ePACE with min-eVOLVER

Overview

This page only details differences from the general min-eVOLVER experimental protocol. You are also expected to have an understanding of PACE and how it is normally run before doing ePACE.

• ePACE described initially in Huang, Heins et al. 2022 Nature Biotech.

• For general PACE methods see Miller, Wang 2020 Nature Protocols.

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

  • 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

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.

Vials set up for reservoir (left) and lagoon (right).

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

4. Alter USER DEFINED VARIABLES using the guide below:

ePACE user defined variables as of 2023-11-20

lower_thresh and upper thresh

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

• Format: [vial 0, vial 1]

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

• Format: rate_config = [reservoir, lagoon]

• 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

• Do not set too high or your cells will be unable to grow fast enough and wash out

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 = [1, 1]

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

3. Setting to rate_config = [0.4, 1.2]

   1. 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

   2. 1.2 V/h * 10mL = 12mL/h into lagoon

   3. 0.4 V/h * 30mL = 12mLh into reservoir

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

• Format [pump 5, pump 6]

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

4. If you are not using another inducer, inducer_concentration = [100, 0]

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 number of host cell reservoir

• It is a turbidostat and a chemostat. Read why here.

lagoon_vial

• Vial number of lagoon

• Only a chemostat, can have up to two inducers