This page is for version 1.1 min-eVOLVERs ONLY! It does not apply if you have a sheet metal case
Overview
This page only details differences from the general min-eVOLVER experimental . You are also expected to have an understanding of PACE and how it is normally run before doing ePACE.
ePACE described initially in.
For general PACE methods see .
Experimental Overview
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
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.
Fluidic Lines
Hook up pump lines in the configuration shown below
custom_script.py
Copy the ePACE template under /dpu/experiment/epace-template/
"USER DEFINED GENERAL SETTINGS"
Most likely you should not need to alter any settings in this section, other than EVOLVER_PORT
Use the
Collapse or ignore growth_curve, turbidostat
Alter settings in the hybrid function=
vial 0 is the host cell reservoir
It is a turbidostat and a chemostat. Read why .
We are setting OD for vial 0
start_time
chemostats will not pump until this amount of hours has elapsed
rate_config
Default format: rate_config = [reservoir, lagoon]
In vial volumes per hour (V/h)
Reservoir
Inducer
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
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
Chemostat for keeping volume constant
, and
chemostat
functions
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
