Automated Microbial Cultivation and Adaptive Evolution using Microbial Microdroplet Culture System (MMC)

Xingjin Jian; Xiaojie Guo; Jia Wang; Zheng Lin Tan; Xin-hui Xing; Liyan Wang; Chong Zhang

doi:10.3791/62800

Method Article

Automated Microbial Cultivation and Adaptive Evolution using Microbial Microdroplet Culture System (MMC)

DOI:

10.3791/62800

⸱

February 18th, 2022

Xingjin Jian*¹^,² , Xiaojie Guo*¹^,²^,³ , Jia Wang⁴ , Zheng Lin Tan¹^,²^,⁶ , Xin-hui Xing¹^,²^,⁵ , Liyan Wang³ , Chong Zhang¹^,²^,⁵

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

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This protocol describes how to use the Microbial Microdroplet Culture system (MMC) to conduct automated microbial cultivation and adaptive evolution. MMC can cultivate and sub-cultivate microorganisms automatically and continuously and monitor online their growth with relatively high throughput and good parallelization, reducing labor and reagent consumption.

Abstract

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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.

Introduction

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Microbial cultivation is an important foundation for microbiological scientific research and industrial applications, which is widely used in the isolation, identification, reconstruction, screening, and evolution of microorganisms¹^,²^,³. Conventional microbial cultivation methods mainly use test tubes, shake flasks, and solid plates as cultivation containers, combined with shaking incubators, spectrophotometers, microplate readers, and other equipment for microbial cultivation, detection, and screening. However, these methods have many problems, such as cumbersome operations,....

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Protocol

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1. Instrument and software installation

Choose a clean and sterile environment (such as a clean bench) as a dedicated permanent space for MMC. Install the MMC steadily in the space.
NOTE: Keep the MMC away from the interference of strong electric fields, magnetic fields, and strong heat radiation sources. Avoid severe vibration from affecting the optical detection components. Provide the power supply of AC220 V, 50 HZ to the MMC. For details on MMC refer to the Table of Materials and the website for MMC¹⁶.
Install the operation software from the MMC.zip file
NOTE: Contact the aut....

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Results

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This protocol uses E. coli MG1655 and a MeSV2.2 strain as examples to demonstrate the microbial cultivation and methanol-essential adaptive evolution with an automated and relatively high-throughputstrategy in MMC. The growth curve measurement was mainly used to characterize microbial cultivation. The adaptive evolution was conducted by automated continuous sub-cultivation and adding a high concentration of methanol as the selective pressure during each sub-cultivation. Whether adaptive evolution had been realiz.......

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Discussion

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This protocol presents how to use the Microbial Microdroplet Culture system (MMC) to perform automated microbial cultivation and long-term adaptive evolution. MMC is a miniaturized, automated, and high-throughput microbial cultivation system. Compared with conventional microbial high-throughput cultivation methods and instruments, MMC has many advantages such as low labor and reagent consumption, simple operation, online detection (OD and fluorescence), high-time-resolution data collection, and superior parallelization. .......

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Disclosures

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The authors have nothing to disclose.

Acknowledgements

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This study was supported by the National Key Research and Development Program of China (2018YFA0901500), the National Key Scientific Instrument and Equipment Project of the National Natural Science Foundation of China (21627812), and the Tsinghua University Initiative Scientific Research Program (20161080108). We also thank Prof. Julia A. Vorholt (Institute of Microbiology, Department of Biology, ETH Zurich, Zurich 8093, Switzerland) for the provision of the methanol-essential E. coli strain version 2.2 (MeSV2.2).

....

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Materials

List of materials used in this article
Name	Company	Catalog Number	Comments
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.

References

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Lewis, W. H., et al. Innovations to culturing the uncultured microbial majority. Nature Reviews Microbiology. 19 (4), 225-240 (2020).
Feist, A. M., Herrgard, M. J., Thiele, I., Reed, J. L., Palsson, B. O. Reconstruction of biochemical networks in microorganisms.

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Automated Microbial Cultivation and Adaptive Evolution using Microbial Microdroplet Culture System (MMC)

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Summary

Abstract

Introduction

Protocol

Results

Discussion

Disclosures

Acknowledgements

Materials

References

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