ePACE described initially in Huang, Heins et al. 2022 Nature Biotech.
For general PACE methods see Miller, Wang 2020 Nature Protocols.
For running ePACE, OD calibration via growth curve is required because S2060 cells have significantly different scattering properties while growing vs during stationary phase.
This section constitutes changes from Huang, Heins et al. 2022 Nature Biotech.
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
Last updated 1 year ago
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