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

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.