Name: automated microbial cultivation and adaptive evolution using microbial microdroplet culture system (mmc)
Uploaded: 2022-02-18T12:30:00
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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).

1. Instrument and software installation
<ol>
	<li>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 Materia...

<ol>
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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet-based+high-throughput+cultivation+for+accurate+screening+of+antibiotic+resistant+gut+microbes.">Droplet-based high-throughput cultivation for accurate screening of antibiotic resistant gut microbes.</a> eLife. 9, 56998 (2020).</li><li>Baret, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surfactants+in+droplet-based+microfluidics.">Surfactants in droplet-based microfluidics.</a> Lab on a Chip. 12 (3), 422-433 (2012).</li><li>Nitschke, M., Pastore, G. 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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. ...

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, 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 microorganisms4,5,6,7; 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 detection8,9.
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 (&#956;L)), 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 &#181;L 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&#160;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#956;m PVDF filter membrane</td>
			<td>Merck Millipore Ltd.</td>
			<td>SLGPR33RB</td>
			<td>Sterilize the MMC oil</td>
		</tr>
		<tr>
			<td>4 &#176;C refrigerator</td>
			<td>Haier</td>
			<td>BCD-289BSW</td>
			<td>For reagent storage</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Becton, Dickinson and Company</td>
			<td>214010</td>
			<td>For solid plate preparation</td>
		</tr>
		<tr>
			<td>CaCl2&#183;2H2O</td>
			<td>Sinopharm Chemical Reagent Beijing Co., Ltd.</td>
			<td>20011160</td>
			<td>Component of the special medium for MeSV2.2.</td>
		</tr>
		<tr>
			<td>Clean bench</td>
			<td>Beijing Donglian Har Instrument Manufacture Co., Ltd.</td>
			<td>DL-CJ-INDII</td>
			<td>For aseptic operation and UV sterilization</td>
		</tr>
		<tr>
			<td>CoCl2&#183;6H2O</td>
			<td>Sinopharm Chemical Reagent Beijing Co., Ltd.</td>
			<td>10007216</td>
			<td>Component of the special medium for MeSV2.2.</td>
		</tr>
		<tr>
			<td>Computer</td>
			<td>Lenovo</td>
			<td>E450</td>
			<td>Software installation and MMC control</td>
		</tr>
		<tr>
			<td>Constant temperature incubator</td>
			<td>Shanghai qixin scientific instrument co., LTD</td>
			<td>LRH 250</td>
			<td>For the microbial cultivation using solid medium</td>
		</tr>
		<tr>
			<td>CuSO4&#183;5H2O</td>
			<td>Sinopharm Chemical Reagent Beijing Co., Ltd.</td>
			<td>10008218</td>
			<td>Component of the special medium for MeSV2.2.</td>
		</tr>
		<tr>
			<td>Electronic balance</td>
			<td>OHAUS</td>
			<td>AR 3130</td>
			<td>For reagent weighing</td>
		</tr>
		<tr>
			<td>EP tube</td>
			<td>Thermo Fisher</td>
			<td>1.5 mL</td>
			<td>For droplet collection</td>
		</tr>
		<tr>
			<td>FeCl3&#183;6H2O</td>
			<td>Sinopharm Chemical Reagent Beijing Co., Ltd.</td>
			<td>10011928</td>
			<td>Component of the special medium for MeSV2.2.</td>
		</tr>
		<tr>
			<td>Freezing Tube</td>
			<td>Thermo Fisher</td>
			<td>2.0 mL</td>
			<td>For strain preservation</td>
		</tr>
		<tr>
			<td>Gluconate</td>
			<td>Sigma-Aldrich</td>
			<td>S2054</td>
			<td>Component of the special medium for MeSV2.2.</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>GENERAL-REAGENT</td>
			<td>G66258A</td>
			<td>For strain preservation</td>
		</tr>
		<tr>
			<td>High-Pressure Steam Sterilization Pot</td>
			<td>SANYO Electric</td>
			<td>MLS3020</td>
			<td>For autoclaved sterilization</td>
		</tr>
		<tr>
			<td>isopropyl-&#946;-d-thiogalactopyranoside (IPTG)</td>
			<td>Biotopped</td>
			<td>420322</td>
			<td>Component of the special medium for MeSV2.2.</td>
		</tr>
		<tr>
			<td>Kanamycin sulfate</td>
			<td>Solarbio</td>
			<td>K8020</td>
			<td>Component of the special medium for MeSV2.2.</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>MACKLIN</td>
			<td>P815661</td>
			<td>Component of the special medium for MeSV2.2.</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>MACKLIN</td>
			<td>M813895</td>
			<td>Component of the special medium for MeSV2.2.</td>
		</tr>
		<tr>
			<td>MgSO4&#183;7H2O</td>
			<td>BIOBYING</td>
			<td>1305715</td>
			<td>Component of the special medium for MeSV2.2.</td>
		</tr>
		<tr>
			<td>Microbial Microdroplet Culture System (MMC)</td>
			<td>Luoyang TMAXTREE Biotechnology Co., Ltd.</td>
			<td>&#160;MMC-I</td>
			<td>Performing growth curve determination and adaptive evolution. Please refer to http://www.tmaxtree.com/en/index.php?v=news&#38;id=110</td>
		</tr>
		<tr>
			<td>Microfluidic chip</td>
			<td>Luoyang TMAXTREE Biotechnology Co., Ltd.</td>
			<td>MMC-ALE-OD</td>
			<td>For various droplet operations. Please refer to http://www.tmaxtree.com/en/</td>
		</tr>
		<tr>
			<td>MMC oil</td>
			<td>Luoyang TMAXTREE Biotechnology Co., Ltd.</td>
			<td>MMC-M/S-OD</td>
			<td>The oil phase for droplet microfluidics. Please refer to http://www.tmaxtree.com/en/</td>
		</tr>
		<tr>
			<td>MnCl2</td>
			<td>Sinopharm Chemical Reagent Beijing Co., Ltd.</td>
			<td>20026118</td>
			<td>Component of the special medium for MeSV2.2.</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>GENERAL-REAGENT</td>
			<td>G81793J</td>
			<td>Component of the LB medium</td>
		</tr>
		<tr>
			<td>Na2HPO4&#183;12H2O</td>
			<td>GENERAL-REAGENT</td>
			<td>G10267B</td>
			<td>Component of the special medium for MeSV2.2.</td>
		</tr>
		<tr>
			<td>NH4Cl</td>
			<td>Sinopharm Chemical Reagent Beijing Co., Ltd.</td>
			<td>10001518</td>
			<td>Component of the special medium for MeSV2.2.</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Corning Incorporated</td>
			<td>90 mm</td>
			<td>For the preparation of solid medium</td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td>eppendorf</td>
			<td>2.5 &#956;L, 10 &#956;L, 100&#956;L, 1000&#956;L</td>
			<td>For liquid handling</td>
		</tr>
		<tr>
			<td>Quick connector A</td>
			<td>Luoyang TMAXTREE Biotechnology Co., Ltd.</td>
			<td>&#8212;</td>
			<td>For the connection of each joint. Please refer to http://www.tmaxtree.com/en/</td>
		</tr>
		<tr>
			<td>Reagent bottle</td>
			<td>Luoyang TMAXTREE Biotechnology Co., Ltd.</td>
			<td>MMC-PCB</td>
			<td>Sampling and storage of bacteria solution and reagents. Please refer to http://www.tmaxtree.com/en/</td>
		</tr>
		<tr>
			<td>Shake flask</td>
			<td>Union-Biotech</td>
			<td>50 mL</td>
			<td>For microbial cultivation</td>
		</tr>
		<tr>
			<td>Shaking incubator</td>
			<td>Shanghai Sukun Industrial Co., Ltd.</td>
			<td>SKY-210 2B</td>
			<td>For the microbial cultivation in shake flask</td>
		</tr>
		<tr>
			<td>Streptomycin sulfate</td>
			<td>Solarbio</td>
			<td>S8290</td>
			<td>Component of the special medium for MeSV2.2.</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>JIANGSU ZHIYU MEDICAL INSTRUCTMENT CO., LTD</td>
			<td>10 mL</td>
			<td>Draw liquid and inject it into the reagent bottle</td>
		</tr>
		<tr>
			<td>Syringe needle</td>
			<td>OUBEL Hardware Store</td>
			<td>22G</td>
			<td>Inner diameter is 0.41 mm and outer diameter is 0.71 mm.</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Oxoid Ltd.</td>
			<td>LP0042</td>
			<td>Component of the LB medium</td>
		</tr>
		<tr>
			<td>Ultra low temperature refrigerator</td>
			<td>SANYO Ultra-low</td>
			<td>MDF-U4086S</td>
			<td>For strain preservation (-80 &#176;C)</td>
		</tr>
		<tr>
			<td>UV&#8211;Vis spectrophotometer</td>
			<td>General Electric Company</td>
			<td>Ultrospec 3100 pro</td>
			<td>For the measurement of OD values</td>
		</tr>
		<tr>
			<td>Vitamin B1</td>
			<td>Solarbio</td>
			<td>SV8080</td>
			<td>Component of the special medium for MeSV2.2.</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Oxoid Ltd.</td>
			<td>LP0021</td>
			<td>Component of the LB medium</td>
		</tr>
		<tr>
			<td>ZnSO4&#183;7H2O</td>
			<td>Sinopharm Chemical Reagent Beijing Co., Ltd.</td>
			<td>10024018</td>
			<td>Component of the special medium for MeSV2.2.</td>
		</tr>
	</tbody>
</table>

automated microbial cultivation and adaptive evolution using microbial microdroplet culture system (mmc)

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&#160;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.

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

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.

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

Conventional microbial cultivation methods usually have cumbersome operations, low throughput, low efficiency, and large consumption of labor and ...

This method can help solve the problems with the conventional microbial cultivation methods. It provides a low-cost, operation-friendly and result-reliable experimental platform for automated microbial cultivation and adaptive evolution. This technology automates operations and greater reduces labor consumption.And at the same time, it has good culture performance with reliable results and simple operations. The technology is mainly used in the microbiology research fields, such as growth curve measurement, adaptive laboratory evolution, and a single factor multi-level analysis. Start the preparations of the experiment by connecting the syringe needle with an inner diameter of 0.41 millimeters and an outer diameter of 0.71 millimeters to a quick connector A and reagent bottle.Then autoclave all the equipment at 121 degree Celsius for 15 minutes. To install the microfluidic chip, open the door of the operation chamber and lift the optical fiber probe. After aligning the electrical field holes with the needles, 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.Next, connect the quick connector A on the chip to the corresponding port of the microbial microdroplet culture system or MMC according to the position number as described in the manuscript. When done, close the door of the operation chamber. Dilute the cultured Escherichia coli MG1655 suspension with the medium to an OD600 of 0.05 to 0.1 to obtain 10 milliliters of initial bacteria solution.To initialize the MMC, click on the Initialization tab. When the initialization interface appears, set the cultivation temperature as 37 degrees Celsius and the photoelectric signal value as 0.6. Initialization will take about 20 minutes.Later, place a sterilized reagent bottle on the clean bench and tighten the cap. Use a 10 milliliters sterile syringe to inject three to five milliliters of MMC oil from the syringe needle to the side tube into the reagent bottle. Then tilt and rotate the reagent bottle slowly to make the oil fully infiltrate the inner wall.After injecting five milliliters of an initial bacteria solution into the bottle, fill the reagent bottle by injecting five to seven milliliters of the MMC oil. Pull out the independent quick connector A of the reagent bottle and insert the quick connector A into the bottle's quick connector B to complete the sample injection operation. Once done, open the door of the operation chamber to put the reagent bottle into the metal bath.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.To choose the function of growth curve measurement, click on Growth Curve in the parameter setting interface input the number as 15. Then turn on the OD detection switch and set the wavelength as 600 nanometers. Click on the Start tab to start droplet generation.The process will take 15 minutes to complete. When a popup window appears on the main interface prompting a message, open the door of the operation chamber to take out the reagent bottle and connect the C2 and O2 connectors. After closing the door, click the OK button in the popup window to automatically cultivate the droplets and detect the OD values.As 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 CVS format To plot the growth curve, use mapping software such as Excel and Origin 9.0. Inject required volumes of the initial bacteria solution, fresh medium, and MMC oil into the separate sterilized reagent bottles for the initial bacteria solution in the fresh medium as explained earlier.To choose the function of adaptive evolution, click on ALE in the software. In the parameter setting interface, turn on the OD detection switch, then set all the parameters described in the manuscript and hit the Start tab to start droplet generation. The process will take about 25 minutes.During each sub-cultivation period, observe whether the maximum OD values of the droplets have increased significantly. If the increase occurs and meets the experiment requirements, click on the data export button to export the OD data. To extract the target droplets from the MMC, click on the screening tab to select the function of droplet extraction.Then choose the Collect option and click the numbers of target droplets. When done, click on OK.Wait for the popup window prompting a message. Then put the CF quick connector into the microcentrifuge tube for collection and click on OK.After one to two minutes when the software interface will pop up a new window prompting a message, insert the CF quick connector back and click on OK to make MMC continue to run.When the next target droplet reaches the droplet recognition site, collect it as specified before. Use a 2.5 microliter pipette to take out and place the droplet on a 90 millimeter solid plate, followed by spreading the drop evenly with a glass triangular coated rod with a side length of three centimeters, then cultivate the drop in a 37 degree Celsius constant temperature incubator for 72 hours. Later, pick three to five independent colonies of bacteria to cultivate each colony separately in a 50 milliliter shake flask with 10 milliliters of fresh medium in a shaking incubator at 200 rotations per minute and 37 degrees Celsius for 48 to 72 hours.After the incubation, follow the related standard regulations to store the cultured bacteria solution in the glycerol tube. The representative analysis shows the growth curves of 50 droplets in the whole adaptive evolution process. It was observed that the methanol essential Escherichia coli strain or MeSV2.2 displayed initial slow growth followed by fast growth.The growth curve of droplet six in the whole adaptive evolution process was plotted separately. The maximum OD600 value in the first generation was 0.37, which increased to 0.58 in the last sub-cultivation period, indicating that the strain in the droplet six has realized an obvious adaptive evolution. Furthermore, the growth curves of the droplet six strain and the initial strain were compared.The droplet six strain exhibited a higher maximum specific growth rate and cell concentration in the stationary phase than the initial strain. The most important thing is to ensure that the connection of the instrument chip and the reagent bottle is correct, which is the droplets for operations to proceed normally. This method provides researchers with a new idea for the cultivation of microorganisms and also provides efficient and low-cost platform for the adaptive evolution of strains.

Research

JoVE Journal

Bioengineering

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