Overview

Implementing Controlled Host Cell Density in Reservoirs

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

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

Fluidic Lines

Hook up pump lines in the configuration shown below

Alter Settings in custom_script.py

1. Copy the whole ePACE template folder (/dpu/experiment/epace-template/)

2. Rename the copied folder to your experiment name

3. Change the EVOLVER_PORT to your eVOLVER's port

lower_thresh and upper thresh

• The lower and upper OD threshold of the turbidostat that is running on the reservoir vial

start_time

• chemostats will not pump until this amount of hours has elapsed

• Useful to allow cells in reservoir to grow up before starting experiment

rate_config

• In vial volumes per hour (V/h)

For the Reservoir

• Replaces volume in turbidostat that is removed via vial to vial

• Must be greater than the volume you are taking out

• Turbidostat controls will separately preventing reservoir from increasing in OD too much

For the Lagoon

• Set based off of phage replication rate

Example Settings:

1. If you have a 30mL reservoir and 10mL lagoon

2. Setting to rate_config: reservoir=1 and lagoon=1

   1. 30mL media into reservoir and 10mL from reservoir into lagoon per hour

Inducer

inducer_on

• 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

inducer_concentration

• Times greater (X) the concentration of your inducer in its bottle compared to its final concentration in the lagoon

For example:

1. Your arabinose stock is 1 M

2. The final lagoon concentration you want is 10 mM

3. Therefore 1000 mM / 10 mM = 100 X your final concentration

Optional Settings

You do not need to alter these settings

Swapping Lagoon and Reservoir Vials

If you do want to alter these variables, you also need to swap the vial locations in the turbidostat and chemostat settings of:

• lower_thresh and upper thresh

• rate_config

reservoir_vial

• Vial numbers of host cell reservoirs

• Each reservoir vial is a turbidostat and a chemostat. Read why .

lagoon_vial

• Vial numbers of lagoons

• Only a chemostat, can have up to two inducers