid medium
CuSO4·5H2O Sinopharm Chemical Reagent Beijing Co., Ltd. 10008218 Component of the special medium for MeSV2.2.
Electronic balance OHAUS AR 3130 For reagent weighing
EP tube Thermo Fisher 1.5 mL For droplet collection
FeCl3·6H2O Sinopharm Chemical Reagent Beijing Co., Ltd. 10011928 Component of the special medium for MeSV2.2.
Freezing Tube Thermo Fisher 2.0 mL For strain preservation
Gluconate Sigma-Aldrich S2054 Component of the special medium for MeSV2.2.
Glycerol GENERAL-REAGENT G66258A For strain preservation
High-Pressure Steam Sterilization Pot SANYO Electric MLS3020 For autoclaved sterilization
isopropyl-β-d-thiogalactopyranoside (IPTG) Biotopped 420322 Component of the special medium for MeSV2.2.
Kanamycin sulfate Solarbio K8020 Component of the special medium for MeSV2.2.
KH2PO4 MACKLIN P815661 Component of the special medium for MeSV2.2.
Methanol MACKLIN M813895 Component of the special medium for MeSV2.2.
MgSO4·7H2O BIOBYING 1305715 Component of the special medium for MeSV2.2.
Microbial Microdroplet Culture System (MMC) Luoyang TMAXTREE Biotechnology Co., Ltd.  MMC-I Performing growth curve determination and adaptive evolution. Please refer to http://www.tmaxtree.com/en/index.php?v=news&id=110
Microfluidic chip Luoyang TMAXTREE Biotechnology Co., Ltd. MMC-ALE-OD For various droplet operations. Please refer to http://www.tmaxtree.com/en/
MMC oil Luoyang TMAXTREE Biotechnology Co., Ltd. MMC-M/S-OD The oil phase for droplet microfluidics. Please refer to http://www.tmaxtree.com/en/
MnCl2 Sinopharm Chemical Reagent Beijing Co., Ltd. 20026118 Component of the special medium for MeSV2.2.
NaCl GENERAL-REAGENT G81793J Component of the LB medium
Na2HPO4·12H2O GENERAL-REAGENT G10267B Component of the special medium for MeSV2.2.
NH4Cl Sinopharm Chemical Reagent Beijing Co., Ltd. 10001518 Component of the special medium for MeSV2.2.
Petri dish Corning Incorporated 90 mm For the preparation of solid medium
Pipette eppendorf 2.5 μL, 10 μL, 100μL, 1000μL For liquid handling
Quick connector A Luoyang TMAXTREE Biotechnology Co., Ltd. — For the connection of each joint. Please refer to http://www.tmaxtree.com/en/
Reagent bottle Luoyang TMAXTREE Biotechnology Co., Ltd. MMC-PCB Sampling and storage of bacteria solution and reagents. Please refer to http://www.tmaxtree.com/en/
Shake flask Union-Biotech 50 mL For microbial cultivation
Shaking incubator Shanghai Sukun Industrial Co., Ltd. SKY-210 2B For the microbial cultivation in shake flask
Streptomycin sulfate Solarbio S8290 Component of the special medium for MeSV2.2.
Syringe JIANGSU ZHIYU MEDICAL INSTRUCTMENT CO., LTD 10 mL Draw liquid and inject it into the reagent bottle
Syringe needle OUBEL Hardware Store 22G Inner diameter is 0.41 mm and outer diameter is 0.71 mm.
Tryptone Oxoid Ltd. LP0042 Component of the LB medium
Ultra low temperature refrigerator SANYO Ultra-low MDF-U4086S For strain preservation (-80 °C)
UV–Vis spectrophotometer General Electric Company Ultrospec 3100 pro For the measurement of OD values
Vitamin B1 Solarbio SV8080 Component of the special medium for MeSV2.2.
Yeast extract Oxoid Ltd. LP0021 Component of the LB medium
ZnSO4·7H2O Sinopharm Chemical Reagent Beijing Co., Ltd. 10024018 Component of the special medium for MeSV2.2.
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Tags

Keywords: Automated Microbial CultivationAdaptive EvolutionMicrobial Microdroplet Culture System (MMC)Low-costOperation-friendlyReliable ResultsSimple OperationsMicrobiology ResearchGrowth Curve MeasurementAdaptive Laboratory EvolutionSingle Factor Multi-level AnalysisMicrofluidic ChipEscherichia Coli MG1655Cultivation TemperaturePhotoelectric SignalMMC OilInitial Bacteria Solution
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Jian, X., Guo, X., Wang, J., Tan, Z. L., Xing, X., Wang, L., Zhang, C. Automated Microbial Cultivation and Adaptive Evolution using Microbial Microdroplet Culture System (MMC). J. Vis. Exp. (180), e62800, doi:10.3791/62800 (2022).
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