This study demonstrates a method for cultivating bacterial cultures engineered for methanol tolerance using a microfluidic system. The process involves injecting bacteria into a stress medium, allowing for the selection of methanol-adapted strains through adaptive evolution.
Inject a bacterial culture engineered for methanol tolerance into one reagent bottle and a stress medium with high methanol concentration into another.
Add oil to both and place them in a microbial microdroplet culture system to reach the incubation temperature.
Using a pump, push the liquids through a microfluidic chip.
The bacterial culture mixes with the stress medium. The oil separates the mixture into microdroplets, which move back and forth for cultivation.
Initially, high methanol stress slows bacterial growth.
Over time, some cells acquire mutations that improve methanol utilization and proliferate.
The system measures bacterial density and selects droplets showing higher growth.
These are remixed with the stress medium to enrich for greater methanol utilization.
After several cycles, extract droplets with high bacterial density, spread them on agar, and incubate.
Isolate and culture the formed colonies in a liquid medium to obtain methanol-adapted strains.
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 degrees 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.
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