# ePACE with min-eVOLVER

## Overview

This page only details differences from the general min-eVOLVER experimental [protocol](/evolver/extensions/min-evolver/starting-an-experiment.md). You are also expected to have an understanding of PACE and how it is normally run before doing ePACE.

{% hint style="info" %}

* ePACE described initially in[ Huang, Heins et al. 2022 *Nature Biotech*](https://www.nature.com/articles/s41587-022-01410-2#Sec15)*.*
* For general PACE methods see [Miller, Wang 2020 *Nature Protocols*](https://www.nature.com/articles/s41596-020-00410-3)*.*
  {% endhint %}

{% hint style="warning" %}
For running ePACE, OD calibration via [growth curve](/evolver/extensions/min-evolver/calibrations/od-calibration-via-growth-curve.md) is *required* because S2060 cells have significantly different scattering properties while growing vs during stationary phase.
{% endhint %}

## Implementing Controlled Host Cell Density in Reservoirs

{% hint style="info" %}
This section constitutes changes from [Huang, Heins et al. 2022 *Nature Biotech*](https://www.nature.com/articles/s41587-022-01410-2#Sec15)*.*
{% endhint %}

#### 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


---

# Agent Instructions: Querying This Documentation

If you need additional information that is not directly available in this page, you can query the documentation dynamically by asking a question.

Perform an HTTP GET request on the current page URL with the `ask` query parameter:

```
GET https://khalil-lab.gitbook.io/evolver/extensions/min-evolver/epace-with-min-evolver.md?ask=<question>
```

The question should be specific, self-contained, and written in natural language.
The response will contain a direct answer to the question and relevant excerpts and sources from the documentation.

Use this mechanism when the answer is not explicitly present in the current page, you need clarification or additional context, or you want to retrieve related documentation sections.