[v1.1] ePACE with min-eVOLVER

This page is for version 1.1 min-eVOLVERs ONLY! It does not apply if you have a sheet metal case

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.

Two min-eVOLVERs set up for ePACE. They are connected to a dedicated laptop (preliminary picture)

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

custom_script.py

Copy the ePACE template under /dpu/experiment/epace-template/

"USER DEFINED GENERAL SETTINGS"

1. Most likely you should not need to alter any settings in this section, other than EVOLVER_PORT

2. Use the "hybrid" function

3. Collapse or ignore growth_curve, turbidostat, and chemostat functions

Alter settings in the hybrid function=

vial 0 is the host cell reservoir

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

• We are setting OD for vial 0

start_time

chemostats will not pump until this amount of hours has elapsed

rate_config

1. Default format: rate_config = [reservoir, lagoon]

2. In vial volumes per hour (V/h)

3. Reservoir

   1. Set to greater than the volume you are taking out

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

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

4. Lagoon - set based off of phage replication rate

5. For example:

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

   2. Setting to rate_config = [1, 1]

      1. 30mL into reservoir and 10mL 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

1. Set inducer_concentration to X the final concentration in the lagoon

2. Turn inducer off to start (inducer_on = False)

3. Wait for host cells to grow up before starting induction (inducer_on = True) and inoculating with phage

Picture of default min-eVOLVER settings as of 04/14/23.