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.
eVOLVER gives researchers experimental freedom, the ability to define their own culture growth experiments with automated feedback on multiple parameters and scale it to whatever size they need. eVOLVER is more flexible than other devices. Users can easily customize software in Python as well as the hardware, by modifying the smart sleeves that hold sensors for each culture.
The system can appear complicated at first glance with multiple components and connections. A visual guide gives first time users more confidence in running experiments on their own. Proper setup is critical.
Be sure to understand what each component does. So you can confirm that they are working. Ensure everything is routed correctly and connected tightly.
While designed for aerobic culturing of bacteria and yeast in varying conditions, eVOLVER could also be modified for anaerobic fermentation or to maintain mammalian cells in suspension. This video will demonstrate how to setup, initialize, and run a high throughput continuous culture experiment using the eVOLVER platform. The day before the experiment, prepare media, vials, and inoculum according to the manuscript.
In three large beakers, prepare two beakers of 10%bleach and one beaker of 70%ethanol. Using the switches on the eVOLVER device, turn on the five volt power supply on the eVOLVER, wait for five seconds, then turn on the 12 volt power supply. If running several vials from the same media bottle, connect multiple media input lines with tubing splitters.
Custom tubing splitters can be constructed with lower components. Submerge the media input lines in the first bleach beaker and the media eflux lines in the second bleach beaker. Connect the downstream end of the media input lines to the second beaker with the eflux lines.
Place waste lines into a waste carboy. Add one to two liters of bleach into the waste carboy to sterilize waste generated during the experiment. Using the touchscreen on the eVOLVER, navigate to the setup section and run all pumps for 20 seconds to fill the fluid lines with 10%bleach.
Allow bleach to sit in the lines for at least 30 minutes to sterilize. Run the pumps again using the touchscreen until the lines are no longer submerged, pushing air through the lines to get as much of the bleach out as possible. Place media input lines in the ethanol beaker, fill the lines with ethanol, flush them with air.
Then, attach media input lines to the media bottles with the lower connectors and run the pumps until the media fully runs through the lines flushing out any residual ethanol. Partially insert sterilized vials into the eVOLVER smart sleeves and hook up the input lines to the short influx straw and the waste lines to the long eflux strawq according to the color coding. Adjust on the eVOLVER touchscreen to run all pumps in 10 second increments to fill the vials with media.
Ensure by visual inspection, eflux pumps are efficiently removing media through the eflux straws to prevent overflows. Then, push vials down until fully encased by the smart sleeve. On the eVOLVER touchscreen interactive setup page, drag a finger across the touchscreen or press the Select All button to select all of the vials and set the initial conditions of temperature at 30 degrees Celsius and the stir to 10.
On a computer, specify the eVOLVER device that the experiment will be run on. Be sure that the desired calibration settings have been selected. Open the eVOLVER dashboard and navigate to the Experiment Manager page.
Select the Base experiment in the Experiment Navigator panel or an existing experiment as a starting point and click Clone. Navigate to the Experiment Editor page. Use the Vial Selector and Parameter Definition panels to adjust the temperature to 30 degrees Celsius and set an upper and lower threshold of OD for each vial according to the experimental design.
Save the experiment by clicking Save. Then click the arrow to go back to the Experiment Manager page and click the Play button to run. Once the experiment is underway, navigate to the Real Time Data panel in the interactive dashboard.
For each vial, check that OD values are holding at zero. Browse through the temperature graphs to check that temperatures are at or progressing towards 30 degrees Celsius. To prepare the inoculum, first measure the OD of overnight cultures and calculate the desired full dilution for overnight cultures assuming a vial volume of 25 milliliters and a desired starting OD of about 0.05.
Through the sampling port, pipette the calculated volume of inoculum or mixture of inocula. Check the graphs on the dashboard to see that the OD is increasing to 0.05. Cell growth and vial conditions can be monitored in real time as the experiment is ongoing, allowing the researcher to intervene if any issues occur.
eVOLVER is an automated continuous culture platform allowing multiparameter control of culture conditions. Qualitative differences were shown between the wild type, temperature sensitive, and salt sensitive groups when the log ratios were plotted against generations interpolated from eVOLVER growth rate data. Color indicates the temperature of selection, while dashes distinguish salt concentrations.
A slope fit through the linear portion of each plot using a minimum of three points, yields a quantitative fitness value that describes how the variant strain competes against the reference strain. Negative fitness values indicate that the variant strain is out-competed by the reference strain. Always check that all pumps are running efficiently and that fluid lines are clear of any clogs.
An overflow can ruin an experiment and potentially damage the smart sleeves. Samples taken from eVOLVER can be subsequently sequenced to check for genomic alterations as a result of the experiment. This information can be used to engineer new strains or systems.
For researchers using lab evolution or library selection, it's easy to apply unique selection pressures in each eVOLVER experiment. Feedback control and dynamic environments are now very accessible as well. The waste carboy will contain small amounts of bleach and ethanol after an experiment.
Consult your lab safety coordinator for proper disposal of waste.
