# Experiment Setup

## Vial Setup

{% hint style="info" %}
Levels of liquid in the vials are set by the height of the efflux needle.
{% endhint %}

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

{% hint style="info" %}
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.
{% endhint %}

<figure><img src="https://3394537773-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FYy5AdmKzUTn7D8n357Md%2Fuploads%2FzDqHeyqgVmX2LlGQWsCr%2Fimage.png?alt=media&#x26;token=3e56b991-5d79-494a-9038-2f2e3ec0e683" alt=""><figcaption><p>Vials set up for reservoir (left) and lagoon (right).</p></figcaption></figure>

## Fluidic Lines

Hook up pump lines in the configuration shown below

<figure><img src="https://3394537773-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FYy5AdmKzUTn7D8n357Md%2Fuploads%2F6iHp0wxRIujoS36PpHIh%2Fimage.png?alt=media&#x26;token=803a83ec-6bbd-44d6-be6b-b336d53ae692" alt=""><figcaption></figcaption></figure>

<figure><img src="https://3394537773-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FYy5AdmKzUTn7D8n357Md%2Fuploads%2FMb2VNXkaNJhpbMAOCtn6%2Fimage.png?alt=media&#x26;token=e0e1e94f-3a32-4323-ada8-e6ac4754d6bf" alt=""><figcaption></figcaption></figure>

## 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:

<figure><img src="https://3394537773-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FYy5AdmKzUTn7D8n357Md%2Fuploads%2FAB7zQjiqvavEdC1WqyGF%2Fimage.png?alt=media&#x26;token=91b58574-e062-42d4-91ae-7c0c55e20817" alt=""><figcaption><p>Image of the ePACE settings as of 7/9/25</p></figcaption></figure>

### `lower_thresh` and `upper thresh`

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

### `start_time`&#x20;

* 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
* 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: reservoir=1 and lagoon=1`
   1. 30mL media into reservoir and 10mL from reservoir into lagoon per hour
3. Setting to `rate_config: reservoir=0.4 and lagoon=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

#### 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`&#x20;

* Vial numbers of host cell reservoirs
* Each reservoir vial is a turbidostat *and* a chemostat. Read why [here](https://khalil-lab.gitbook.io/evolver/extensions/custom-experiments/epace/..#implementing-controlled-host-cell-density-in-reservoirs).

#### `lagoon_vial`&#x20;

* Vial numbers of lagoons
* Only a chemostat, can have up to two inducers