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

Summary

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

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

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 microorganisms^1,2,3. 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, low throughput, low efficiency, and large consumption of labor and reagents. The high-throughput cultivation methods developed in recent years are mainly based on the microplate. But the microplate has a low level of dissolved oxygen, poor mixing property, and severe evaporation and thermal effect, which often lead to poor growth status and experiment parallelization of microorganisms^4,5,6,7; on the other hand, it needs to be equipped with expensive equipment, such as liquid-handling workstations and microplate readers, to achieve automated cultivation and process detection^8,9.

As an important branch of microfluidic technology, droplet microfluidics has been developed in recent years based on traditional continuous-flow microfluidic systems. It is a discrete flow microfluidic technology that uses two immiscible liquid phases (usually oil-water) to generate dispersed micro-droplets and operate on them¹⁰. Because micro-droplets have the characteristics of small volume, large specific surface area, high internal mass transfer rate, and no cross-contamination caused by compartmentalization, and the advantages of strong controllability and high throughput of droplets, there have been many kinds of research applying droplet microfluidic technology in high-throughput cultivation, screening, and evolution of microorganisms¹¹. However, there are still a series of key issues to make droplet microfluidic technology popularized and widely applied. Firstly, the operation of droplet microfluidics is cumbersome and intricate, resulting in high technical requirements for operators. Secondly, droplet microfluidic technology combines optical, mechanical, and electrical components and needs to be associated with biotechnology application scenarios. It is difficult for a single laboratory or team to build efficient droplet microfluidic control systems if there is no multi-disciplinary collaboration. Thirdly, on account of the small volume of micro-droplet (from picoliter (pL) to microliter (μL)), it takes much difficulty to realize the precise automated control and real-time online detection of droplets for some basic microbial operations such as sub-cultivation, sorting, and sampling, and it is also difficult to construct an integrated equipment system¹².

In order to address the above problems, an automatic Microbial Microdroplet Culture system (MMC) was successfully developed based on droplet microfluidic technology¹³. The MMC consists of four functional modules: a droplet recognition module, a droplet spectrum detection module, a microfluidic chip module, and a sampling module. Through the system integration and control of all the modules, automated operation system including the generation, cultivation, measurement (optical density (OD) and fluorescence), splitting, fusion, sorting of droplets is accurately established, achieving the integration of functions such as inoculation, cultivation, monitoring, sub-cultivation, sorting and sampling required by the process of microbial droplet cultivation. MMC can hold up to 200 replicate droplet cultivation units of 2-3 µL volume, which is equivalent to 200 shake flask cultivation units. The micro-droplet cultivation system can satisfy the requirements of non-contamination, dissolved oxygen, mixing, and mass-energy exchange during the growth of microorganisms, and meet the various needs of microbial research through multiple integrated functions, for instance, growth curve measurement, adaptive evolution, single factor multi-level analysis, and metabolite research and analysis (based on fluorescence detection)^13,14.

Here, the protocol introduces how to use the MMC to conduct automated and microbial cultivation and adaptive evolution in detail (Figure 1). We took wild-type Escherichia coli (E. coli) MG1655 as an example to demonstrate the growth curve measurement and a methanol-essential E. coli strain MeSV2.2¹⁵ to demonstrate the adaptive evolution in MMC. An operation software for MMC was developed, which makes the operation very simple and clear. In the whole process, the user needs to prepare the initial bacteria solution, set the conditions of the MMC, and then inject the bacteria solution and related reagents into the MMC. Subsequently, the MMC will automatically perform operations such as droplet generation, recognition and numbering, cultivation, and adaptive evolution. It also will perform online detection (OD and fluorescence) of the droplets with high time resolution and display the related data (which can be exported) in the software. The operator can stop the cultivation process at any time according to the results and extract the target droplets for subsequent experiments. The MMC is easy to operate, consumes less labor and reagents, and has relatively high experimental throughput and good data parallelity, which has significant advantages compared with conventional cultivation methods. It provides a low-cost, operation-friendly, and robust experimental platform for researchers to conduct related microbial research.

Protocol

1. Instrument and software installation

1. 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.
2. Install the operation software from the MMC.zip file
   NOTE: Contact the authors for the MMC.zip file.
   1. Create a dedicated folder and save the zip file in it.
   2. Create another dedicated folder as the "Installation Directory". Unzip the MMC.zip and save the files in the new folder.
      NOTE: The computer configuration is best to meet: (1) Windows 7 64-bit operating system or above; (2) CPU: i5 or above; (3) memory: 4 GB or above; (4) hard disk: 300 GB or above (rotational speed greater than 7200 rpm or solid-state disk).

2. Preparations

1. Connect the syringe needle (inner diameter is 0.41 mm and outer diameter is 0.71 mm), quick connector A, and reagent bottle (Figure 2C), and autoclave them at 121 °C for 15 min.
   NOTE: Unscrew the cap of the reagent bottle slightly during sterilization. A few more reagent bottles can be prepared each time for use.
2. Use a 0.22 µm polyvinylidene fluoride (PVDF) filter to filter MMC oil. Put the microfluidic chip (Figure 2B) and MMC oil into the clean bench in advance and sterilize them by ultraviolet irradiation for 30 min before use.
   NOTE: For the details of Quick connector A, reagent bottle, MMC oil and microfluidic chip refer to the Table of Materials.
3. Install the microfluidic chip
   1. Open the door of the operation chamber (Figure 2A) and lift the optical fiber probe.
   2. Align the electric field holes with the electric field needles and gently place the chip on the chip pedestal. Then insert the two positioning columns into the positioning holes and put down the optical fiber probe (Figure 2D).
   3. Connect the quick connector A on the chip to the corresponding port of the MMC according to the position number (C5-O5, C4-O4, C6-O6, C2-O2, CF-OF, C1-O1, C3-O3). Then close the door of the operation chamber.
4. Replenish the MMC oil (to about 80 mL) in the oil bottle and empty the waste liquid in the waste bottle before use.
   NOTE: The waste liquid is usually organic waste. Please refer to regional law and regulation upon disposal, subject to change based on experimental setup.

3. Growth curve measurement in MMC

1. Preparation for initial bacterial solution
   1. Follow the related standard regulations to prepare Luria-Bertani (LB) medium and autoclave at 121°C for 15 min.
      NOTE: Components of LB medium: NaCl (10 g/L), yeast extract (5 g/L) and tryptone (10 g/L).
   2. Take out the E. coli MG1655 strain from glycerol stock and cultivate it in a 50 mL shake flask with 10 mL of LB medium in a shaking incubator (200 rpm) at 37 °C for 5-8 h.
      NOTE: The cultivation time depends on the specific strains. It is optimal to cultivate the strain to the logarithmic period/phase.
   3. Dilute the cultured E. coli MG1655 solution with fresh medium to an OD₆₀₀ of 0.05-0.1 to obtain an initial bacteria solution (prepare about 10 mL).
2. Click on Initialization to initialize the MMC. After the initialization interface appears, set the cultivation temperature as 37 °C and the photoelectric signal value as 0.6 (Figure 3A). Initialization will take about 20 min.
3. Turn on the UV lamp (wavelength 254 nm) during initialization.
4. Inject the initial bacteria solution and MMC oil into the reagent bottle.
   1. Take out a sterilized reagent bottle on the clean bench and tighten the cap.
   2. Use a 10 mL sterile syringe to inject 3-5 mL of MMC oil from the syringe needle of the side tube. Tilt and rotate the reagent bottle slowly to make the oil fully infiltrate the inner wall.
   3. Inject about 5 mL of initial bacteria solution, and then fill the reagent bottle by injecting 5-7 mL of the oil again.
   4. Pull out the independent quick connector A, and insert the quick connector A of the reagent bottle into its quick connector B to complete the sample injection operation (Figure 4A).
5. Wait for the initialization to end and then turn off the UV lamp (wavelength 254 nm).
6. Open the door of the operation chamber, and put the reagent bottle into the metal bath.
7. Pull out the C2 connector of the chip and the quick connector A of the reagent bottle. Connect the side tube connector of the reagent bottle to the C2 connector and the top tube connector to the O2 connector. Then close the door of the operation chamber.
8. Click on Growth Curve to choose the function of growth curve measurement (Figure 3A). In the parameter setting interface, input the Number as 15, turn on the OD detection switch and set the Wavelength as 600 nm. Click on Start to start droplet generation. It will take about 10 min.
   NOTE: Here, Number refers to the number of droplets to be generated. Wavelength refers to the wavelength of the OD to be detected. Set the Number (maximum 200) and Wavelength (350-800 nm) according to the experiment requirements.
9. When a pop-up window appears on the main interface prompting "Remove the reagent bottle between C2 and O2, then please click the OK button after completion", open the door of the operation chamber to take out the reagent bottle and connect the C2 and O2 connectors.
10. Close the door, and click the OK button in the pop-up window to automatically cultivate the droplets and detect the OD values.
    NOTE: The MMC detects the OD value when the droplet passes the optical fiber probe. Therefore, the detection period depends on the number of droplets generated.
11. When the growth curve reaches the stationary phase, click the Data Export button to export the OD data. Select the data save path and export the OD value recorded during the cultivation period in the .csv format, which can be opened by appropriate software (e.g., Microsoft Excel). Then use a mapping software (e.g., EXCEL and Origin 9.0) to plot the growth curve.
    NOTE: During the cultivation process, it is feasible to click on the Data Export at any time to export the OD data of all the current droplets.

4. Adaptive evolution in MMC

1. Preparation for initial bacterial solution
   1. Follow the related standard regulations to prepare the special liquid medium and solid plates for the MeSV2.2 and autoclave at 121 °C for 15 min.
      NOTE: For the components of the special medium refer to Table 1 and the Table of Materials.
   2. Cultivate the MeSV2.2 using the solid plate (diameter = 90 mm) in a 37 °C constant temperature incubator for 72 h. Then pick an independent colony and cultivate it in a 50 mL shake flask with 10 mL of the special liquid medium in a shaking incubator (200 rpm) at 37 °C for 72 h.
   3. Dilute the cultured MeSV2.2 solution with the medium to an OD₆₀₀ of 0.1-0.2 (ensure that the total volume is not less than 10 mL) and continue cultivating it in the shake flask for 5 h to obtain the initial bacteria solution.
      NOTE: The MeSV2.2 is a methanol-essential E. coli strain. The special liquid medium contains 500 mmol/L methanol, which is a strong stress for MeSV2.2, resulting in very slow growth. Note that obtaining the initial bacteria solution here is different from that described in step 3.1.
2. Initialize the MMC as explained in steps 3.2, 3.3, and 3.5.
3. Take out two sterilized reagent bottles, one of which is for the initial bacteria solution and the other is for the fresh medium. Inject the initial bacteria solution (5 mL), fresh medium (12-15 mL), and MMC oil into the reagent bottles as explained in step 3.4.
   NOTE: As adaptive evolution is a long-term process involving multiple sub-cultivations, store as much fresh medium as possible in MMC. The medium cannot be replenished during the experiment running.
4. Install the two reagent bottles into MMC as explained in step 3.6. Install the one for the initial bacteria solution between the C2 and O2 connector and the other for the fresh medium between the C4 and O4 connector.
5. Click on ALE to choose the function of adaptive evolution (Figure 3B). In the parameter setting interface, turn on the OD Detection switch.
6. Set the Number as 50, Wavelength as 600 nm, Concentration as 0%, Type as Time, Parameter as 30 h, and Repetitions as 99. Click on Start to start droplet generation. It will take about 25 min.
   NOTE: Here, "Concentration" refers to the maximum concentration of chemical factors for adaptive evolution. For different droplets, it is realizable in MMC to introduce different concentrations of chemical factors to provide different growth conditions. Calculate the introduced concentrations using the following equation:
   
   Here "C" refers to the concentration of chemical factors introduced into droplets; "a" refers to the concentration of chemical factors in the reagent bottles between the C4 and O4 connector; "b" refers to the concentration of chemical factors in the reagent bottles between the C6 and O6 connector; and "i" refers to the available concentration. There are eight concentrations available in MMC. Since the chemical factor here has a single concentration (500 mmol/L methanol) and it is one of the ingredients of the medium, only one reagent bottle containing the chemical factor is installed here, and the Concentration is set as 0%. Type refers to the mode of sub-cultivation, which is divided into three types: time mode, OD value mode, and fluorescence mode. The former means to cultivate the droplets for a fixed time and then sub-cultivate, while the latter two means to cultivate the droplets to pre-defined OD value/fluorescence intensity and then sub-cultivate. Parameter refers to the related parameter required when choosing a mode of sub-cultivation. Repetitions refers to the number of sub-cultivations.
7. Remove the reagent bottle placed between the C2 and O2 connector as explained in step 3.8.
8. Observe whether the maximum OD values of the droplets during each sub-cultivation period have increased significantly. If the increase occurs and meets the experiment requirements, click on the Data Export button to export the OD data as explained in step 3.9.
   NOTE: Here, the sub-cultivation period depends on the Parameter. For example, when setting Type as Time and Parameter as 30 h, the sub-cultivation period is 30 h. During each sub-cultivation period, there are the maximum OD values of the droplets. Estimate whether the adaptive evolution meets the experiment requirements by the increase of maximum OD values (The increase depends on the actual cultivation process of the strain, for example, increased by more than 20%).
   CAUTION: Pay attention to whether the stored fresh medium is exhausted. If the significant increase has not occurred even after the medium is exhausted, extract the better-growing droplets and carry out a new round of adaptive evolution.
9. Extract the target droplets from the MMC.
   1. Click on the Screening button to choose the function of droplet extraction (Figure 3C). Choose the Collect option, click on the numbers of target droplets, and then click on OK.
      NOTE: Droplet screening includes "Collect", "Discard" and "Extract seed solution". "Extract seed solution" means to collect the remaining droplets¹³ after the sub-cultivation operation.
   2. Wait for the pop-up window to prompt, "Please pull out the CF quick connector and put it into the EP tube". Put the CF quick connector into the microcentrifuge tube for collection according to the software prompt and then click on OK (Figure 4D).
   3. After 1-2 min, the software interface will pop up a new window prompting, "Please insert the connector back and click OK if finished". Then, insert the CF quick connector back and click on OK to make MMC continue to run (Figure 4D). When the next target droplet reaches the droplet recognition site, repeat 4.9.2-4.9.3 to collect it.
      NOTE: After all the target droplets are collected, the MMC will continue cultivating the remaining droplets. If the cultivation is not necessary, click on Stop to directly terminate the operation.
   4. Extract the droplet using a 2.5 µL pipette, drop it on the 90 mm agarose plate, and spread it evenly with a triangular glass spreading rod with a side length of 3 cm. Then cultivate it in a 37 °C constant temperature incubator for 72 h.
   5. Pick 3-5 independent colonies and separately cultivate them in the 50 mL shake flasks with 10 mL of fresh medium in a shaking incubator (200 rpm) at 37 °C for 48-72 h. Follow the related standard regulations to store the cultured bacteria solution in the glycerol tube for subsequent experiments.

5. Clean of the MMC

1. After completion of the experiment, click on Stop to stop all the operations. Then click on Clean to clean the chip and tubes. It will take about 15 min.

Results

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...

Discussion

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. ...

Materials

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.