[v1.1] ePACE with min-eVOLVER
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This page is for version 1.1 min-eVOLVERs ONLY! It does not apply if you have a sheet metal case
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.
ePACE described initially in Huang, Heins et al. 2022 Nature Biotech.
For general PACE methods see Miller, Wang 2020 Nature Protocols.
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.
Hook up pump lines in the configuration shown below
Copy the ePACE template under /dpu/experiment/epace-template/
Most likely you should not need to alter any settings in this section, other than EVOLVER_PORT
Use the "hybrid" function
Collapse or ignore growth_curve, turbidostat, and chemostat functions
hybrid function=vial 0 is the host cell reservoir
It is a turbidostat and a chemostat. Read why here.
We are setting OD for vial 0
start_time chemostats will not pump until this amount of hours has elapsed
rate_configDefault format: rate_config = [reservoir, lagoon]
In vial volumes per hour (V/h)
Reservoir
Set to greater than the volume you are taking out
Do not set too high or your cells will be unable to grow fast enough and wash out
Turbidostat controls will separately preventing reservoir from increasing in OD too much
Lagoon - set based off of phage replication rate
For example:
If you have a 30mL reservoir and 10mL lagoon
Setting to rate_config = [1, 1]
30mL into reservoir and 10mL into lagoon per hour
Setting to rate_config = [0.4, 1.2]
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
1.2 V/h * 10mL = 12mL/h into lagoon
0.4 V/h * 30mL = 12mLh into reservoir
Set inducer_concentration to X the final concentration in the lagoon
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
