ePACE

Overview

• 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.

Implementing Controlled Host Cell Density in Reservoirs

This section constitutes changes from Huang, Heins et al. 2022 Nature Biotech.

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

  • 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 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

• Chemostat for keeping volume constant