ePACE

Overview

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

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