• 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

Two-week Extractor experiment

Turbidostat extractor experiment

Chemostat extractor experiment

Feedstock experiment

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.

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

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 .

• 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.

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

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

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.

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.

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.

Improper of the fluidic lines can also cause contamination

Incorrect Fluidics setup:

Incorrect set up of the fluidics can cause immediate problems during your experiment make sure to double check your before starting your experiment

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.

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.

Base eVOLVER Requirements

1. From :

Rather than propose a single continuous culture device designed to a specific purpose, our goal with eVOLVER was to demonstrate a design framework that gives the user the freedom to imagine and carry out virtually any type of experiment that uses automated cell growth functionality to study cellular fitness. Here, we comment on reconfiguring eVOLVER for several experiments of significant interest in the community. For further examples, see Supplementary Table 1.

Overview

Implementing Controlled Host Cell Density in Reservoirs

Problem: PACE host cells overgrow

• In previous PACE experiments, host cells would increase growth rate as the experiment wore on

• This caused problems with phage replication in the lagoon and the selection plasmid breaking

• Solution: controlling host cell density in cell reservoirs

Implementing Controlled Host Cell Density in Reservoirs

• We control cell density in eVOLVER by running a turbidostat, which checks cell density and dilutes the culture if it is over a threshold.

• In PACE we remove volume from the cell reservoir and transfer it to the lagoon

  • This changes our turbidostat's volume

Chemostat and Turbidostat on the Same Vial

• We implement a "hybrid" function

• The "hybrid" function uses both a turbidostat and a chemostat on the host cell reservoir

• Turbidostat for keeping the cells from overgrowing

Vial Setup

Needles used were all 16ga

Reservoir volume = 30 mL

• Efflux needle = 3" needle in the tallest vial cap port

• Media in = 2" needle in the shortest port

• Vial to Vial = 3" needle in the second tallest port

Lagoon volume = 10 mL

• Efflux needle = 4" needle in the second tallest vial cap port

• Vial to Vial = 3" needle in the lowest port

• Inducer = 4" needle in the tallest port or 2" needle in the second lowest

Fluidic Lines

Hook up pump lines in the configuration shown below

Alter Settings in custom_script.py

1. Copy the whole ePACE template folder (/dpu/experiment/epace-template/)

2. Rename the copied folder to your experiment name

3. Change the EVOLVER_PORT to your eVOLVER's port

lower_thresh and upper thresh

• The lower and upper OD threshold of the turbidostat that is running on the reservoir vial

start_time

• chemostats will not pump until this amount of hours has elapsed

• Useful to allow cells in reservoir to grow up before starting experiment

rate_config

• In vial volumes per hour (V/h)

For the Reservoir

• Replaces volume in turbidostat that is removed via vial to vial

• Must be greater than the volume you are taking out

• Turbidostat controls will separately preventing reservoir from increasing in OD too much

For the Lagoon

• Set based off of phage replication rate

Example Settings:

1. If you have a 30mL reservoir and 10mL lagoon

2. Setting to rate_config: reservoir=1 and lagoon=1

   1. 30mL media into reservoir and 10mL from reservoir into lagoon per hour

Inducer

inducer_on

• Turn inducer off to start (inducer_on = False)

• Wait for host cells to grow up before starting induction (inducer_on = True) and inoculating with phage

inducer_concentration

• Times greater (X) the concentration of your inducer in its bottle compared to its final concentration in the lagoon

For example:

1. Your arabinose stock is 1 M

2. The final lagoon concentration you want is 10 mM

3. Therefore 1000 mM / 10 mM = 100 X your final concentration

Optional Settings

You do not need to alter these settings

Swapping Lagoon and Reservoir Vials

If you do want to alter these variables, you also need to swap the vial locations in the turbidostat and chemostat settings of:

• lower_thresh and upper thresh

• rate_config

reservoir_vial

• Vial numbers of host cell reservoirs

• Each reservoir vial is a turbidostat and a chemostat. Read why .

lagoon_vial

• Vial numbers of lagoons

• Only a chemostat, can have up to two inducers

Overview

Design Considerations

Overview

Light is calibrated a single vial at a time using an external light probe and data logger.

Files for calibration can be found in the calibration folder of your photo-eVOLVER DPU

Materials

• Light meter and probe

  1. Our light meter is the LI-COR

  2. Our light probe is the Walz US-SQS/L omnidirectional light

Protocol

1. Make sure you are using the calibration conf.yml not the experiment conf.yml

   • Experiment conf.yml will have pauses that cause missing values in the light calibration.

   • See for how to change to the calibration conf.yml

Morbidostat algorithms have been developed that gradually increase the selection pressure of an evolving culture, typically based on measured growth rate23. Previously, this algorithm has been implemented with two media inputs (+ and - drug), requiring three peristaltic pumps per culture (w/ efflux pump). In a 16-vial eVOLVER unit, this setup can easily be implemented by (1) controlling 48 pumps with the auxiliary board or (2) using multiplexed fluidics with the millifluidic devices. The prior being simpler to implement for 2 media inputs and the latter letting one scale to >2 inputs. As currently designed, the auxiliary board can control up to 48 fluidic elements (pumps/ solenoids). To run morbidostat mode, one would need to modify the Python code to the desired growth algorithm (e.g. control rate of drug increase, growth rate threshold to trigger the drug input).

Parts

Construction Protocol