Step-wise Process for Setting Up the Script for Extractor Experiments

1. Select the Type of Experiment

• Decide whether to run a Chemostat or Turbidostat experiment.

• Ensure you choose the correct type when configuring the script.

2. Set Parameters for the Experiment

• The setup process for Chemostat and Turbidostat is identical to non-extractor versions.

• Define the operational parameters as you normally would for the chosen type of experiment.

3. Specify Extractor and Non-Extractor Vials

• Identify which vials will be used as extractor and non-extractor vials:

  • Common Extractor Vials: 4, 5, 6, 7, 12, 13, 14, and 15.

  • Common Non-Extractor Vials: 0, 1, 2, 3, 8, 9, 10, and 11.

• Edit the script to assign these roles to the corresponding vials.

4. Edit Vial Assignments if Needed

• If the vial assignments need to be changed during the experiment:

  • Modify the script accordingly.

  • Ensure all changes are accurately reflected in the script.

5. Restart the Script After Changes

• If any changes are made to the extractor or non-extractor vial assignments:

  • Stop the current running script.

  • Restart the updated script in the terminal to apply the changes.

6. Run the Experiment

• Once the script is set up with the correct parameters and vial assignments:

  • Start running the experiment using the script.

7. Monitor and Adjust as Necessary

• During the experiment, monitor the setup to ensure everything functions as expected.

• If adjustments are needed, repeat the steps to edit the script and restart it.

• Set up your lines using the same method as the traditional eVOLVER

• Use this special method to run the slow pumps and fill them for sterilization

Maintaining the Solvent Layer

The solvent layer on top of the cells / media in the extractor must be maintained. However, in the basic eVOLVER setup, volume in the vial is maintained by setting the efflux straw's height to a certain vial volume and pumping any excess volume away. Therefore in basic eVOLVER, the solvent layer would be the first to go.

Instead, the extractor controls the volume in the vial by using the od90 photodiode - LED pair to monitor fluid levels.

Volume Maintenance Algorithm

1. Experiment started

2. Pump Wait: No pumping until vol_check_wait is exceeded

   Copy

   vol_check_wait = 0.15 # (hours) time to wait before regulating extractor volume

3. Blanking: the value where the volume line is below the volume sensor is taken

   1. The raw od90 values just before vol_check_wait are stored and averaged to use as this threshold 'blank'

4. Continuous Pumping: Cells are pumped from the cell growth bioreactor (chemostat or turbidostat) to the extractor and back from the extractor to the cell growth bioreactor

   1. In cycles based on the eVOLVER broadcast timing

5. Pump Duration Biasing: Pumping out from the extractor is shorter than pumping in

6. Extractor Volume Increases: The volume in the extractor steadily rises towards the volume sensor

7. Volume Threshold Exceeded: When the volume line increases above threshold, the system will only pump OUT one broadcast cycle, decreasing the volume level

8. Steps repeat

1. Prepare and Organize Your Equipment

• Gather all necessary vials, slow pumps, tubing, and any other required equipment.

  • Recommended equipment: clean vials, pumps, tubing, 1:1 extractor/non-extractor caps, appropriate media, clean beakers, 10% Bleach, 70% Ethanol

• Ensure that each extractor vial has a matching non-extractor vial (e.g., vial pair 0 and 4).

2. Connect the Tubing

• Match each pump with one extractor/non-extractor pair.

• For each pair, connect the tubing from the pump to the appropriate vials:

  • For example, if using vial pair 0 and 4, connect the pump to suck up liquid from vial 0 and dispense it into vial 4.

3. Check Tubing Pathways

• Carefully follow the path of the tubing:

  • Ensure the tube is correctly drawing from the extractor vial (e.g., vial 0) and dispensing into the non-extractor vial (e.g., vial 4).

  • Similarly, ensure the tube for the reverse flow (non-extractor to extractor) is correctly set up.

While the tubing set up may look confusing at a glance remember that logically the setup works the same and as long as you understand the job of each pump on the slow pump array and the port to each straw on the cap you can trace back what goes where

4. Volume and Media Preparation

• Prepare 20 mL of media for the Chemostat/Turbidostat vials used in the experiment.

• For extractor columns, use a media volume of 15 mL to prevent the liquid level from reaching the photodiodes/LEDs.

• Use the pumps/slow pumps to fill lines with media

• Fill non-extractor lines and vials with media first and using a secondary set of extractor vials with media in them already run the slow pumps second to prevent bubbles from forming in slow pump lines

  • This is important because the initial blank should be based off of filled straws

5. Positioning of Vials

• Ensure that the vials are placed correctly making sure you account for any added acrylic blocks underneath.

• Double-check that the liquid levels are BELOW the photodiodes/LEDs.

6. Monitor the Pumping Process

• Observe the pumping operation closely:

  • Ensure that the correct volumes are being transferred between vials as intended.

  • Monitor for any signs of malfunction or unexpected behavior.

7. Ensure Cleanliness of Vials

• Clean vials thoroughly to avoid stains, condensation, or drippage.

• Make sure there are no residual liquids that can impact the sensor readings.

9. Adjust if Necessary

• Make adjustments to tubing, pumps, or vial positions if liquid levels are incorrect or if the system does not operate as expected.

9. Start the Experiment

• Once all the checks and setups are completed, start the experiment with your extractor script.

• Regularly monitor the system to ensure it is functioning correctly throughout the experiment.

By following these steps, you can ensure a successful setup and operation of the two-column extractor system, minimizing errors and ensuring accurate experimental results.

The main result of issues with the extractor can lead to extractor or non-extractor vials to being empty or result in overflow.

Some factors that can contribute to this are:

Condensation:

Condensation can occur in the extractor vials depending on ambient conditions in the extractor as well as what organism you are working with. Condensation can obscure the photodiodes tricking them into thinking the vial has more volume then it actually does, triggering unwanted pump events. Generally the best solution for this is to ensure the heat is properly working or to raise the temperature of the vial.

Disturbing the experiment:

The extractor and non-extractor vials are delicate when the experiment is actively running. Vials should NOT be moved or sampled while the experiment is actively running. It is recommended to pause the experiment before handling any vials. Moving the extractor vials while the experiment is running can negatively impact the fluidics set up, or move the liquid into range of the photodiodes triggering an unwanted pump event.

Contamination:

Contamination of vials:

Vial contamination will cause a rapid increase in OD which will rapidly accelerate the rate at which pump events occur.

Contamination of media bottle:

If the non-extractor vial begins to overfill if it reaches the main media influx line the inoculated media can work its way back through the line towards your media bottle. Additionally the same can occur if you have your vial-to-vial line dispensing liquid from above your main media influx line which will again expose the main media line to inoculated media.

Incorrect Fluidics setup:

Droplets on side of vial:

In the extractor vials if the straw dripping culture in is placed too high above the liquid level the drops from the straw can splash and cause droplets to collect on the side of the vials. Causing a similar effect to condensation by obscuring the photodiodes triggering unwanted pump events. When designing your extractor caps measure the straw length so it sits at about 1 inch above your desired volume.

Incorrect volume/block setup:

Remember to double check what volume you are putting into the extractor vial, currently the volume of the extractor vials should be no greater than 15 mL the photodiodes should blank "nothing" at both blanking steps extractor vials exceeding 15 mL can interfere with the blanking steps causing immediate problems with pump events. Similarly if using acrylic blocks to elevate extractor vials remember to not account for how much "volume" each block take up.

Dirty vials:

Improperly cleaned vials can run into the same issues as condensation or droplets on the vial, again tricking the photodiodes into triggering an unwanted pump event.

Two-week Extractor experiment

Turbidostat extractor experiment

Chemostat extractor experiment

Feedstock experiment

Overview

The two column extractor is a type of eVOLVER experiment designed to continuously extract a molecule of interest exuded by a microbe. One set of vials are bioreactors where cells are continuously cultured. Those vials are each paired with an extractor vial, where microbes are continuous dropped through a solvent layer that extracts a biomolecule of interest. This solvent can be sampled to determine amount of biomolecule that is made.

When starting an experiment you'll go through the same process as a normal experiment. Make sure that you have the extractor columns properly set up and that you also include the slow pumps lines in your line sterilization process.