Materials

Nalgene 20L carboy

Nalgene fitting/venting closure

Media Requirements

A primary consideration when running eVOLVER experiments is predicting the media consumption rate, which is typically orders of magnitude higher than in typical batch experiments. The required media for continuous culture can be estimated using the following equation: 𝑀𝑒𝑑𝑖𝑎 𝑅𝑒𝑞𝑢𝑖𝑟𝑒𝑑 [𝑚𝐿] = 𝐶𝑢𝑙𝑡𝑢𝑟𝑒 𝑉𝑜𝑙𝑢𝑚𝑒 [𝑚𝐿] / 𝐴𝑣𝑔. 𝐷𝑜𝑢𝑏𝑙𝑖𝑛𝑔 𝑇𝑖𝑚𝑒 [ℎ] ∗ 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓𝐶𝑢𝑙𝑡𝑢𝑟𝑒𝑠 ∗ 𝐷𝑢𝑟𝑎𝑡𝑖𝑜𝑛 [ℎ] (1) It should be noted that additional media (~20mL) is needed during device setup to flush media input lines.

Protocol

1. Before preparing media, steam clean the carboy by filling with ~2L of water and autoclaving on the liquids cycle for at least 45 minutes. Be sure to keep the lid loose to prevent the carboy from collapsing. Pour out the water.

2. If you are able to prepare a concentrate of your media, you can do so as a 10x 2L solution (or whatever makes sense for your media) in a beaker with a stir bar. It is also possible to mix everything directly in the carboy. Add your media components in small batches and try to mix as you go.

3. Fill the carboy up to 20L - volume of media components that will be supplied after the autoclave step. For example, if you need your media to be 2% glucose, you should fill the carboy to 18L and then add 1L of filter sterilized 40% glucose after autoclaving.

4. Place a male Luer cap on the outlet of the carboy lid. Place a filter on the inlet, and cover it with foil. Loosen the entire lid.

5. Place the carboy into an autoclave bin, then place into an autoclave. Autoclave on the liquids cycle for 2 hours.

6. Take the carboy out of the autoclave.

7. If media components need to be added after autoclave (glucose, antibiotics, supplements, etc.) place the carboy on a cart and move next to a bench flame. Sterilize the area thoroughly with ethanol before quickly unscrewing the lid to make the additions. Mix by placing the carboy on a cart and moving the cart in a circular motion, or by placing on the floor, tilting ~30 degrees, and moving the top in a circular motion.

8. You can place the autoclave on the floor next to your experiment. Be sure to keep the lid loose to prevent negative pressure buildup in the carboy which could prevent media from going into the culture vessels.

9. After the carboy is empty, rinse thoroughly with water, being sure to run water through all of the tubing. Steam clean as described in step 1, then pour out the remaining liquid.

Relevant Forum Posts

About the volume of required media

Making Media Bottles and Media Splitters

1. Place waste lines into carboy

2. We use a small stand to bring the carboy closer to the eVOLVER

3. Have an extra carboy ready to swap in when the first gets full

4. (Optional) You can fill 10% of the carboy with bleach

   1. Or bleach after depending on whether you hate the smell bleach or your culture more

5. (Optional) Shrink heat shrink tubing onto the waste lines to make them easier to deal with

6. (Optional) Drill a hole through the carboy cap to make the opening to the carboy smaller (less smelly)

7. (Optional) Parafilm the opening to the carboy to make it less smelly

Checklist

Before starting experiment prep:

1. Finished Getting Started Page

2. Calibrations

   1. Temperature (must be first, affects OD cals)

   2. OD

   3. Pump calibrations

3. Should things not be as expected

   1. eVOLVER Maintenance

   2. Vial Troubleshooting

Sample Experimental Workflow

Day 0:

Prepare media - especially if using a 20L carboy so it can cool overnight

Prepare vials

Sterilize fluidic lines

Make sure vials / pumps are behaving as expected

• Are all vials stirring, getting up to temperature, all pumps pumping, etc?

• Especially check that efflux is running long enough to remove enough culture from vials

Start overnight cultures

Day 1:

Load vials and set initial conditions

Start experiment via GUI or Command Line

Inoculate cultures into vials

Monitor experiment for correct behavior (over several hours)

• Are the cells growing?

• Is the experiment diluting as expected?

• Are all pumps working?

Day 2 - Onwards:

Monitor media levels - make sure that there is more than enough media to make it to the next time you check the experiment

Monitor biofilming on the vials - this can cause increased OD over time

Finally, end the experiment and clean up

Relevant Forum Posts

Considerations for your first eVOLVER experiment

You should be able to follow the 2019 JoVE paper for most of the process. Differences will be highlighted here.

Running experiments with Dropbox/Google Drive

If you would like your data to be automatically backed up on Dropbox or Google Drive, first install the desktop applications. This will enable any computer with Dropbox/Google Drive and access to the directory to monitor the experiment using the eVOLVER GUI in real-time, while providing all the benefits of using these services (automatic backup of data, ease of data sharing, etc).

We recommend saving your experiments through Dropbox or Google Drive, but you do not have to - it's fine to have everything run locally on your lab computer. Both options will be detailed in the Experiment Manager section below.

Setup

Select the eVOLVER you wish to run an experiment on in the dropdown on the top right, then click the SETUP button.

On left side of the screen, ensure the correct calibrations are selected for Temp, OD, and the pumps. Click the box to bring up a list of available calibrations for each parameter.

Once you have selected the calibrations, you can set the initial stir and temperatures, and sterilize the fluidics as described in the JoVE paper.

Experiment Manager

The JoVE paper is out of date with regards to using the GUI for running and managing an experiment, from 3) Configuring eVOLVER Software and Programming Algorithmic Culture Routines on.

Open the experiment manager on the Home screen of the GUI. You should see something that looks like this if this is the first time you have used the application:

In the top center the Expt Directory is listed out. You can click the pen icon to change this location to be anywhere you like.

If you have Dropbox or Google Drive installed, you can select a location on the cloud to save your experiments. This will enable you to view your data from any computer that has permissions and the Dropbox/Google Drive application installed!

You do not need to make a new folder called experiments as shown below - the GUI will automatically create this directory.

Click the New Experiment button to open a prompt to name your experiment. Type a name then click Submit.

Click the Pen button on the row of the new experiment to bring up with experiment editor page.

Experiment Editor

By default, the Turbidostat editor will appear when you come to the editor page.

1. The name of the experiment. You can click the pen icon to change the experiment name.

2. The parameter selector. The name of the currently selected parameter is listed at the top. You can click the -/+ buttons to change the value (or use the slider), and then click the box in the middle to apply this value to the selected vials.

3. Experiment control panel. From left to right: Start/Stop experiment, View Data, Save Files (Only for File Editor), Copy Experiment, View Files in File browser, Delete experiment.

4. eVOLVER Selector. Select the eVOLVER to run the experiment on.

5. Experiment type. Default will bring up the Turbidostat editor. Chemostat and growth rate editors are also available. You can also bring up the file editor to directly modify the python files within the GUI.

6. Vial selector - select the vials and then apply desired experimental parameters from the buttons on the parameter selector. Values display the currently set parameters.

7. Vial selector control buttons - flip the orientation on the display or select all of the vials.

8. Reset - goes back to default. Save Experiment - saves the parameters for all vials.

The chemostat and growth rate modules function similarly to the turbidostat one.

Clicking File Editor in the experiment type dropdown will allow you to directly modify the python files.

You can select the file you would like to edit on the left, edit it on the right, and save by clicking the Save icon on the bottom left. For most cases you will not need to use this! If you change the files, you will not be able to use the t-stat/c-stat/growth rate modules, and you will need to modify the EVOLVER_IP, OPERATION_MODE, and other variables throughout the code.

You can also click the folder icon on the bottom left to view the files in a file browser and edit them using an editor of your choice.

Starting the experiment

When you're ready to start the experiment, click the Play button either on the Experiment Manager page or the Experiment Editor page. Then you can click the graphs icon to go watch your experiment running in real-time.

When you navigate to the graphs page, the graphs will look fairly empty to start. After a minute or so, data will start to appear as it is collected. You can also click the VIEW LOGS button to see how the experiment is running.

On the left, you can modify how the graphs are displayed by clicking the appropriate radio buttons. You can also look close up at an individual vial and use the data slider at the bottom of the graph to zoom in on an area of interest.

When you click ALL to go back to seeing all vials, the range set on a single vial will be applied to all vials. You can click the radio buttons to set it back to the latest data.

To end the experiment, click the STOP square button on the device running the experiment.

Protocol

1. Rinse vial lids by running DI water from sink through influx and efflux straws (2-3 seconds). Alternatively fill a large beaker with DI water, dump vial lids in and stir.

2. Shake vials caps to remove excess water and set aside on a clean surface

3. Get out enough dishwashed vials (stored upside down), place in white rack

4. Place a small stirbar in each vial

5. If using septa, place a septa on each

6. Screw on rinsed vial lids

7. If vials will be used within 24 hours of prepping, then place white rack in small autoclave bin, cover entire bin with foil (reuse foil if possible), and autoclave on vacuum cycle for 10 minutes

8. Keep foil on bin until right before loading vials into eVOLVER to maintain sterility

9. If vials are being prepped in advance, screw a white or black mini-cap onto every straw, then autoclave in bin without foil

10. Keep mini-caps on until right before loading vials into eVOLVER to maintain sterility

Why

When we start an experiment, the script asks us whether we want to calibrate to blank.

OD is dependent on vial position, vial opacity, media, environmental light conditions, temperature, and more.

Therefore, we take the first OD measurement as a zero and subtract that out from every subsequent measurement.

Details

OD blank is only applied to the calculated OD from calibration, not the raw OD data. The blank is applied for each vial, it just subtracts it out from subsequent measurements for that vial

https://www.evolver.bio/t/where-or-how-is-od-blank-info-stored/182

1. For the next few steps, it is recommended to spray your gloves with 70% ethanol.

2. Partially insert autoclaved vials into the eVOLVER Smart Sleeves such that efflux straw is entirely visible, if possible.

3. Start by attaching input lines (clear) to the short influx straw, avoid touching the tips of the lines to avoid contaminating the inside.

4. Next, attach efflux lines (blue) to the long efflux straw. It is critical to check for loose connections or incorrectly routed lines. Failure to do so will cause overflows and potentially damage the Smart Sleeve.

5. In the setup menu in the electron app, run all pumps in 10 second intervals to fill the vials with media. Run enough media so that the efflux pumps start removing media through the efflux straws.

6. Visually inspect the culture volumes at this point. If the efflux straws do not appear to be functioning efficiently (media levels rise above the efflux straw), inspect the fluidic line connections and the peristaltic pump. Correct or replace parts as needed to prevent overflows.

7. Push vials down until fully encased by the Smart Sleeve. If aluminum sleeves are cut to different lengths, vials may appear to be covered in different amounts.

8. Be sure that the desired calibration files have been selected from the dropdown menu in the setup panel. These files are stored on the device itself, and will default to the most recently used calibration. Temperature and OD calibrations must be repeated if vials are replaced, and OD calibrations are recommended if switching to a new organism (i.e. bacteria vs yeast). Otherwise, calibrations can be used over and over again.

9. Set the initial conditions for experimental parameters using the eVOLVER electron app. This will be overwritten once the experiment code starts running, but can be helpful for preheating media or trying out different stir rates.

10. If stir bars are jumping around (audibly) then the stir rate may be too high for the volume of culture you are using. Commonly used values are between 8 and 12.

Relevant Forum Posts

Recommendations for Septas and Needles?

Using nylon caps to determine culture volumes

Making vial caps w/ laser cutter and pipette tip

Overview

Reasons you might use command line experiment start:

1. Doing a custom experiment

2. Unable to install GUI or getting errors you aren't able to fix easily (post on the forum first)

Protocol

1. In a command line window, enter the dpu virtual environment.

   1. Navigate to the DPU directory

      • Use this command: cd <path_to_dpu>

   2. Enter the command:

      • On Mac OS:

        • source dpu-env/bin/activate

      • On Windows PowerShell:

        • dpu-env\Scripts\Activate.ps1

2. Navigate to your experiment folder

   1. This should be in dpu/experiment

3. Copy the template folder and rename it for each experiment

   1. Navigate into your new experiment folder

4. Alter settings in custom_script.py

   1. EVOLVER_PORT = set correct IP of the eVOLVER

   2. Set initial temp and stir settings

   3. custom_script.py has basic settings for running chemostat and turbidostat experiments, but you can also customize. To learn more click here.

5. Start the experiment using the command:

   1. python3 eVOLVER.py -i <your_evolver's_IP_address>

   2. python3 eVOLVER.py -i 192.168.1.9

6. Calibrate to blank

   1. Temperature should be fully equilibrated before this

   2. DO NOT add cells before blanking, whatever OD value the cells + media have will be subtracted from future measurements

7. Stop the experiment using the keyboard shortcut control+C

   1. One control+C pauses the experiment from recording data

   2. Two control+C stops the experiment

8. To change parameters mid-experiment or restart from blank data files:

   1. Stop the experiment fully

   2. Change parameters in custom_script.py

   3. Restart the experiment

      1. Continue to keep your data

         1. Overwrite to make new blank data files and throw away your old experiment. NOTE: you will lose your OD blank and will need to make a new one (a hassle if you have already inoculated with cells)

9. You can run experiments on more than one eVOLVER at a time

   1. Create a new experiment folder (different custom_script.py) and change IP

   2. Follow this procedure in an additional command line window

Ending an Experiment

1. Clean up the experiment

2. Data is in the data files

Protocol

1. In three large beakers, prepare 500 mL of 10% bleach, 200 mL of 10% bleach and 300 mL 70% ethanol.

2. Using the switches on the eVOLVER device, turn on the 5 V power supply on the eVOLVER, wait for >3 seconds, then turn on the 12 V power supply.

3. If running several vials from the same media bottle, connect multiple media input lines with tubing splitters. Custom tubing splitters can be constructed with Luer components.

4. Submerge the media input lines (clear, media in) in the first bleach beaker, and the media efflux lines (blue) in the second bleach beaker. Place the downstream end of the media input lines (clear, to vial) in the second beaker with the efflux lines. Since the lines may be different lengths, a hairclip is useful to keep the ends of each lines close to eachother in order to ensure all remain submerged.

5. Add 1 L bleach into large empty waste carboy, which will sterilize waste generated during experiment. Consult with a safety coordinator to ensure proper disposal of waste.

6. Using the switches on the eVOLVER device, turn on the 5 V power supply on the eVOLVER, wait for 5 seconds, then turn on the 12 V power supply.

7. If running several vials from the same media bottle, connect multiple media input lines with tubing splitters (drawer under eVOLVER computer) before sterilizing. Custom sizes can be made if necessary.

8. Submerge the media input lines in the first bleach beaker, and the media efflux lines in the second bleach beaker.

9. Place the downstream end of the media input lines in the second beaker with the efflux lines. Place waste lines into waste carboy.

10. Open the eVOLVER electron app, select the appropriate eVOLVER from the dropdown menu.

11. Navigate to setup, then fluidics. Highlight the appropriate vials, select the influx and the efflux pumps, and run pumps for 20 seconds to fill lines with 10% bleach.

12. While running, ensure by visual inspection that all lines have been filled and that pumps are operating normally. Allow bleach to sit in the lines for at least 30 minutes to sterilize. This is a good pause point, can wait up to overnight. This is a good time to make code edits if necessary.

13. Run pumps again using the touchscreen app until lines are no longer submerged, pushing air through the lines to get as much of the bleach out as possible.

14. Place media input lines in the ethanol beaker (or pour ethanol into the first beaker) and repeat as in above, filling the lines with ethanol and flushing with air. Note: ethanol sterilizes quickly, so no need to wait 30 mins here.

15. It is also recommended to dip the downstream ends of the influx lines in ethanol to rinse off residual bleach, then drape on side of beaker rather than resubmerging in the remaining bleach. Residual bleach can affect culture viability, though washing with ethanol and media should be sufficient.

16. Attach media input lines to the media bottles with the Luer connectors and run the pumps until the media fully runs through the lines (10-20 seconds, depending on tubing), flushing out any residual ethanol.

Stopping Experiment and Bleaching

1. In the GUI electron app or command line, click to stop your experiment.

2. In large beakers, prepare 500mL of 10% bleach and 200 mL of 10% bleach.

3. Disconnect media bottles and place lines into 10% bleach solution

4. To use pumps to bleach the cultures, keep tubing connected, but move vials to a white eVOLVER rack. Prepare an additional 1L of 10% bleach and add to the media input beaker.

5. Run 10% bleach through vials until final concentration in the vials reaches ~10% bleach (~60 seconds for 25mL volume). Disconnect influx and efflux lines from vials, set vials aside for 30 mins.

6. To manually clean vials, disconnect influx and efflux lines and skip steps 39 and 40

7. Submerge the disconnected influx and efflux lines in the 200mL bleach beaker, then run all pumps for 20 seconds to fill all lines with 10% bleach. Let sit for no less than 30 minutes, no more than 48 hours.

8. Remove media input lines from bleach, and run air though the lines to remove bleach. If not planning on using the device again right away, run diH2O through all lines for 20 seconds to remove residual bleach from the lines. Dried bleach can form salt crystals and clog the lines. If this happens, break up the crystals manually using pliers to squeeze the line.

9. Turn off the 12V power, then the 5V power.

10. Proceed to vial & bottle cleaning protocol.

Clean Vials After Bleaching Cultures

1. Prepare a beaker ~50% full of 10% bleach

2. Remove vial lids and place in 10% bleach solution

3. Using the magnetic stir bar grabber, remove the stir bars from each vial, place in the 10% bleach solution

4. Let vial caps and stir bars sit for 30 minutes-overnight in the 10% bleach solution

5. Meanwhile, dump bleached culture down the drain, rinse with diH2O

6. Use a test tube brush to scrub inside of vial, particularly important if you grew bacterial cultures

7. Place bleached/scrubbed vials right-side-up in the vial racks to indicate they have not yet been dishwashed

8. Once vial lids/stir bars have been soaked for sufficient time, carefully pour out the 10% bleach solution, and replace with diH2O

9. Let vial lids and stir bars soak for another 30 mins-overnight.

10. Once vial lids/stir bars have been soaked for sufficient time, carefully pour out the diH2O.

11. Run diH2O through influx and efflux straws for 2-3 seconds at the sink to remove any residual bleach

12. Place stir bars in the stir bar tray.

13. Once 60+ vials have accumulated, vials should be dishwashed.

14. Place vials open side up in 3 white eVOLVER racks.

15. Take one rack, cover the vials using the wire mesh from the bottom rack of the dishwasher, then flip upside down. Repeat for the other two racks.

16. Add any other glassware or eVOLVER bottles to the top rack as desired, then run on cycle 3.

17. Store vials upside down in vial boxes to indicate that they have been dishwashed.