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 Materials and the website for MMC16.</li>
	<li>Install the operation software from the MMC.zip file 
	NOTE: Contact the authors for the MMC.zip file.
	<ol>
		<li>Create a dedicated folder and save the zip file in it.</li>
		<li>Create another dedicated folder as the &#34;Installation Directory&#34;. 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).</li>
	</ol>
	</li>
</ol>
2. Preparations
<ol>
	<li>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 &#176;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.</li>
	<li>Use a 0.22 &#181;m 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.</li>
	<li>Install the microfluidic chip
	<ol>
		<li>Open the door of the operation chamber (Figure 2A) and lift the optical fiber probe.</li>
		<li>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).</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
</ol>
3.&#160;Growth curve measurement in MMC
<ol>
	<li>Preparation for initial bacterial solution
	<ol>
		<li>Follow the related standard regulations to prepare Luria-Bertani (LB) medium and autoclave at 121&#176;C for 15 min. 
		NOTE: Components of LB medium: NaCl (10 g/L), yeast extract (5 g/L) and tryptone (10 g/L).</li>
		<li>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 &#176;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.</li>
		<li>Dilute the cultured E. coli MG1655 solution with fresh medium to an OD600&#160;of 0.05-0.1 to obtain an initial bacteria solution (prepare about 10 mL).</li>
	</ol>
	</li>
	<li>Click on Initialization to initialize the MMC. After the initialization interface appears, set the cultivation temperature as 37 &#176;C and the photoelectric signal value as 0.6 (Figure 3A). Initialization will take about 20 min.</li>
	<li>Turn on the UV lamp (wavelength 254 nm) during initialization.</li>
	<li>Inject the initial bacteria solution and MMC oil into the reagent bottle.
	<ol>
		<li>Take out a sterilized reagent bottle on the clean bench and tighten the cap.</li>
		<li>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.</li>
		<li>Inject about 5 mL of initial bacteria solution, and then fill the reagent bottle by injecting 5-7 mL of the oil again.</li>
		<li>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).</li>
	</ol>
	</li>
	<li>Wait for the initialization to end and then turn off the UV lamp (wavelength 254 nm).</li>
	<li>Open the door of the operation chamber, and put the reagent bottle into the metal bath.</li>
	<li>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.</li>
	<li>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.</li>
	<li>When a pop-up window appears on the main interface prompting &#34;Remove the reagent bottle between C2 and O2, then please click the OK button after completion&#34;, open the door of the operation chamber to take out the reagent bottle and connect the C2 and O2 connectors.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
4. Adaptive evolution in MMC
<ol>
	<li>Preparation for initial bacterial solution
	<ol>
		<li>Follow the related standard regulations to prepare the special liquid medium and solid plates for the MeSV2.2 and autoclave at 121 &#176;C for 15 min. 
		NOTE: For the components of the special medium refer to Table 1&#160;and the Table of Materials.</li>
		<li>Cultivate the MeSV2.2 using the solid plate (diameter = 90 mm) in a 37 &#176;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 &#176;C for 72 h.</li>
		<li>Dilute the cultured MeSV2.2 solution with the medium to an OD600&#160;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.</li>
	</ol>
	</li>
	<li>Initialize the MMC as explained in steps 3.2, 3.3, and 3.5.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Click on ALE to choose the function of adaptive evolution (Figure 3B). In the parameter setting interface, turn on the OD Detection switch.</li>
	<li>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, &#34;Concentration&#34; 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: 
	<img alt="Equation 1" src="/files/ftp_upload/62800/62800eq01.jpg" /> 
	Here &#34;C&#34; refers to the concentration of chemical factors introduced into droplets; &#34;a&#34; refers to the concentration of chemical factors in the reagent bottles between the C4 and O4 connector; &#34;b&#34; refers to the concentration of chemical factors in the reagent bottles between the C6 and O6 connector; and &#34;i&#34; 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.</li>
	<li>Remove the reagent bottle placed between the C2 and O2 connector as explained in step 3.8.</li>
	<li>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&#160;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.</li>
	<li>Extract the target droplets from the MMC.
	<ol>
		<li>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 &#34;Collect&#34;, &#34;Discard&#34; and &#34;Extract seed solution&#34;. &#34;Extract seed solution&#34; means to collect the remaining droplets13&#160;after the sub-cultivation operation.</li>
		<li>Wait for the pop-up window to prompt, &#34;Please pull out the CF quick connector and put it into the EP tube&#34;. Put the CF quick connector into the microcentrifuge tube for collection according to the software prompt and then click on OK (Figure 4D).</li>
		<li>After 1-2 min, the software interface will pop up a new window prompting, &#34;Please insert the connector back and click OK if finished&#34;. 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.</li>
		<li>Extract the droplet using a 2.5 &#181;L pipette, drop it on the 90 mm agarose plate, and spread it evenly with a triangular glass&#160;spreading rod with a side length of 3 cm. Then cultivate it in a 37 &#176;C constant temperature incubator for 72 h.</li>
		<li>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 &#176;C for 48-72 h. Follow the related standard regulations to store the cultured bacteria solution in the glycerol tube for subsequent experiments.</li>
	</ol>
	</li>
</ol>
5. Clean of the MMC
<ol>
	<li>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.</li>
</ol>

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The authors have nothing to disclose.

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. MMC also has some special advantages different from the conventional droplet microfluidic technology, which usually uses the pL and nL droplets. Most previously reported systems that used pL and nL droplets have poor cultivation performance and few detectable parameters (usually only fluorescence)18,19,20,21. Although there have been some platforms that can achieve better cultivation performance and multiple parameter detection, it is difficult and requires a lot of effort. For example, some researchers reported the OD detection of pL droplets. It is based on image recognition, which has not only false positives but also needs further verification of accuracy22. However, MMC can accomplish these in a relatively simple manner. MMC uses microliter (&#181;L) droplets that are rarely reported. The superior microbial cultivation performance of MMC has been verified, and it can also directly detect OD and fluorescence. Due to the large volume of the &#181;L droplets, the droplet generation is less susceptible to interference, which has higher stability. Meanwhile, more diverse operations can be performed in the microliter droplets, conducive to the realization of automated operations. Furthermore, because the droplets are enclosure spaces, the volatility of the contents can be suppressed (Supplementary&#160;Table 2), conducive to performing the long-term microbial cultivation and adaptive evolution when volatile substances exist in the medium14. This is difficult to achieve in shake flasks and microplates.
However, certain critical points in the protocol are worth emphasizing. Firstly, it should be noted that the OD value measured by MMC is different from that of a spectrophotometer because their optical path lengths of OD measurement are different (1 mm and 10 mm, respectively). Therefore, when comparing the OD value of MMC with that of shake flask, it is necessary to measure the calibration curve13. Fortunately, the adaptive evolution process does not require calibration curves because we focus on the relative trends among the growth curves. Next, certain microorganisms are uncultivated in MMC. The droplets rely on the surface tension of the oil-water interface to maintain stability23. If the microorganisms produce certain substances that disrupt the surface tension of the oil-water interface, such as some Bacillus subtilis strains producing surfactants24, the droplets cannot maintain stability. Furthermore, if the medium itself is an obstacle to the generation of droplets, it is not viable to be used in MMC, for example, very viscous medium or medium containing large particles. At present, the species we have successfully cultivated in MMC includes E. coli, Lactobacillus plantarum, Corynebacterium glutamicum, yeasts, Methylobacterium extorquens, Aspergillus oryzae, microalgae and so on. It is recommended to cultivate the strain in MMC for preliminary experiment. Finally, the connectors and ports between the chip, the reagent bottle, and the MMC must be connected in strict accordance with the protocol. Otherwise, the bacteria solution may flow into the MMC and contaminate the interior. Additionally, it must be pointed out that the current throughput of MMC is limited (0-200), due to the time taken for the operations of sub-cultivation. In the future, we will optimize the control software and the size of the chip to shorten the time and improve the throughput. Since MMC is a modular system, only related parts or software need to be replaced without the requirement of new equipment.
At present, MMC can not only conduct growth curve measurement, adaptive laboratory evolution, and single-factor multi-level analysis but also be used to customize different droplet operation procedures to meet experimental needs. In the future, it is necessary to further enrich the application functions of the MMC system in response to the different needs of microbial research, such as conducting the multi-factor multi-level orthogonal experiments, multi-sample automatic sampling technology to simultaneously measure the growth curves of multiple bacterial species, and accurately detect and control more parameters (e.g., dissolved oxygen (DO) and pH). At the same time, it is also necessary to develop more functions in the field of microbiology to apply MMC to more practical scenarios, such as optimization of medium compositions, determination of minimum inhibitory concentration (MIC), co-cultivation of microorganisms25, etc.

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><tbody><tr><td>0.22 &amp;mu;m PVDF filter membrane</td><td>Merck Millipore Ltd.</td><td>SLGPR33RB</td><td>Sterilize the MMC oil</td></tr><tr><td>4 &amp;deg;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>CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O</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>CoCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;6H&lt;sub&gt;2&lt;/sub&gt;O</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>CuSO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;5H&lt;sub&gt;2&lt;/sub&gt;O</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>FeCl&lt;sub&gt;3&lt;/sub&gt;&amp;middot;6H&lt;sub&gt;2&lt;/sub&gt;O</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-&amp;beta;-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>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</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>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O</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>&amp;nbsp;MMC-I</td><td>Performing growth curve determination and adaptive evolution. Please refer to http://www.tmaxtree.com/en/index.php?v=news&amp;amp;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>MnCl&lt;sub&gt;2&lt;/sub&gt;</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>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;12H&lt;sub&gt;2&lt;/sub&gt;O</td><td>GENERAL-REAGENT</td><td>G10267B</td><td>Component of the special medium for MeSV2.2.</td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;Cl</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 &amp;mu;L, 10 &amp;mu;L, 100&amp;mu;L, 1000&amp;mu;L</td><td>For liquid handling</td></tr><tr><td>Quick connector A</td><td>Luoyang TMAXTREE Biotechnology Co., Ltd.</td><td>&amp;mdash;</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 &amp;deg;C)</td></tr><tr><td>UV&amp;ndash;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>ZnSO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O</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.

Watch this Scientific Journal Video about Automated Microbial Cultivation and Adaptive Evolution using Microbial Microdroplet Culture System MMC at JoVE.com

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 ...

automated-microbial-cultivation-adaptive-evolution-using-microbial

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

3.7K Views.  Tsinghua University. Deze methode kan helpen bij het oplossen van de problemen met de conventionele microbiële teeltmethoden. Het biedt een goedkoop, bedrijfsvriendelijk en resultaatbetrouwbaar experimenteel platform voor geautomatiseerde microbiële teelt en adaptieve evolutie. Deze technologie automatiseert bewerkingen en vermindert het arbeidsverbruik aanzienlijk.En tegelijkertijd heeft het goede cultuurprestaties met betrouwbare resultaten en eenvoudige bewerkingen. De technologie wordt voornamelijk g...

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

Design and Use of a Low Cost, Automated Morbidostat for Adaptive Evolution of Bacteria Under Antibiotic Drug Selection

We describe a low cost, configurable morbidostat for characterizing the evolutionary pathway of antibiotic resistance. The morbidostat is a bacterial culture device that continuously monitors bacterial growth and dynamically adjusts the drug concentration to constantly challenge the bacteria as they evolve to acquire drug resistance. The device features a working volume of ~10 ml and is fully automated and equipped with optical density measurement and micro-pumps for medium and drug delivery. To validate the platform, we measured the stepwise acquisition of trimethoprim resistance in Escherichia coli MG 1655, and integrated the device with a multiplexed microfluidic platform to investigate cell morphology and antibiotic susceptibility. The approach can be up-scaled to laboratory studies of antibiotic drug resistance, and is extendible to adaptive evolution for strain improvements in metabolic engineering and other bacterial culture experiments.

We describe a low cost, configurable morbidostat that enables the characterization of antibiotic drug resistance by dynamically adjusting the drug concentration. The device can be integrated with a multiplexed microfluidic platform. The approach can be scaled up for laboratory antibiotic drug resistance studies.

We describe a low cost, configurable morbidostat for characterizing the evolutionary pathway of antibiotic resistance. The morbidostat is a bacterial ...

Genetics

Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat

Natural evolution involves genetic diversity such as environmental change and a selection between small populations. Adaptive laboratory evolution (ALE) refers to the experimental situation in which evolution is observed using living organisms under controlled conditions and stressors; organisms are thereby artificially forced to make evolutionary changes. Microorganisms are subject to a variety of stressors in the environment and are capable of regulating certain stress-inducible proteins to increase their chances of survival. Naturally occurring spontaneous mutations bring about changes in a microorganism's genome that affect its chances of survival. Long-term exposure to chemostat culture provokes an accumulation of spontaneous mutations and renders the most adaptable strain dominant. Compared to the colony transfer and serial transfer methods, chemostat culture entails the highest number of cell divisions and, therefore, the highest number of diverse populations. Although chemostat culture for ALE requires more complicated culture devices, it is less labor intensive once the operation begins. Comparative genomic and transcriptome analyses of the adapted strain provide evolutionary clues as to how the stressors contribute to mutations that overcome the stress. The goal of the current paper is to bring about accelerated evolution of microorganisms under controlled laboratory conditions.

Here, we present a protocol to obtain adaptive laboratory evolution of microorganisms under conditions using chemostat culture. Also, genomic analysis of the evolved strain is discussed.

Natural evolution involves genetic diversity such as environmental change and a selection between small populations. Adaptive laboratory evolution (ALE) ...

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (&ge;105) in milliliter-scale volumes (&ge;0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 &mu;l). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

We have developed an automated cell culture and interrogation platform for micro-scale cell stimulation experiments. The platform offers simple, versatile, and precise control in cultivating and stimulating small populations of cells, and recovering lysates for molecular analyses. The platform is well suited to studies that use precious cells and/or reagents.

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

Designing Automated, High-throughput, Continuous Cell Growth Experiments Using eVOLVER

Continuous culture methods enable cells to be grown under quantitatively controlled environmental conditions, and are thus broadly useful for measuring fitness phenotypes and improving our understanding of how genotypes are shaped by selection. Extensive recent efforts to develop and apply niche continuous culture devices have revealed the benefits of conducting new forms of cell culture control. This includes defining custom selection pressures and increasing throughput for studies ranging from long-term experimental evolution to genome-wide library selections and synthetic gene circuit characterization. The eVOLVER platform was recently developed to meet this growing demand: a continuous culture platform with a high degree of scalability, flexibility, and automation. eVOLVER provides a single standardizing platform that can be (re)-configured and scaled with minimal effort to perform many different types of high-throughput or multi-dimensional growth selection experiments. Here, a protocol is presented to provide users of the eVOLVER framework a description for configuring the system to conduct a custom, large-scale continuous growth experiment. Specifically, the protocol guides users on how to program the system to multiplex two selection pressures &#8212; temperature and osmolarity &#8212; across many eVOLVER vials in order to quantify fitness landscapes of Saccharomyces cerevisiae mutants at fine resolution. We show how the device can be configured both programmatically, through its open-source web-based software, and physically, by arranging fluidic and hardware layouts. The process of physically setting up the device, programming the culture routine, monitoring and interacting with the experiment in real-time over the internet, sampling vials for subsequent offline analysis, and post experiment data analysis are detailed. This should serve as a starting point for researchers across diverse disciplines to apply eVOLVER in the design of their own complex and high-throughput cell growth experiments to study and manipulate biological systems.

The eVOLVER framework enables high-throughput continuous microbial culture with high resolution and dynamic control over experimental parameters. This protocol demonstrates how to apply the system to conduct a complex fitness experiment, guiding users on programming automated control over many individual cultures, measuring, collecting, and interacting with experimental data in real-time.

Continuous culture methods enable cells to be grown under quantitatively controlled environmental conditions, and are thus broadly useful for measuring ...

Daily Transfers, Archiving Populations, and Measuring Fitness in the Long-Term Evolution Experiment with Escherichia coli

The Long-Term Evolution Experiment (LTEE) has followed twelve populations of Escherichia coli as they have adapted to a simple laboratory environment for more than 35 years and 77,000 bacterial generations. The setup and procedures used in the LTEE epitomize reliable and reproducible methods for studying microbial evolution. In this protocol, we first describe how the LTEE populations are transferred to fresh medium and cultured each day. Then, we describe how the LTEE populations are regularly checked for possible signs of contamination and archived to provide a permanent frozen "fossil record" for later study. Multiple safeguards included in these procedures are designed to prevent contamination, detect various problems when they occur, and recover from disruptions without appreciably setting back the progress of the experiment. One way that the overall tempo and character of evolutionary changes are monitored in the LTEE is by measuring the competitive fitness of populations and strains from the experiment. We describe how co-culture competition assays are conducted and provide both a spreadsheet and an R package (fitnessR) for calculating relative fitness from the results. Over the course of the LTEE, the behaviors of some populations have changed in interesting ways, and new technologies like whole-genome sequencing have provided additional avenues for investigating how the populations have evolved. We end by discussing how the original LTEE procedures have been updated to accommodate or take advantage of these changes. This protocol will be useful for researchers who use the LTEE as a model system for studying connections between evolution and genetics, molecular biology, systems biology, and ecology. More broadly, the LTEE provides a tried-and-true template for those who are beginning their own evolution experiments with new microbes, environments, and questions.

Daily Transfers, Archiving Populations, and Measuring Fitness in the Long-Term Evolution Experiment with Escherichia coli

This protocol describes how to maintain the Escherichia coli Long-Term Evolution Experiment (LTEE) by performing its daily transfers and periodic freeze-downs and how to conduct competition assays to measure fitness improvements in evolved bacteria. These procedures can serve as a template for researchers starting their own microbial evolution experiments.

The Long-Term Evolution Experiment (LTEE) has followed twelve populations of Escherichia coli as they have adapted to a simple laboratory environment for ...

Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular processes. There is a need for culturing and measuring systems that incorporate programmed illumination and stimulation of optogenetic systems. We present a protocol for building and using a continuous culturing apparatus to illuminate microbial cells with programmed doses of light, and automatically acquire and analyze images of cells in the effluent. The operation of this apparatus as a chemostat allows the growth rate and the cellular environment to be tightly controlled. The effluent of the continuous cell culture is regularly sampled and the cells are imaged by multi-channel microscopy. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses in the fluorescence intensity and cellular morphology of cells sampled from the culture effluent are measured over multiple days without user input. We demonstrate the utility of this culturing apparatus by dynamically inducing protein production in a strain of Saccharomyces cerevisiae engineered with an optogenetic system that activates transcription.

We designed a continuous culturing apparatus for use with optogenetic systems to illuminate cultures of microbes and regularly image cells in the effluent with an inverted microscope. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses to illumination can be measured over several days.

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular ...

Generic Protocol for Optimization of Heterologous Protein Production Using Automated Microbioreactor Technology

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of bioprocess conditions. One important aspect is the improvement of cultivation medium to provide an optimal environment for microbial formation of the product of interest. It is well accepted that the media composition can dramatically influence overall bioprocess performance. Nutrition medium optimization is known to improve recombinant protein production with microbial systems and thus, this is a rewarding step in bioprocess development. However, very often standard media recipes are taken from literature, since tailor-made design of the cultivation medium is a tedious task that demands microbioreactor technology for sufficient cultivation throughput, fast product analytics, as well as support by lab robotics to enable reliability in liquid handling steps. Furthermore, advanced mathematical methods are required for rationally analyzing measurement data and efficiently designing parallel experiments such as to achieve optimal information content.
The generic nature of the presented protocol allows for easy adaption to different lab equipment, other expression hosts, and target proteins of interest, as well as further bioprocess parameters. Moreover, other optimization objectives like protein production rate, specific yield, or product quality can be chosen to fit the scope of other optimization studies. The applied Kriging Toolbox (KriKit) is a general tool for Design of Experiments (DOE) that contributes to improved holistic bioprocess optimization. It also supports multi-objective optimization which can be important in optimizing both upstream and downstream processes.

This manuscript describes a generic approach for tailor-made design of microbial cultivation media. This is enabled by an iterative workflow combining Kriging-based experimental design and microbioreactor technology for sufficient cultivation throughput, which is supported by lab robotics to increase reliability and speed in liquid handling media preparation.

A core business in industrial biotechnology using microbial production cell factories is the iterative process of strain engineering and optimization of ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

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

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