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ePACE with min-eVOLVER
This page only details differences from the general min-eVOLVER experimental protocol. You are also expected to have an understanding of PACE and how it is normally run before doing ePACE.
- 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
- 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
- The amount we dilute will therefore be incorrect (adding 5mL of media to 30mL decreases OD less than adding 5mL of media to 20mL)
- We rely on host cells to get to a threshold cell density before we dilute
- They may not reach this threshold before we remove more volume
- This causes a feedback loop of little volume being added and more being taken out
- Therefore our turbidostat will get lower and lower volume and eventually break
- Solution: put in the amount of volume we take out of the cell reservoir
- 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
- Chemostat for keeping volume constant
Levels of liquid in the vials are set by the height of the efflux needle.
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
To have high accuracy when using the low volume pumps it is important to avoid individual drops. Therefore we want needles to abut inside of the vials to get a constant stream of fluid when pumping.

Vials set up for reservoir (left) and lagoon (right).
Hook up pump lines in the configuration shown below


- 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 - 4.Alter
USER DEFINED VARIABLES
using the guide below:

ePACE user defined variables as of 2023-11-20
- The lower and upper OD threshold of the turbidostat that is running on the reservoir vial
- Format:
[vial 0, vial 1]
- chemostats will not pump until this amount of hours has elapsed
- Useful to allow cells in reservoir to grow up before starting experiment
- Format:
rate_config = [reservoir, lagoon]
- In vial volumes per hour (V/h)
- 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
- Do not set too high or your cells will be unable to grow fast enough and wash out
- Set based off of phage replication rate
- 1.If you have a 30mL reservoir and 10mL lagoon
- 2.Setting to
rate_config = [1, 1]
- 1.30mL media into reservoir and 10mL from reservoir into lagoon per hour
- 3.Setting to
rate_config = [0.4, 1.2]
- 1.If we set lagoon rate to 1.2 V/h, we should not set reservoir rate to lower than 0.4 V/h to avoid draining the reservoir
- 2.1.2 V/h * 10mL = 12mL/h into lagoon
- 3.0.4 V/h * 30mL = 12mLh into reservoir
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
- Format
[pump 5, pump 6]
- 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
- 4.If you are not using another inducer,
inducer_concentration
= [100, 0]
You do not need to alter these settings
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
andupper thresh
rate_config
- Vial number of host cell reservoir
- Vial number of lagoon
- Only a chemostat, can have up to two inducers
Last modified 9d ago