eVOLVER was initially developed by the Khalil Lab at Boston University. The software and hardware designs are available under an open-source license on GitHub. There are users around the world using and developing eVOLVER for a wide range of scientific interests.

Khalil Lab

The Khalil lab uses eVOLVER to enable fundamental eco-evolutionary studies of cellular systems, and to evolve proteins with new and biomedically-useful functions at scale.

• Brandon Wong

• Zachary Heins

• Daniel Hart

• Ezira Wolle

• Nathaniel Borders

Alumni

• Chris Mancuso

Buying

If you wish to purchase an eVOLVER, email account@fynchbio.com for a quote.

Upgrades

There is one essential upgrade to the Fynch Bio eVOLVER (as of April 2024).

eVOLVER Community Code of Conduct

The eVOLVER community follows the CNCF Code of Conduct.

Instances of abusive, harassing, or otherwise unacceptable behavior may be reported by contacting heinsz@bu.edu and ccing evolvercommunity@gmail.com

Check out the forum!

New to eVOLVER, or have any questions about how something works or how to do something? Head over to our Discourse forum! We have many guides and resources available and are happy to start a conversation about your eVOLVER and science! https://evolver.bio

What are the different platforms used for?

Forum

For questions, discussion, and knowledge building

Should users find a problem, they should characterize it and submit it to group discussion on the forum

GitHub

For tracking and distributing code and hardware development.

Not for discussion

Wiki - Documentation

Basic information about the eVOLVER (about the project, contributing, where to buy)

Guides

In-depth hardware / software documentation

How to Edit the Wiki

Publications

Background, final data and experiments

Extensive documentation in the supplements, meant for reference

Useful Links

Forum for asking questions, posting guides, and discussing eVOLVER related topics.

Nature Biotechnology Paper

Nature Biotechnology Supplement

JoVE Paper - Visual protocol for running an eVOLVER continuous culture experiment.

Software Repositories

Server (RPi) Code Repository

Data Processing Unit (DPU) Code Repository

Arduino Code Repository

Electron GUI Code Repository - Graphical UI for interacting with the eVOLVER platform

Hardware Repositories

eVOLVER Hardware Repository - eVOLVER Hardware (PCB, 3D printed parts)

Page under construction!

Link to tutorial on forum for DPU installation

How to add multiple evolvers on the same computer

Can go through building router, but contact institution IT (tell them it's RPi)

Page under construction

Forum post

Log in to router and make it have a static IP

otherwise IP will change each time it's power cycled

Then when connecting via ssh it will warn you and you need to change the file

You can then change the name, MAC address, and IP of the evolver in the evolver-config.json file

Router IP: 192.168.1.1

Login: admin

pw: password

You can run eVOLVER experiment culture routines directly on the command line, or through the Electron graphical interface. This page documents how to install the DPU for use on the command line.

(Mac only) Homebrew and openssl/sqlite installation

In Terminal, install homebrew:

/bin/bash -c "$(curl -fsSL https://raw.githubusercontent.com/Homebrew/install/HEAD/install.sh)"

Afterwards, run the two following commands:

brew install openssl

brew install sqlite

Install Python 3.9

Python 3.9 is required for dpu operation

Mac:

brew install python@3.9

Windows

1. Go to the official python website to download python 3.9.

2. Follow these instructions to install python on your machine.

3. If you have multiple versions of python3 on your machine, you can set up an alias to manage the usage between the different versions.

DPU Installation

1. Navigate to the DPU Github page

2. Click Releases on the right side of the page

3. Click the Source Code link (zip) to download the latest release.

4. Move the downloaded .zip file (which should be in your Downloads directory for your browser) to the location where you want your experiments to run. Documents or Desktop are good locations. This is also where your data will be saved.

5. Unzip the file.

6. Open a terminal. On Mac, you can use the built in Terminal application (press CMD + Space then type terminal to find it). I recommend iTerm2 if you want a terminal with some quality of life enhancements. On Windows, you can use PowerShell.

7. We use poetry to manage our python dependencies. Go to their website to download and install it on your machine.

• If you are using a Mac and have installed homebrew (described above) you can run the following command: brew install poetry

• Windows Powershell:

(Invoke-WebRequest -Uri https://install.python-poetry.org -UseBasicParsing).Content | py -

8. Navigate to the DPU directory you just downloaded and unzipped:

• Use this command: cd <path_to_dpu>

• Replace the <path_to_dpu> with the location you put your dpu

9. Create a new virtual environment then activate it with the following command:

Mac:

python3.9 -m venv venv

source venv/bin/activate

Windows PowerShell:

py -3.9 -m venv venv

venv\Scripts\Activate.ps1

10. Use poetry to install all necessary dependencies.

poetry install

If you can't find poetry on Windows "poetry : The term 'poetry' is not recognized as the name of a cmdlet" and it is installed, use the following command (replace <Your_User_Name> with your user name)

C:\Users\<Your_User_Name>\AppData\Roaming\Python\Scripts\poetry.exe install

Algorithm

1. OD is constantly monitored

2. When OD goes above the an upper OD threshold

3. A dilution is calculated to bring OD to the lower threshold

   1. Dilutions are capped at 20 seconds to avoid vial overflow

4. Both influx and efflux pumps are run simultaneously for the calculated length

5. Efflux pumps are run extra to make sure volume stays constant

   1. The efflux straw sets volume

Turbidostat Dilution

Pumps

• Influx and efflux are both run at the same time

• Like chemostat, vial volume is set by efflux straw height

  • Efflux is run extra time to make sure that enough volume is removed

• Pumps run at roughly 0.75mL/second

• Pump calibration is important for turbidostat otherwise dilutions will not hit the lower OD threshold accurately

Dilution Calculation Example

Example Dilution

time_in = -(np.log(lower_thresh[x]/average_OD) *VOLUME)/flow_rate[x]

However, the mox dilution is 20 seconds to avoid overflow.

More Info

Guidelines

1. Use the below Google Sheet to source parts for each of the eVOLVER subsystems (click on the tabs for different parts of the eVOLVER)

2. Parts may not be available due to backorder, discontinuation, or regional differences

3. If you are able to find an alternative vendor for a part, please post to the forum so it can be added to the spreadsheet

4. Should you not be able to order a part, post to the forum

eVOLVER Specific Parts

Many eVOLVER parts must be 3D printed or are custom PCBs made to control eVOLVER hardware. For these, please see the hardware GitHub for CAD files and electronics parts lists for eVOLVER PCBs.

Vial Caps

Nylon Caps should be 3D printed from the hardware GitHub or ordered from Fynch Bio

• Once nylon caps arrive, clean excess nylon powder out of the tubes with a drill bit or small pipe cleaner

Old caps: Making vial caps w/ laser cutter and pipette tip

Relevant Forum Posts

eVOLVER Consumables

Vial Racks

Waste Containers

If you have any questions about this process, feel free to reach out on the forum!

Congrats on getting your eVOLVER and supporting our open-sourced community. We rely on support from you guys to help sustain this ecosystem so we can let everyone do the experiments that they want to! In this tutorial, we are going to teach you how to put this system together:

If you ordered the standard kit, this is the tutorial for you! The kit contains the following major components:

• Vial Platform

• 16 Smart Sleeves

• 2x Pump Arrays (total 32 pumps)

• Fluidic Box to for control of 32 Pumps (can expand to 48 w/ additional hardware)

1) First Unboxing the System

There are several components with protective foam. Insure all the components were delivered intact.

16 Smart Sleeves and a Box of Accessories

Pump Array and Fluidics Box

Vial Platform

2) Set up power and ethernet for the vial platform

The Vial Platform is the unit on which all the Smart Sleeves will be screwed onto. This system contains the eVOLVER Motherboard, Raspberry Pi, and Power Modules that control the Smart Sleeves. Parts used to setup the Vial Platform should be labeled with a black dot.

Contents of the Vial Platform Components Bag:

First, plug in Power and the Ethernet Connection to the system and ensure all the components turn ON properly (e.g. the touch screen turns ON). For more information on how to configure the router, please read the tutorial on router setup.

The Ethernet should plug into your router, more information about assigning a static IP in this tutorial.

3) Plug in and screw on Smart Sleeves

If the system turns ON properly, turn off the eVOLVER and start fastening the Smart Sleeves. Use the 4-40 screws to fasten down each Smart Sleeve (2 screws/ sleeve). Vial 0 is in the front left (closest to the touch screen) with vials 1, 2, and 3 on the right of vial 0, in that order. Vial 15 is in the back right (farthest from the touch screen).

4) Setup pump rack

The Fluidics Platform is a separate module than the Vial Platform, typically is placed next to the Vial Platform. For this next part, we are setting up a pump rack that goes on top of the Fluidics Platform.

First we want to set up the components for the pump rack. The parts for this is located in the Fluidic Box Components Bag (Red Dot).

We want to fasten (w/ M6 screws) in aluminum extrusion into 3D printed part for the front pieces first. The front piece are the ones with the longer feet. See below:

Next, insert back piece. Make sure the two feet are offset in the same direction. Do not screw the screws in yet. We will screw those in when we mount everything together with the acrylic piece.

Repeat for the other side.

Next, line up the acrylic piece to the back holes and fasten together with M6 screws.

Screw in 10-32 screws to the feet of the mounts to fasten the pump rack on top of the Fluidics Box. Repeat to fasten all four mounts

5) Plug in Power + Communications to Fluidics Platform

Next, let’s setup the fluidics box. The fluidic box requires several connections:

• Two Power Entry Cables (one for 12V Power, other for 5V)

• Serial Communication Cable (To communicate with Raspberry Pi in Vial Platform)

Power the 5V power in the Fluidic Box from the USB port of the Raspberry Pi.

Connect Serial Communication Cable between the Vial Platform and the Fluidics Platform.

6) Attach Pump Arrays to the Fluidics Box

The fluidic box is meant to be flexible, to power whatever fluidic components you want. The standard setup assumes the following setup (back of fluidics box):

Attach ribbon cables to the appropriate slots on the PCB the pumps are mounted on.

Top down view of ribbon cable connections.

Finally, attach the tubing to the appropriate pumps.

7) Label Smart Sleeves with Appropriate Label

Labeling the vials with the appropriate color coded labels help minimize human error (I just print it and tape it on the vial). You can download a PDF of the file here 17. The front left position (row closest to touchscreen) is vial 0.

Checklist

Before starting experiment prep:

1. Finished Getting Started Page

2. Calibrations

   1. Temperature (must be first, affects OD cals)

   2. OD

   3. Pump calibrations

3. Should things not be as expected

Sample Experimental Workflow

Day 0:

Make sure vials / pumps are behaving as expected

• Are all vials stirring, getting up to temperature, all pumps pumping, etc?

• Especially check that efflux is running long enough to remove enough culture from vials

Start overnight cultures

Day 1:

Inoculate cultures into vials

Monitor experiment for correct behavior (over several hours)

• Are the cells growing?

• Is the experiment diluting as expected?

• Are all pumps working?

Day 2 - Onwards:

Monitor media levels - make sure that there is more than enough media to make it to the next time you check the experiment

Monitor biofilming on the vials - this can cause increased OD over time

Relevant Forum Posts

Protocol

1. Rinse vial lids by running DI water from sink through influx and efflux straws (2-3 seconds). Alternatively fill a large beaker with DI water, dump vial lids in and stir.

2. Shake vials caps to remove excess water and set aside on a clean surface

3. Get out enough dishwashed vials (stored upside down), place in white rack

4. Place a small stirbar in each vial

5. If using septa, place a septa on each

6. Screw on rinsed vial lids

7. If vials will be used within 24 hours of prepping, then place white rack in small autoclave bin, cover entire bin with foil (reuse foil if possible), and autoclave on vacuum cycle for 10 minutes

8. Keep foil on bin until right before loading vials into eVOLVER to maintain sterility

9. If vials are being prepped in advance, screw a white or black mini-cap onto every straw, then autoclave in bin without foil

10. Keep mini-caps on until right before loading vials into eVOLVER to maintain sterility

Materials

Media Requirements

A primary consideration when running eVOLVER experiments is predicting the media consumption rate, which is typically orders of magnitude higher than in typical batch experiments. The required media for continuous culture can be estimated using the following equation: 𝑀𝑒𝑑𝑖𝑎 𝑅𝑒𝑞𝑢𝑖𝑟𝑒𝑑 [𝑚𝐿] = 𝐶𝑢𝑙𝑡𝑢𝑟𝑒 𝑉𝑜𝑙𝑢𝑚𝑒 [𝑚𝐿] / 𝐴𝑣𝑔. 𝐷𝑜𝑢𝑏𝑙𝑖𝑛𝑔 𝑇𝑖𝑚𝑒 [ℎ] ∗ 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓𝐶𝑢𝑙𝑡𝑢𝑟𝑒𝑠 ∗ 𝐷𝑢𝑟𝑎𝑡𝑖𝑜𝑛 [ℎ] (1) It should be noted that additional media (~20mL) is needed during device setup to flush media input lines.

Protocol

1. Before preparing media, steam clean the carboy by filling with ~2L of water and autoclaving on the liquids cycle for at least 45 minutes. Be sure to keep the lid loose to prevent the carboy from collapsing. Pour out the water.

2. If you are able to prepare a concentrate of your media, you can do so as a 10x 2L solution (or whatever makes sense for your media) in a beaker with a stir bar. It is also possible to mix everything directly in the carboy. Add your media components in small batches and try to mix as you go.

3. Fill the carboy up to 20L - volume of media components that will be supplied after the autoclave step. For example, if you need your media to be 2% glucose, you should fill the carboy to 18L and then add 1L of filter sterilized 40% glucose after autoclaving.

4. Place a male Luer cap on the outlet of the carboy lid. Place a filter on the inlet, and cover it with foil. Loosen the entire lid.

5. Place the carboy into an autoclave bin, then place into an autoclave. Autoclave on the liquids cycle for 2 hours.

6. Take the carboy out of the autoclave.

7. If media components need to be added after autoclave (glucose, antibiotics, supplements, etc.) place the carboy on a cart and move next to a bench flame. Sterilize the area thoroughly with ethanol before quickly unscrewing the lid to make the additions. Mix by placing the carboy on a cart and moving the cart in a circular motion, or by placing on the floor, tilting ~30 degrees, and moving the top in a circular motion.

8. You can place the autoclave on the floor next to your experiment. Be sure to keep the lid loose to prevent negative pressure buildup in the carboy which could prevent media from going into the culture vessels.

9. After the carboy is empty, rinse thoroughly with water, being sure to run water through all of the tubing. Steam clean as described in step 1, then pour out the remaining liquid.

Relevant Forum Posts

For all operating systems, navigate to the section of the Electron App GitHub page.

For instructions on updating the GUI on the RPi, see .

Mac Installation

1. Download the evolver-electron-X.X.X.dmg file, where X denotes the version number in the latest release. The latest release should be the first one you see at the top of the page.

2. When the download finishes, open the file (it should be in your Downloads directory) and drag the eVOLVER app into your Applications directory.

3. Open the eVOLVER app from the Applications directory.

4. Using an editor of your choice (TextEdit, Nano, Vim, TextWrangler, etc.) open the file config.json located at /Users/<your username>/Library/Application Support/eVOLVER/config.json

5. Add a line in this file between the braces: "dpu-env": "/Users/<your username>/Document/dpu/venv"

6. Save the file and you're done!

Windows Installation

1. Download the evolver-electron-X.X.X.exe file, where X denotes the version number in the latest release. The latest release should be the first one you see at the top of the page.

2. Open the .exe file.

3. Using an editor of your choice open the file config.json located at C:\Users\<Your-UserName>\AppData\Roaming\eVOLVER\

4. Add a line in this file between the braces: "dpu-env": "C:\\Users\\<Your-UserName>\\Desktop\\dpu\\venv"

Note the escaped \ , be sure to put double \\ throughout the path.

5. Save the file and you're done!

Linux Installation

1. Download the evolver-electron-X.X.X.AppImage or evolver-electron-X.X.X.deb file, where X.X.X denotes the version number in the latest release. The latest release should be the first one you see at the top of the page.

2. If downloading evolver-electron-X.X.X.deb, open your Downloads directory and right-click evolver-electron-X.X.X.deb to open the file with Software Install .

3. A new window should open, allowing you to install the eVOLVER GUI. Click Install and follow the onscreen instructions to install the eVOLVER GUI.

4. Open the eVOLVER app by either clicking Show Applications from the home screen or through the command line. To open via command line, open Terminal and enter to access installed applications:

Run the eVOLVER GUI by entering:

5. Using an editor of your choice, open the file ~/.config/eVOLVER/config.json .

6. Add a line in this file between the braces to denote the location of the installed DPU virtual environment:

"dpu-env": <path_to_dpu>

7. Save the file and you're done!

About

In order to appropriately interpret data from and actuate physical elements connected to the eVOLVER framework, a relationship must be established between the electrical signals generated by attached sensors and the physical phenomenon they are measuring. This is done by manually measuring values on a controlled set of data via some gold-standard assay or measurement device and comparing these data to the eVOLVER generated data. By doing this across a range of different values, a function can be fit to this relationship to allow eVOLVER to interpret data accurately.

For more information see .

Procedure

Calibrations should proceed in the following order:

1. Place waste lines into carboy

2. We use a small stand to bring the carboy closer to the eVOLVER

3. Have an extra carboy ready to swap in when the first gets full

4. (Optional) You can fill 10% of the carboy with bleach

   1. Or bleach after depending on whether you hate the smell bleach or your culture more

5. (Optional) Shrink onto the waste lines to make them easier to deal with

6. (Optional) Drill a hole through the carboy cap to make the opening to the carboy smaller (less smelly)

7. (Optional) Parafilm the opening to the carboy to make it less smelly

You should be able to follow the 2019 JoVE for most of the process. Differences will be highlighted here.

Running experiments with Dropbox/Google Drive

If you would like your data to be automatically backed up on Dropbox or Google Drive, first install the desktop applications. This will enable any computer with Dropbox/Google Drive and access to the directory to monitor the experiment using the eVOLVER GUI in real-time, while providing all the benefits of using these services (automatic backup of data, ease of data sharing, etc).

We recommend saving your experiments through Dropbox or Google Drive, but you do not have to - it's fine to have everything run locally on your lab computer. Both options will be detailed in the section below.

Setup

Select the eVOLVER you wish to run an experiment on in the dropdown on the top right, then click the SETUP button.

On left side of the screen, ensure the correct calibrations are selected for Temp, OD, and the pumps. Click the box to bring up a list of available calibrations for each parameter.

Once you have selected the calibrations, you can set the initial stir and temperatures, and sterilize the fluidics as described in the JoVE paper.

Experiment Manager

The JoVE paper is out of date with regards to using the GUI for running and managing an experiment, from 3) Configuring eVOLVER Software and Programming Algorithmic Culture Routines on.

Open the experiment manager on the Home screen of the GUI. You should see something that looks like this if this is the first time you have used the application:

In the top center the Expt Directory is listed out. You can click the pen icon to change this location to be anywhere you like.

If you have Dropbox or Google Drive installed, you can select a location on the cloud to save your experiments. This will enable you to view your data from any computer that has permissions and the Dropbox/Google Drive application installed!

You do not need to make a new folder called experiments as shown below - the GUI will automatically create this directory.

Click the New Experiment button to open a prompt to name your experiment. Type a name then click Submit.

Click the Pen button on the row of the new experiment to bring up with experiment editor page.

Experiment Editor

By default, the Turbidostat editor will appear when you come to the editor page.

1. The name of the experiment. You can click the pen icon to change the experiment name.

2. The parameter selector. The name of the currently selected parameter is listed at the top. You can click the -/+ buttons to change the value (or use the slider), and then click the box in the middle to apply this value to the selected vials.

3. Experiment control panel. From left to right: Start/Stop experiment, View Data, Save Files (Only for File Editor), Copy Experiment, View Files in File browser, Delete experiment.

4. eVOLVER Selector. Select the eVOLVER to run the experiment on.

5. Experiment type. Default will bring up the Turbidostat editor. Chemostat and growth rate editors are also available. You can also bring up the file editor to directly modify the python files within the GUI.

6. Vial selector - select the vials and then apply desired experimental parameters from the buttons on the parameter selector. Values display the currently set parameters.

7. Vial selector control buttons - flip the orientation on the display or select all of the vials.

8. Reset - goes back to default. Save Experiment - saves the parameters for all vials.

The chemostat and growth rate modules function similarly to the turbidostat one.

Clicking File Editor in the experiment type dropdown will allow you to directly modify the python files.

You can select the file you would like to edit on the left, edit it on the right, and save by clicking the Save icon on the bottom left. For most cases you will not need to use this! If you change the files, you will not be able to use the t-stat/c-stat/growth rate modules, and you will need to modify the EVOLVER_IP, OPERATION_MODE, and other variables throughout the code.

You can also click the folder icon on the bottom left to view the files in a file browser and edit them using an editor of your choice.

Starting the experiment

When you're ready to start the experiment, click the Play button either on the Experiment Manager page or the Experiment Editor page. Then you can click the graphs icon to go watch your experiment running in real-time.

When you navigate to the graphs page, the graphs will look fairly empty to start. After a minute or so, data will start to appear as it is collected. You can also click the VIEW LOGS button to see how the experiment is running.

On the left, you can modify how the graphs are displayed by clicking the appropriate radio buttons. You can also look close up at an individual vial and use the data slider at the bottom of the graph to zoom in on an area of interest.

When you click ALL to go back to seeing all vials, the range set on a single vial will be applied to all vials. You can click the radio buttons to set it back to the latest data.

To end the experiment, click the STOP square button on the device running the experiment.

Protocol

1. In three large beakers, prepare 500 mL of 10% bleach, 200 mL of 10% bleach and 300 mL 70% ethanol.

2. Using the switches on the eVOLVER device, turn on the 5 V power supply on the eVOLVER, wait for >3 seconds, then turn on the 12 V power supply.

3. If running several vials from the same media bottle, connect multiple media input lines with tubing splitters. Custom tubing splitters can be with Luer components.

4. Submerge the media input lines (clear, media in) in the first bleach beaker, and the media efflux lines (blue) in the second bleach beaker. Place the downstream end of the media input lines (clear, to vial) in the second beaker with the efflux lines. Since the lines may be different lengths, a hairclip is useful to keep the ends of each lines close to eachother in order to ensure all remain submerged.

5. Add 1 L bleach into large empty waste carboy, which will sterilize waste generated during experiment. Consult with a safety coordinator to ensure proper disposal of waste.

6. Using the switches on the eVOLVER device, turn on the 5 V power supply on the eVOLVER, wait for 5 seconds, then turn on the 12 V power supply.

7. If running several vials from the same media bottle, connect multiple media input lines with tubing splitters (drawer under eVOLVER computer) before sterilizing. Custom sizes can be made if necessary.

8. Submerge the media input lines in the first bleach beaker, and the media efflux lines in the second bleach beaker.

9. Place the downstream end of the media input lines in the second beaker with the efflux lines. Place waste lines into waste carboy.

10. Open the eVOLVER electron app, select the appropriate eVOLVER from the dropdown menu.

11. Navigate to setup, then fluidics. Highlight the appropriate vials, select the influx and the efflux pumps, and run pumps for 20 seconds to fill lines with 10% bleach.

12. While running, ensure by visual inspection that all lines have been filled and that pumps are operating normally. Allow bleach to sit in the lines for at least 30 minutes to sterilize. This is a good pause point, can wait up to overnight. This is a good time to make code edits if necessary.

13. Run pumps again using the touchscreen app until lines are no longer submerged, pushing air through the lines to get as much of the bleach out as possible.

14. Place media input lines in the ethanol beaker (or pour ethanol into the first beaker) and repeat as in above, filling the lines with ethanol and flushing with air. Note: ethanol sterilizes quickly, so no need to wait 30 mins here.

15. It is also recommended to dip the downstream ends of the influx lines in ethanol to rinse off residual bleach, then drape on side of beaker rather than resubmerging in the remaining bleach. Residual bleach can affect culture viability, though washing with ethanol and media should be sufficient.

16. Attach media input lines to the media bottles with the Luer connectors and run the pumps until the media fully runs through the lines (10-20 seconds, depending on tubing), flushing out any residual ethanol.

Code Breakdown and Explanation of Flow Rates

This section is assuming you already have gone through the , which outlines basic parts of the script, and lets one run an initial pilot experiment.

Here, I will explain chemostat functionality. This is the segment where the chemostat function begins on

• OD_data: The OD_data variable saved from the transformed values from the input_data variable. The data is transformed via calibration scripts called from eVOLVER.py.

• start_OD: This variable determines if you want the culture to be above a certain OD before starting dilutions

• start_time: This variable determines when the dilutions should start. If set to 0, the dilutions start immediately

• OD_values_to_average: The script will average N recorded OD values before it triggers the dilution via the start_OD variable.

• chemostat_vials: Modify this script to to turn off vials (e.g. pumps). There is a known bug on the rate definition that might not turn off the pumps there (see below).

The peristaltic pumps are fixed flow rates, does that mean I have to use different pumps for each flow rate setting?

No, with the eVOLVER setup, we approximate continuous perfusion by frequent small boluses of media. By doing this, a faster pump can achieve tunable, slower net flow rates. In our experience, this compromise does not significantly impact the culture conditions of common laboratory microbes.

Here is an example calculation one would make:

• The culture volume is 30 mL/ vial.

• The typical pumps eVOLVER comes with have a fixed flow rate of 0.5 - 1.0 mL/s. We typically recommend a minimum bolus of 0.5 mL per dilution, meaning 60 boluses would turnover the culture once.

  • FynchBio also sells slower pumps that flow at ~1 mL/ min

• If we want our culture to double every 3 hours, the pumps will fire every 3 minutes (180 min/ 60 boluses). In a chemostat, nutrient is limiting thus growth rate == dilution rate.

• A doubling time of 3hr has a growth rate of 0.231/hr via the following equation:

Below, we set the script such that the cultures will all have a 3 hour doubling:

If one wanted to set the V0-3 at a doubling time of 3 hours but the rest of the 14 vials with a doubling time of 6 hours, the rate_config would be set as the following:

NOTE (5/12/2023): There is a bug that does not turn OFF pumps if defined to 0 on rate_config variable.

Code for interfacing with eVOLVER GUI

Below is code that grabs values from the GUI and populates it in the code. If you aren't using the GUI, please ignore this portion of the code:

Rest of the Chemostat Configuration Settings

The following parameters initialize variables that will calculate how much time to turn the pumps ON to maintain the flow rate set in rate_config

• flow_rate: get the calibrated flow rates for each pump

• period_config: how often to pump (seconds)

• bolus_in_s: how long to pump during a given pump event (seconds)

Chemostat Pump Commands

When we set vials to a new flow rate in the chemostat function, we see the experiment script send a comma separated pump command. Each pump assigned to the a chemostat gets a command that looks like this:

1.18|144

Which is in the format of:

bolus_in_s|period_config

If the above pump was influx, its corresponding efflux pump would be 2.36|144 because we pump out double the time to avoid overflows.

Stopping Experiment and Bleaching

1. In the GUI electron app or command line, click to stop your experiment.

2. In large beakers, prepare 500mL of 10% bleach and 200 mL of 10% bleach.

3. Disconnect media bottles and place lines into 10% bleach solution

4. To use pumps to bleach the cultures, keep tubing connected, but move vials to a white eVOLVER rack. Prepare an additional 1L of 10% bleach and add to the media input beaker.

5. Run 10% bleach through vials until final concentration in the vials reaches ~10% bleach (~60 seconds for 25mL volume). Disconnect influx and efflux lines from vials, set vials aside for 30 mins.

6. To manually clean vials, disconnect influx and efflux lines and skip steps 39 and 40

7. Submerge the disconnected influx and efflux lines in the 200mL bleach beaker, then run all pumps for 20 seconds to fill all lines with 10% bleach. Let sit for no less than 30 minutes, no more than 48 hours.

8. Remove media input lines from bleach, and run air though the lines to remove bleach. If not planning on using the device again right away, run diH2O through all lines for 20 seconds to remove residual bleach from the lines. Dried bleach can form salt crystals and clog the lines. If this happens, break up the crystals manually using pliers to squeeze the line.

9. Turn off the 12V power, then the 5V power.

10. Proceed to vial & bottle cleaning protocol.

Clean Vials After Bleaching Cultures

1. Prepare a beaker ~50% full of 10% bleach

2. Remove vial lids and place in 10% bleach solution

3. Using the magnetic stir bar grabber, remove the stir bars from each vial, place in the 10% bleach solution

4. Let vial caps and stir bars sit for 30 minutes-overnight in the 10% bleach solution

5. Meanwhile, dump bleached culture down the drain, rinse with diH2O

6. Use a test tube brush to scrub inside of vial, particularly important if you grew bacterial cultures

7. Place bleached/scrubbed vials right-side-up in the vial racks to indicate they have not yet been dishwashed

8. Once vial lids/stir bars have been soaked for sufficient time, carefully pour out the 10% bleach solution, and replace with diH2O

9. Let vial lids and stir bars soak for another 30 mins-overnight.

10. Once vial lids/stir bars have been soaked for sufficient time, carefully pour out the diH2O.

11. Run diH2O through influx and efflux straws for 2-3 seconds at the sink to remove any residual bleach

12. Place stir bars in the stir bar tray.

13. Once 60+ vials have accumulated, vials should be dishwashed.

14. Place vials open side up in 3 white eVOLVER racks.

15. Take one rack, cover the vials using the wire mesh from the bottom rack of the dishwasher, then flip upside down. Repeat for the other two racks.

16. Add any other glassware or eVOLVER bottles to the top rack as desired, then run on cycle 3.

17. Store vials upside down in vial boxes to indicate that they have been dishwashed.

Ways you might accidentally change the OD reading

1. Moving vials once experiment has started

2. Altering

FAQs

Q: Can the eVOLVER do very long-term growth experiments?

A: Yes. Experiments with over a month of growth have been done. An example of a 250h experiment was reported in Supplemental Figure 14 of the 2018 NBT paper.

1. For the next few steps, it is recommended to spray your gloves with 70% ethanol.

2. Partially insert autoclaved vials into the eVOLVER Smart Sleeves such that efflux straw is entirely visible, if possible.

3. Start by attaching input lines (clear) to the short influx straw, avoid touching the tips of the lines to avoid contaminating the inside.

4. Next, attach efflux lines (blue) to the long efflux straw. It is critical to check for loose connections or incorrectly routed lines. Failure to do so will cause overflows and potentially damage the Smart Sleeve.

5. In the setup menu in the electron app, run all pumps in 10 second intervals to fill the vials with media. Run enough media so that the efflux pumps start removing media through the efflux straws.

6. Visually inspect the culture volumes at this point. If the efflux straws do not appear to be functioning efficiently (media levels rise above the efflux straw), inspect the fluidic line connections and the peristaltic pump. Correct or replace parts as needed to prevent overflows.

7. Push vials down until fully encased by the Smart Sleeve. If aluminum sleeves are cut to different lengths, vials may appear to be covered in different amounts.

8. Be sure that the desired calibration files have been selected from the dropdown menu in the setup panel. These files are stored on the device itself, and will default to the most recently used calibration. Temperature and OD calibrations must be repeated if vials are replaced, and OD calibrations are recommended if switching to a new organism (i.e. bacteria vs yeast). Otherwise, calibrations can be used over and over again.

9. Set the initial conditions for experimental parameters using the eVOLVER electron app. This will be overwritten once the experiment code starts running, but can be helpful for preheating media or trying out different stir rates.

10. If stir bars are jumping around (audibly) then the stir rate may be too high for the volume of culture you are using. Commonly used values are between 8 and 12.

Relevant Forum Posts

Recommendations for Septas and Needles?

Using nylon caps to determine culture volumes

Making vial caps w/ laser cutter and pipette tip

Overview

Reasons you might use command line experiment start:

1. Doing a custom experiment

2. Unable to install GUI or getting errors you aren't able to fix easily (post on the forum first)

Protocol

1. In a command line window, enter the dpu virtual environment.

   1. Navigate to the DPU directory

      • Use this command: cd <path_to_dpu>

   2. Enter the command:

      • On Mac OS:

        • source dpu-env/bin/activate

      • On Windows PowerShell:

        • dpu-env\Scripts\Activate.ps1

2. Navigate to your experiment folder

   1. This should be in dpu/experiment

3. Copy the template folder and rename it for each experiment

   1. Navigate into your new experiment folder

4. Alter settings in custom_script.py

   1. EVOLVER_PORT = set correct IP of the eVOLVER

   2. Set initial temp and stir settings

   3. custom_script.py has basic settings for running chemostat and turbidostat experiments, but you can also customize. To learn more click here.

5. Start the experiment using the command:

   1. python3 eVOLVER.py -i <your_evolver's_IP_address>

   2. python3 eVOLVER.py -i 192.168.1.9

6. Calibrate to blank

   1. Temperature should be fully equilibrated before this

   2. DO NOT add cells before blanking, whatever OD value the cells + media have will be subtracted from future measurements

7. Stop the experiment using the keyboard shortcut control+C

   1. One control+C pauses the experiment from recording data

   2. Two control+C stops the experiment

8. To change parameters mid-experiment or restart from blank data files:

   1. Stop the experiment fully

   2. Change parameters in custom_script.py

   3. Restart the experiment

      1. Continue to keep your data

         1. Overwrite to make new blank data files and throw away your old experiment. NOTE: you will lose your OD blank and will need to make a new one (a hassle if you have already inoculated with cells)

9. You can run experiments on more than one eVOLVER at a time

   1. Create a new experiment folder (different custom_script.py) and change IP

   2. Follow this procedure in an additional command line window

Ending an Experiment

1. Clean up the experiment

2. Data is in the data files

Why

When we start an experiment, the script asks us whether we want to calibrate to blank.

OD is dependent on vial position, vial opacity, media, environmental light conditions, temperature, and more.

Therefore, we take the first OD measurement as a zero and subtract that out from every subsequent measurement.

Details

OD blank is only applied to the calculated OD from calibration, not the raw OD data. The blank is applied for each vial, it just subtracts it out from subsequent measurements for that vial

https://www.evolver.bio/t/where-or-how-is-od-blank-info-stored/182

About

The GUI will run calibrations automatically upon completion of the calibration protocols. However, you can still manually run a calibration if you would like to change calibration settings. Can be useful if your GUI calibration fails for some reason.

Located in /dpu/calibration/.

Requirements

1. You have run calibration and logged raw values already

2. You are in your DPU virtual environment in command line. This is set up when you install the DPU.

Commands

List raw calibration files on eVOLVER

Mac

python3 calibration/calibrate.py -a <ip_address> -g

For Windows, use py instead of python for all commands.

Calibrate Temperature

python3 calibration/calibrate.py -a <ip_address> -n <file_name> -t linear -f <name_after_fit> -p temp

List raw OD JSON files logged on evolver

OD135

python3 calibration/calibrate.py -a <ip_address> -n <file_name> -t sigmoid -f <name_after_fit> -p od_135

OD90 (Check to ensure mode is configured properly)

python3 calibration/calibrate.py -a <ip_address> -n <file_name> -t sigmoid -f <name_after_fit> -p od_90

3D FIT (Check to ensure mode is configured properly)

python3 calibration/calibrate.py -a <ip_address> -n <file_name> -t 3d -f <name_after_fit> -p od_90,od_135

Page Under Construction

Design

Eagle

• Using Eagle to drag and drop elements + make layers

• Problem with Eagle

• • Only outputs dxf

  • We then have to trace as a png in illustrator

  • This creates a double line

  • Less accurate + goes over each area twice

Zack defines width of the pumps, but doesn't worry about depth

Width of channel / valves

• Too small = hard to align + valves will no longer stay closed

• Larger is more robust

Barb spacing

• Want enough space between each barb for tool

• Far enough from the edge

• • Prevent leaking from the port

About Server Logs

We use supervisor to run and manage the logs for the eVOLVER server. eVOLVERs should come pre-configured with this.

We also provide a utility monitoring and restart script, evolvercron and server_monitor.sh. To use, add the crontab job line in evolvercron into your cron installation.

View Server Logs

1. Connect to your server via the following command (replace <eVOLVER_IP> with your eVOLVER's IP)

   1. ssh pi@<eVOLVER_IP>

2. Enter the eVOLVER server's password

[Option 1] View the server log live

1. Enter the supervisor environment via the following command:

   1. sudo supervisorctl

2. Use the following command to follow the eVOLVER server log:

   1. tail -f evolver

3. Ctrl+C to exit

[Option 2] View the whole server log file

The server monitor stores the log file here: /var/log/supervisor/

You can either view there or move it to your computer for more analysis

Restart the eVOLVER Server

1. If you are currently following the eVOLVER server in supervisorctl, use ctrl+C to exit. Otherwise, follow steps to view server log.

2. Enter the following command:

   1. restart evolver

3. If you quickly follow the server via tail -f evolver you should see it restart

We have monitored the lifetime of eVOLVER components since the invention of the device (~3 years). As in most systems, mechanical parts have the highest possibility of wear and thus need replacement most frequently. Likewise, in eVOLVER, the peristaltic pumps used have a lifetime of 6 months of typical use during continuous culture. Specifically, the silicone tubing within the head of the pump is frequently compressed when actuating peristaltic pumps and will tear over time. The head can easily and inexpensively be replaced ($4/ pump). The computer fan used for stirring has robustly operated continuously for > 3 years, whereas the magnetic stir bars are rated for a limited number of autoclave cycles and need eventual replacement. All other components (e.g. heaters, thermistors, LEDs, diodes, PCB, power sources) have been stably operating for > 3 years.

https://www.evolver.bio/t/replacing-photodiodes-led-on-smart-sleeve/155

Summary

The eVOLVER system is highly complex and it is easy to feel lost about what happened when something went wrong.

For example, the behavior of optical density readings can vary widely and the underlying cause can be complex.

Please look through these troubleshooting guides and the relevant documentation before posting to the forum about your problem. It could also be the case that your problem is a bug.

Where to start

1. Physically Check the connections

2. Logs - errors

Page under construction

Protocol

Control Layer (1/4" acrylic)

Cut outlines first

• 0.1pt

• 3 copies

Keep full stock in place + outline on computer

• Acts as a frame for the device

• Remove board from stock with tweezers

Add adhesive

Put adhesive covered outline back in place on stock

On the computer

• Align control vector cuts (ports) to the outlines

• Delete outlines

• Print (3 copies for 1/4")

Keep board in place

Rastering control channel

• Undo deletion of outline, delete previous vector cuts

• Align raster outline to old outline + delete outline

• Load control_channel 2 in advanced settings

  • 1200 dpi

  • Deep to get through adhesive

• 1 copy => print

Remove board + stock from laser cutter

Flow Layer (1/8" acrylic)

Cut adhesive sheet

• Copy/paste in design with scooped out region around bridge

• Cut adhesive out first

• This way we don't have to move the acrylic stock later to put adhesive on the laser cutter

• Check adhesive stickiness

  • • Remove corner from adhesive

    • One side will stay attached to adhesive, one will remove cleanly

• Put adhesive sheet on laser cutter, with side that easily removes down

• Focus to adhesive

• Vector cut Full speed + 80 - 90% power

Cutting acrylic

• Delete scoops, we just want the outlines

• Print vector cut for outline

• Refocus laser cutter to 1/8th inch + cut

Plasma clean acrylic

Put adhesive on

Rastering acrylic

• Place device back in stock on laser cutter

• Copy/paste raster of flow route

• Remove outline

• Raster cut

• While rastering, start tapping control board

Assembly

Tap control board

• 2 turns one way, little turn the other way

• Use a countersink to remove burrs around the holes

  • A couple of turns

  • Both sides of board

  • Allows silicone to sit flush + barbs to insert easily

• Blow out small bits of acrylic from threading using air before plasma treating

Remove backing from all acrylic (helps with alignment later)

Silicone

• Sam from Densmore lab bought a big roll

• Some in drawer under cutting station

• Cut larger size than device

• Peal one side of silicone

• Plasma treat 30 seconds

Apply silicone to control board

• Align and press onto control board

• Take off plastic from other side of silicone

• Wrap in paper towel

• Put between aluminum plates in vice

• Turn tight + one small more turn

• Wait 10 minutes

Punch holes in silicone

• Use biopsy punch (green on shelf above 3D printers) to make holes silicone for flow

• Don't need to do the control

• Put pressure and turn

Clean silicone + adhere to flow layer

• Use piece of packing tape to remove dust

• Plasma clean silicone 30 seconds

• Align flow and control

  • Take special care to align IPP flow and control

  • Easier to align flow layer on top

• Clamp full device in paper towel for 10 minutes

  • Avoid over tightening - this can overstretch the silicone layer

Put loctite on barbs + screw in to board

• Barbs are in Dan's drawer

Subprotocols

Laser Cutter

Different colors of line produce different depth / intensity of cut

• Use black for everything

Make all lines hairline outline

• Otherwise it won't cut!

Open up a new page

• Paste whatever you're cutting into this

• This will be the correct size of bed

Printing

USB Bullshit

• Open up printers / devices

• Have to reconnect USB multiple times for it to be recognized

1. Open up print

2. Select correct printer you figured out

3. Load the correct settings

4. Set the correct copy number

5. 1. For millifluidics this is 2 for vector cutting (we have the double line problem)

   2. 1 for raster probably

6. Press print

On the Laser Cutter

Make sure it's on / connected

Focus first using metal thing attached to laser

Run the job with lid open to double check it's doing what you hope

Run the job

Plasma Cleaning

Plasma treat surfaces (except adhesive)

• Take off one side of acrylic backing

• Vacuum knob arrow left

• Metal valve tight, then quarter turn loose

• Pump on + power on to remove air 1 minute

• Then switch on upper right knob to high

  • Acrylic = 1 minute treatment

  • Silicone = 30 seconds treatment

• Turn off pump and power

• Slowly open black knob and let vacuum fill

Adhesive

Adhesive is in a box above 3D printers

Cut adhesive to larger size than acrylic

• Remove adhesive backing and place face up on surface

• Remove acrylic from plasma cleaner and firmly press clean side onto adhesive

• Squeegee the bubbles out as much as you can (and away from where the channels will be)

• Cut excess adhesive off with razor blade

Vial overflow is one of the most common problems to plague eVOLVER experiments. Overflow events can cause damage to internal components such as the PCB motherboard, sensor actuator (SA) boards, power supplies, etc. If you experience overflow during an experiment, it is important to immediately stop the eVOLVER and evaluate potential causes of overflow, as well as any damage that the overflow may have caused.

If you catch an overflow that happened recently (i.e. an overflow that happened overnight), the best thing you can do after stopping the experiment is turn off and unplug the eVOLVER, determine the extent of overflow spilling, and wash any components that have media on them. There are no components on the vial board or motherboard that retain significant charge (i.e. no batteries or large capacitors), so they are safe to wash gently in soap and water. Once clean, they should be dried quickly with a paper towel.

The main reason for electronics failure after an overflow is current shorting through media (most microbial media is salty and conductive), or connections and copper corroding from media that has been left to dry. If you can clean off all the media immediately after an overflow, you will have a much better chance of saving electrical components, and saving yourself many headaches down the road.

If you did not catch an overflow quickly, or a large amount of media spilled, follow the workflow below.

Troubleshooting Workflow for Overflow

Inspecting internal components

Overflowed media can enter into the vial platform through the ports that connect the Smart Vials to the motherboard. This can cause damage to the internal circuitry, which is should first be evaluated by visually inspecting the motherboard, SA boards, power supplies, and Raspberry Pi. Damage to any of these components can look like burned or melted spots or loose/broken connections. If you see visible damage to any of these parts, they will need to be fully replaced. See pages below for specific instructions on how to replace these various parts.

Motherboard Troubleshooting/Replacement

12V Power Supply Troubleshooting/Replacement

Old evolver pumps (solid black plastic head)

Pump is not actuating / spinning

Relevant Forum Posts

Pump failure and spill, lessons learned

While batch culture techniques often utilize biosafety hoods or flame convection currents to keep workspaces sterile, these are rarely amenable for automated cell culture devices. Prevention of contamination in eVOLVER is achieved at three levels: 1) sterilization of media, culture vessels, and fluidic lines, 2) attention to sterile technique, and 3) physical and chemical barriers to contaminants. First, all media bottles and their adapters, and all components of the culture vessel (e.g. borosilicate glass vial, magnetic stir bar, cap with fluidic adapters) are designed to be autoclaved before each use. Fluidic lines on the device are sterilized before and after each experiment using bleach and ethanol (see Online Methods). Second, following sterilization, sterile technique should be practiced when attaching media lines to culture vials by working quickly, avoiding physical contact with the ends of fluidics lines, and taking care to spray gloves with ethanol. Finally, additional physical and chemical measures may be taken depending on the organism and experiment. For example, the sampling port may be covered by a sterile membrane for long-term culture. For slow-growing eukaryotic cultures, antibiotics can be added to the media to exclude bacterial contaminants. UV sterilization of components or surfaces is another preventative measure to consider.

Page under construction

Page under construction

Description

As IPP devices are sensitive to changes in pressure at valves and in connected media bottles, we developed an 8-channel pressure regulator that can be used to regulate these pressures through the eVOLVER framework. The device consists of sets of two proportional valves that can limit air flow from a high-pressure source and a vent at atmospheric pressure. By connecting an electronic pressure gauge to the output of this valve configuration, it is possible to implement proportional-integral-derivative (PID) control over the valves in order to set the output pressure to any desired level between the input and atmospheric pressure. We validated the functionality of this device by regulating pressure at 1.5 psi over 24 hours, and compared the performance of our device with that of a fixed, manually set regulator (PARKER-WATTS R25-02A) connected to the benchtop air supply (Supplementary Figure 3). The average pressure with PID control was 1.498 psi with an RMS error of 0.0086 psi, while the fixed regulator had an average pressure of 1.706 psi with an RMS error of 0.2220 psi. Large pressure deviations (>0.5 psi) that can affect the performance of the devices were observed with the fixed regulator, but were successfully eliminated with our automated pressure regulator scheme. We further characterized the effects of pressure changes at various locations in the system in order to optimize performance of the IPP devices for the course of a PACE experiment (Supplementary Figure 3).

[Strongly Recommended] Better 3D Printed Vial Holder

Info

If your eVOLVER is stock from Fynch, then you do not have the upgraded part (as of April 2024).

Download the latest version here. Compare to the version online here to determine which version you have.

Benefits

• [Versions >= 5.1.1] More consistent OD calibration

  • You can tell if you have this part by pulling out the OD LED or photodiode from your vial holder and seeing if there is a smaller circle of plastic inside the hole

    • If there is simply a flat area on the bottom of the hole, you have an older version

  • Because of variation in depth of photodiode and OD LED. See here

  • Otherwise you will have vials with poor calibrations

• [Versions >= 5.2] More resistant to overflow

  • You can tell if you have this part because there are triangle-shapes around the screws holding the Smart Sleeve together.

  • On the old 3D printed part (v5.1.1 and below), overflows could easily short the heating resistors

  • The latest version has cutaways to prevent this

Guide

1. Print out your vial holder by downloading the latest version here

   1. Send the .stl file to a manufacturer (such as hubs.com) and choose FDM printing

   2. Choose black plastic (optics change otherwise)

2. Remove any extra plastic in the photodiode and LED holes

3. Use this guide to help you rebuild your Smart Sleeve with the new vial holder

4. Make sure to firmly push the OD LED and photodiodes all the way in (see figure below)

5. Recalibrate temperature and OD

Stronger Stirring

More stirring leads to better aeration and thus better growth in many cases. Try this stir bar for more vigorous stirring: Length 20 mm, Diameter 3 mm (# SBM-2003-MIC).

Try different stir speeds to see if you can get stable and intense stirring.

1. Background

2. Guide

If you need additional aid or have questions about this process, feel free to ask on the forum!

Background

eVOLVER hardware is primarily divided between the Smart Sleeves and Motherboard. The Motherboard is where all the sensitive control hardware is, whereas the Smart Sleeve is nice modular way of integrating all the sensors to control your culture conditions.

Goal

The goal of this tutorial is to show how one can build an eVOLVER Smart Sleeve using commonly found tools and easily customizable parts.

Time/Cost

In this post, we are describing the Smart Sleeve built for continuous culture documented in our Nature Biotechnology paper. It typically takes 10 to 15 minutes for construction of this version of the Smart Sleeve with all the appropriate parts (e.g. soldered PCB, constructed aluminum tube). Raw materials cost roughly $40/ vial however cost for 3D printing, machining, and coloring (spray painting/ anodizing) parts vary due to accessibility to appropriate tools.

That all sounds great, but I have never soldered or 3D printed before, how do I start?

Here is a great introductory tutorial on soldering from our friends at Sparkfun. Also, you can easily outsource your 3D printed parts to be produced by folks at 3DHubs by uploading the appropriate STL file (if you’re a student, don’t forget the student discount!). Generally, building a Smart Sleeve is simple because we only use through hole components and screw terminals, so it is much easier to make than surface mount designs.

How can I modify or change the design?

We built our PCB designs on KiCAD, an open-sourced software for circuit board design, and our 3D models on Solidworks. Here is a schematic of the PCB here and the Solidworks part here. There are many other programs for vial board design, including a bunch of tutorials on Eagle from Sparkfun, another popular CAD tool for the open-source community.

Does this tutorial include how to build the Motherboard?

No. This tutorial only provides instructions on how to build a Smart Sleeve.

Guide

1. Order all of the necessary parts and tools.

List of supplies needed ( displayed in photograph above ):

1. Laser Cut 1/4" Acrylic Base

2. 2 pieces of laser cut 1/8" Acrylic Fan Spacers

3. 5/64" Hex Key

4. IR LED

5. IR Photodiode

6. Thermistor

7. 2x Socket head screw

8. 12 V DC Computer Fan with Magnets Glued

9. Vial Board

10. Lab Tape

11. 3D Printed Part

12. Aluminum Tube

13. Soldering Iron + Solder

14. 2x Stainless Steel Screws, 2.5" 4-40 threading (Not Shown)

2. Familiarize yourself with how the Vial Board, 3D printed part, and aluminum tube should be assembled.

Take notice if all the components line up properly:

• Tapped 2-56 holes on the Aluminum Tube lines up with holes on heating resistors (2 black rectangular components soldered on vial board)

• The heating resistors should sit on two rectangular holes on the 3D printed part

• Green screw terminals line up with the LED holes on the 3D printed part

Take note of where the two holes labeled “thermistor” are on the PCB.

3. Tape thermistor on the aluminum tube near where the two vias were located on the PCB.

When taping the thermistor, ensure the writing on the thermistor is facing away from the aluminum tube. Tape the thermistor parallel to the length of the tube about .5 cm away from the bottom of the tube. The leads should be exposed when slotted into the 3D printed part.

4. Slot the aluminum tube into the 3D printed part. Verify that the holes for the LEDs on both the aluminum tube and 3D printed part are properly aligned.

Make sure 3D printed part is slotted tightly. Add additional pieces of tape if is loose. Avoid damaging the thermistor by twisting or rotating the parts.

5. Align the PCB over the 3D printed part, with the two thermistor leads threading through the vias on the PCB.

Also verify that the two small 2-56 holes on the aluminum sleeve align with the holes on the heating resistor.

6. Use the 2x Socket head screws to fasten the heating resistors to the aluminum sleeve.

Aligning the hole with the heating resistor makes screwing the socket cap in much easier. Thermal paste can also be applied before this step to form better contact between the heaters and aluminum tube.

7. Place the 1/8" laser cut acrylic pieces onto of the fan to act as spacers between the magnets and glass vessel.

8. Use the two 4-40 stainless steel screws to fasten the assembled 3D printed part and the computer fan with spacers. Fasten both screws onto the 1/4" acrylic piece.

9. Coil the wires from the computer fan around the Smart Sleeve and connect to appropriate ports on the screw terminal labeled “fan”.

The red wire should go into PWR and the black wire should go into the GND ports.

10. Attach the LED (clear packaging) and photodiode (dark packaging) for OD measurement to the appropriately labeled slots.

Both the photodiode and LED should have a short and a long lead. This corresponds to the polarity of components. Make sure for both components, the longer lead is connected to PWR . If this is done improperly, you will not get a reliable signal.

11. Bend the LED and diode into the corresponding slots.

12. Finally, solder the thermistor onto the PCB.

That’s it! You’re done.

Following a similar framework for building the Smart Sleeves enables adding different parameters, adaptation to different vial sizes, and other creative eVOLVER applications! Have fun! Let me know if anything is unclear on the forum.

• What is UNIX?

• Key Concepts

• The Shell

• Command Line Usage

• Current Working Directory and Paths

• Useful commands and tips

  • whoami

  • which

  • pwd

  • ls

  • cd

  • mkdir

  • cp

  • mv

  • rm

  • touch

  • echo

  • cat

  • head

  • tail

  • less

  • grep

  • find

  • sed

• Pretty colors and Text editors

What is UNIX?

UNIX is an operating system originally developed in the 1960s that is the base for modern operating systems such as macOS X, GNU/Linux, Sun Solaris, and many more. While modern UNIX systems usually have a graphical user interface (GUI) that you can use to interact with the system and run programs, the most direct and powerful way to interact with the kernel (operating system core) is through the shell (command line).

Key Concepts

Pretty much everything in UNIX is either a file or a process. Files are simply collections of data – they can be plain text, programs, binary machine code, or even directories. A process is a program that is being executed by the kernel.

The file-system on UNIX is very simple – it’s a tree that starts at root, which is written as /. Everything then branches off of the root with a location specified by a path from the root to the file. For example, if there were a user on a UNIX system with the username unixuser1, the home directory would typically be located at /home/unixuser1. If this user had written a hello world program in c and put it in a directory called myprograms in their home directory, that path would look like /home/unixuser1/myprograms/hello.c. It’s useful to think of the file-system as a tree that you navigate up and down through, and create routes from one spot to another. More on this later!

The Shell

The shell is the interface between the user and the kernel – it is a command line interpreter (CLI) which takes commands that the user types in and sends them off to the kernel to be carried out. Commands in UNIX are actually programs, which means that they are files on the system. When a user enters a command, the shell goes and finds that program and arranges for it to be executed by the kernel. The shell is configured through some other files that it looks at when it starts up to know where these programs are located so that the user doesn’t have to type out the path each time they want to use them.

Command Line Usage

When you open your command line terminal, you should see something like this:

This line is called the prompt – it is the shell waiting for the user to enter a command to run. Typically, the prompt will end with a $, meaning the shell is ready to accept a command.

Your prompt will probably look a little bit different, as this system has some customization and configurations set that print some extra information and changes the colors. This will be explained at the end! Just make sure you see a $ at the end of the line.

To execute a command, simply type it out on the command line after the prompt and press enter! Some commands can accept additional arguments or flags that will change the way they print things out or how they execute. Arguments are passed to a command as space separated strings of text. Because they are space separated, you cannot include spaces in your arguments - they will be interpreted as separate arguments. To pass in a string with spaces, you must either escape the space character by placing a \ in front of the space, or put the entire string in double quotes ("). Be careful though, as not all commands behave the same! It is best practice to not use spaces and other special characters in filenames if it can be helped. More on passing arguments will be shown in the following examples.

Current Working Directory and Paths

The shell has a concept of a current working directory, which can be thought of as the location in the file-system that the shell is currently “looking” at. When you start a terminal, usually the current working directory is the home directory of the user, typically at /home/username, or /Users/username on mac.

As stated earlier, a path is a string that tells the shell where to look for a specified file. There are two types of paths, absolute paths and relative paths. An absolute path starts at root (/) and lists out the entire file-system route to a file. For example:

/home/unixuser1/myprograms/hello.c

A relative path is the route through the file-system from one location to another. These are useful as they require fewer characters, less typing, and allow programs to be written that interact with the file-system that can run from anywhere and not have to worry about where the current working directory was when it was called.

When writing out paths on the shell, if you do not write an absolute path, the shell will interpret the path as relative starting from the current working directory. For example, let’s say a user wants to do something with the hello.c file in their /home/unixuser1/myprograms directory, and their current working directory is their home directory (/home/unixuser1), as in this figure:

The relative path to their c file would be the following:

myprograms/hello.c

This would be interpreted the same as the absolute path /home/unixuser1/myprograms/hello.c, but it requires less typing!

It is also possible to write relative paths to things further up or in a different branch of the file-system. For example, if the current working directory is /home/unixuser1/Desktop, and the user wants to reference the same c file as above, visually it would look like this:

and the relative path would be:

../myprograms/hello.c

The double dot (..) means up one level in the file-system. You can go up more than one level by stringing them together. For example, given a current working directory of /home/unixuser1/Desktop/another_directory, to reference the same file the relative path would be

../../myprograms/hello.c

Useful commands and tips

• Double dot .. means the directory above the current one. A single dot . means this current location.

• Tab can be to autocomplete a path or filename. Start typing the first letters, then hit Tab. USE THIS! It will prevent you from making typos or looking for something that doesn’t exist, and saves time overall.

  • Hitting Tab twice in quick succession will print out all files matching up to the point typed, then allows you to keep typing.

• The star character * matches 0 or more characters in a filename. For example, *.txt would match all files ending in .txt.

• The tilde character ~ stands for the home directory of a user.

• Ctrl + C: kills the current foreground running process in the terminal and returns control back to the user (prints a new prompt).

• Ctrl + L: Clear the screen. Alternatively, use the clear command.

• Ctrl + A or Home: Moves the cursor to the end of the line.

• Ctrl + E or End: Moves the cursor to the end of the line.

• Ctrl + K: Deletes from the current cursor position to the end of the line. String this together with Ctrl + A to delete everything on the line!

• The up arrow and down arrow on your keyboard will let you scroll through your recent commands so you don’t have to retype them.

• Ctrl + R: Recall the last command matching the characters you provide. Press repeatedly to search past the last matching to previously matching commands.

• command > file redirects the output of a command to a file, overwriting existing content.

• command >> file redirects and appends output of command into a file (does not overwrite).

• first_command | second_command is a pipeline, (the | character is called a pipe). This will send the output of the first command as input to the second command.

Here are a few useful commands for navigation, inspection, and basic file manipulation on the command line. This is by far from being exhaustive! Most operating systems will have documentation listing out available commands and how to use them. You can also use the command man <command> to bring up the documentation (or manual) for a command within the shell. Press q to quit the manual after running. You can type /<search_string> to search through the manual.

whoami

Displays the username of the current user.

Notice that after executing the command, the shell will print a new prompt for the user to enter another command.

which

Identifies the location of an executable. Use this to determine if a command or program is installed on your system, and also to see where it actually resides on the file-system. If the program cannot be found, nothing will be returned.

pwd

Stands for Print Working Directory. It prints the absolute path of the current working directory from root. The current working directory is the location on the file-system where the shell is currently focused.

ls

Stands for list. This command will list out all files in a given directory.

You can provide a path to ls to list files not in the current working directory. Some useful flags are -l (long listing format), which can show you file permissions, owner, size, and last modified date, and -a (shows all files, including hidden files).

cd

Stands for Change Directory. Moves the current working directory to the path specified as an argument to the command. The syntax is cd <path>, where <path> can be any relative or absolute path on the file-system. If the path is invalid, the current working directory will stay the same.

mkdir

Stands for make directory. Creates a directory at the stated path if it does not already exist.

cp

Stands for copy. Copies file at one path to another specified path. If the path to be copied to is a directory, cp will copy the file and keep the same filename. You can also specify a new filename. The syntax is cp <file_to_copy> <new_location>

If you want to copy an entire directory, pass the -r flag.

mv

Stands for move. Works exactly like cp, but will remove (rename) the file from the original location.

rm

Stands for remove. Delete a file at a given path. To delete an entire directory, pass the -r flag. You can also pass the -f flag which will not prompt for confirmation regardless of the files permissions. Remember, there is no undo when using this command. The syntax is rm <filename>

touch

Updates file timestamp information. Can be used to create an empty file.

echo

Outputs the strings being passed to it as arguments. You would usually pipe this command into other commands or use it to write data into a file through a single command. If you pass the -e flag, you can parse escaped characters. You can use this to print out Unicode symbols or different colors.

To append a line into a file:

echo "This is a string I want to append" >> myfile.txt

cat

Stands for concatenate. Displays the contents of a file. You can use it to concatenate by passing it multiple files, and it will print them to some output location.

head

Displays the first 10 lines of a file. Pass the -n argument to specify how many lines to display.

tail

Displays the last 10 lines of a file. Pass the -n argument to specify how many lines to display. Pass the -f flag to follow, or to not stop when end of file is reached, but rather to wait for additional data to be appended to the input. Press Ctrl + C to quit afterwards. Useful for watching log files in real time.

less

Like cat, but only displays contents from the beginning up to what will fit on the screen. Use the arrow keys (or j and k) to move the display through the file. Press q to quit.

grep

Searches a file or files for lines that match a regular expression or string. There are lots of flags and options for this command depending on what you’re trying to do, run man grep to see everything it can do.

For example, say you have a complicated project with many directories and want to find where a specific variable name or library is used. You could use grep to recursively look through all directories and look for that variable name. Grep will print the filename and contents of the line it found it on. In this example, the -r flag means to look recursively through any directory it finds, the -I means to ignore binary files, the -n will make it also print the line number, and the . means to start the search at this current location (the current working directory).

find

Finds files with properties that match a specified pattern. To find a file with a specific name from the current working directory, type the following:

find . -name "myfilename.txt "

You can also pass regex patterns to find multiple files that match a certain pattern. To find all c files, try the following:

find . -name "*.c"

sed

Stands for Stream Editor. Useful for searching, finding and replacing, inserting, or deleting things in files. As an example, maybe you want to replace all the tabs in every file within a directory to four spaces. You could do this all at once with one command:

sed -i .bak $'s/\t/ /g' *.c

The -i means to edit the files in-place, and backs up the files with an extension .bak. The characters within the quotes are what we are searching and replacing (basic regular expression), with / as a delimiter between what we are searching for, replacing, and any other options in the regex. So in this case, we are searching for \t (tab), and replacing it with four spaces. The g signifies to do this globally across the entire file and not just on the first occurrence. You can combine commands like find with sed to modify files across your file-system or in specific projects all at once.

If you really want to do some interesting command line text processing, check out the awk command!

Pretty colors and text editors

If you want the same coloring scheme I have in my terminal, edit the file ~/.bash_profile using a simple text editor. If you want to do it in the terminal, try using nano (a very simple text editor) or vim (an extremely powerful editor with a learning curve). Look up the usage of these on google before you open them! If you want to learn vim, you can use the built-in program vimtutor that will guide you through the basics.

To turn on some basic coloring, add this line anywhere in the file:

export CLICOLOR=1

The export command is setting the value of the environment variable CLICOLOR to 1, turning on colors. You can change how these colors look by changing some other environment variables. For example:

export LSCOLORS=GxFxCxDxBxegedabagaced

This setting makes the ls command print in a different color scheme. You can look up how to customize this on your own.

Lastly, to make your username/hostname on the prompt have different colors or display different things, you can modify the PS1 environment variable, which contains the value of the default prompt:

export PS1='\[\033[01;32m\]\u@\h\[\033[00m\]:\[\033[01;34m\]\w\[\033[00m\]\$ '

This makes my username (the \u) bold and green, prints an @, then the host name (\h) and a colon, then prints the current working directory in blue (\w) followed by a $ in white.

You can modify the .bash_profile file to do lots of other useful things as well, such as making aliases for different commands, modifying or appending to environment variables, or setting up other things. Do your own research to see what kinds of things you can do!

Some terminals also have some color settings applied by default, or the ability to load up different profiles. Keep this in mind when trying to modify the appearance of your terminal.

If you have any questions or concerns not addressed here, reach out on this forum post.

Updating the RPi GUI

1. SCP File to Pi

On Mac: scp evolver-electron-2.0.0.AppImage pi@<your_evolver_ip>:.

On Windows, use WinSCP or Filezilla to drag the file to /users/pi/home/

2. Change Permissions to AppImage

First, ssh to the Pi:

ssh pi@<your_evolver_ip>

Next, make the file executable using chmod:

chmod +x evolver-electron-2.0.0.AppImage

3. Change What AppImage the Pi uses at startup

sudo nano /etc/xdg/openbox/autostart

Scroll down and change the following line to correspond with the version you are using.

# Start Electron App in Kiosk Mode
./evolver-electron-2.0.0.AppImage

4. Reboot the eVOLVER

You can either power cycle (turn off the eVOLVER completely, wait a few seconds, then turn it back on) or you can use the following command:

sudo reboot now

Updating the eVOLVER Server Code

Mac/UNIX

Windows

Mac/Unix

1. Open up a terminal of your choice (I use iTerm2).

2. Use the cd command to navigate to the eVOLVER server software.

3. Use git to pull the latest version of the code. I have my own fork, so I need to pull the code from an upstream master branch. git pull origin master should work for most setups.

4. I like to use the scp command to transfer the files over to the eVOLVER. scp stands for secure copy - it usesssh to transfer files between computers on a network. You can also use any GUI based tool - Filezilla is a popular one. I also normally only transfer the python files, and not the entire evolver directory. The server saves calibration files and other configurations which we don't want to modify by accident. To transfer only the relevant python files, run the following command:

scp evolver/evolver/*.py pi@<eVOLVER_IP>:/home/pi/evolver/evolver

5. Now that the files have been updated, the last step is to restart the server so the changes will take effect. ssh over to the evolver and run sudo supervisorctl. Then enter restart evolver, and you're done!

Windows

Follow steps 1-3 as described above.

4. Use WinSCP or Filezilla to drag the .py files into the correct locations. Be careful not to overwrite other types of files.

5. Most Windows 10 computers have a Windows 10 SSH client (such as OpenSSH Client) so you can log into the Raspberry Pi from Command Prompt in order to restart the server. Simply type ssh pi@<eVOLVER_IP> (replace <eVOLVER_IP> with your actual eVOLVER IP) and log in. From here you can run sudo supervisorctl as normal.

Overview

While large carboys (autoclavable plastic jugs >10L) are useful for long-term experiments or particularly fast-growing bugs, the best typical media storage is standard Pyrex/Corning bottles, which range from 100 mL to 10 L. The 1-2L bottles are the most convenient size for media prep, short experiments, and experiments with many media types, so we'll be sharing the process for those bottles here.

Per media bottle, you will need (see parts list, fluidics page):

Parts:

1 ft of 1/8" ID silicone tubing 2 Female luer-lock connector with 1/8" barb, polypropylene 1 Male luer-lock connector with 1/8" barb, polypropylene 1 Male luer cap Tools: Scissors/razor Drill gun 3/16" drill bit

Step-by-step

First, drill a hole directly down through the cap. Be sure to have something safe to drill into on the other side. If having trouble with the 3/16 bit catching on the plastic, drill a pilot hole with a smaller bit.

Next, push one of the barbed female luer connectors through the top of the cap, so the luer side is facing up.

Next, cut two pieces of tubing. One is for the cap above, and can be as short as 3". Put the two remaining luer adapters (one male, one female) on this piece. This flexible piece makes it a little easier to connect lines to bottles. The other should be at least 9" so that it makes it from the barb on the cap all the way to the bottom of the bottle (for 1L bottles). Connect the longer piece of tubing to the barb poking through the bottom of the lid.

The long straw should reach the bottom of the bottle and bend slightly. If it’s too long it will be annoying to screw the bottle cap on, and might get stuck against the wall. If it's too short, it won’t be able to reach media at the bottom of the bottle. Your bottles may vary: for 2L bottles, you need a 13" straw.

Rinse out the straw with diH2O, and add a male luer cap to the top of the straw before autoclaving, and you’re good to go!

You can also make splitters with the same 1/8" tubing and corresponding luer-barb connectors and barbed T-connectors, allowing you to split one media bottle to multiple input lines. We usually sterilize the splitters using 10% bleach and ethanol (by connecting them before sterilizing all the lines when beginning an eVOLVER experiment), but everything shown here is autoclavable as well.

Turn on the eVOLVER 5V power supply by flipping the associated switch. IMPORTANT: Wait for 5 seconds before turning on the 12V power supply.

Ensure the correct eVOLVER is selected by consulting the upper right hand corner of the GUI. It will display the eVOLVER name (default is evolver-darwin), followed by the IP address in parentheses. This will be the IP address you assigned it when setting up the router connecting the DPU to the vial platform.

In the PARAMETER box, ensure the correct calibrations are selected for Temp, OD, and the pumps. Click the box for each to bring up a list of available calibrations for that parameter.

Select all vials by tapping Select All in the bottom right corner of the screen. The vial circles on the right side of the display should become orange.

You can also click and drag to select as many vials as you want.

Tap the NEXT → on the box that says parameter to get to the Stir tab. Tap the - button or drag the slider to a Stir of 8. Press the Stir: 8 button to alter the stirring of all vials.

Tap the NEXT → on the box that says parameter to get to theTemperature tab. Tap the - button or drag the slider until the middle button says 37.0 °C and tap the button. The EXECUTED COMMANDS box should state that a stir and then temp command was sent to all vials.

Pumps

Refer to the JoVE video for how to use pumps and sterilize fluidic lines

If you have any questions or hit a roadblock, check this post on the forum.

1. Download the Arduino IDE

Their website should be fairly easy to navigate.

2. Install SAMD libraries

The Sparkfun website has a very good blog on this. For more details, please refer to that link. The following is information copied from there.

Navigate to your board manager ( Tools > Board > Boards Manager… ), then find an entry for Arduino SAMD Boards (32-bits ARM Cortex-M0+) . Select it, and install the latest version.

3. Install the Sparkfun libraries for the SAMD21

First, open your Arduino preferences ( File > Preferences ). Then find the Additional Board Manager URLs text box, and paste the below link in

https://raw.githubusercontent.com/sparkfun/Arduino_Boards/master/IDE_Board_Manager/package_sparkfun_index.json

Then hit “OK”, and travel back to the Board Manager menu. You should (but probably won’t) be able to find a new entry for SparkFun SAMD Boards . If you don’t see it, close the board manager and open it again.

4. Choose the correct BOARD and PORT to upload into

1. Plug the SAMD21 into your computer via a data transfer micro-USB cable.

2. The boards used in eVOLVER are the SAMD21 Mini boards.

3. The port changes based on which USB the Arduino is connected to. The wrong port is typically the most common reason why the Arduino won’t upload.

   1. If you are unsure which port is the correct one, plug and unplug the SAMD21 board and see which port appears and disappears.

Choosing the board:

Choosing the port:

5. Download the Arduino scripts and copy eVOLVER libraries into Arduino folder

1. Download the scripts from our Github Page

   1. Download by clicking "Code" and downloading as a zip file

2. Extract the files from the zipped file

3. Copy the libraries folder into your local Arduino libraries folder

   1. For example, on my computer, it is under Documents > Arduino > libraries

6. Upload script to Arduino!

This should be all. Open up the files and upload your scripts to the microcontroller via microUSB! The program should tell you that it has uploaded successfully.

Please see the Raspberry Pi infrastructure page for additional information on the RPi we use for the eVOLVER.

RPi Imager Guide - Recommended

Command Line Guide (Mac/UNIX only)

Using Raspberry Pi Imager

1. Download the pre-configured eVOLVER RPi image here.

2. Download and install the Rapberry Pi Imager Tool.

3. Plug an 8GB micro-SD for the RPi into an adapter, then into a the computer.

4. Click the Choose OS button, then scroll to the bottom and click Use Custom.

5. In the popup at the dropdown at the bottom of the window, select All files(*.*). Then select the image file downloaded in Step 1.

6. Click the Choose Storage button and select the SD Card.

7. Click the Write button. A notification might appear warning about erasing all existing data on the card. Click Yes. If it asks for your computer password afterwards, enter it. It might take a while to finish (~40m).

8. When it completes, you can plug the SD card into the RPi. You will need to update the server code and potentially the conf.yml with the latest code from GitHub.

Using command line utilities (Mac/UNIX only)

1. Download the pre-configured eVOLVER RPi image here.

2. Plug the SD card into the computer.

3. Open a terminal of your choice (I recommend iTerm2).

4. Run the following command:

diskutil list

Identify the SD card name from this (it should be obvious). Next run:

diskutil unmountdisk <diskname>

where <diskname> is the name you identified from the previous command - it should look like

/dev/disk3/ or something similar.

5. Use the dd command to copy the image onto the SD card:

sudo dd bs=4m if=<diskimage.img> of=<diskname>

This command can take a while, and it doesn't show any status or progress updates - be patient. If it fails, try decreasing the bs argument to 1m instead of 4m - this will slow things down but will decrease the chance of failure. If you still have issues reach out on the forum.

6. When it completes, you can plug the SD card into the RPi. You will need to update the server code and potentially the conf.yml with the latest code from GitHub.

Backing Up Raspberry Pi (Make a Custom Image)

Writing an image to save your full Raspberry Pi configuration.

Useful to save time when reimaging your Raspberry Pi if you want to go back to your custom Python code and calibrations as quickly as possible.

Follow this guide: https://www.tomshardware.com/how-to/back-up-raspberry-pi-as-disk-image

Page under construction

Burst Tube at Barb Connection

When there is an obstruction in the line, from dried media or biofilm, the peristaltic pumps can produce up to 45 psi of pressure, which is enough to exceed the barbed connector tolerance and burst barb connections. This is clearly undesirable, so if you find that one of your barb connections has burst, check the length of the line for obstructions. If it's an efflux line to the waste container (opaque), assume it has an obstruction.

Obstructions

For the pumps that we typically use for eVOLVER, 1/16" ID tubing does not resist flow in any appreciable amount. If a pump is not pulling liquid, it is likely blocked or obstructed.

Unblocking With Syringe

Biofilm obstructions can happen during long experiments with a biofilm-forming organism like E. coli - your best bet is to pause the experiment and bleach sterilize all lines before restarting. If your line is completely blocked, either replace the line or clear the obstruction by pushing water through the line with a 50 mL syringe (shown below).

Unblocking by Soaking the Lines

If the syringe method does not immediately work to clear the blockage, the blocked lines can be soaked in water for a few days to help rehydrate the dried media/biomass - the silicone tubing is quite permeable, so water can slowly enter the tubing and help loosen any dried blockage so that it can be flushed out with a syringe.

Stuck luer-lok connectors

Residual or leaked media on the ends of luer-lok connectors can dry and leave the connectors stuck. Rather than reach for a pair of plyers, soak the connection in hot water to dissolve the media. This can also help for any stubborn fluidic connection, and make tubing much more pliable.

Set Up

Plug in 5V power supply and 5V fan

Changing Pressure Settings

Option 1: Manually change via Arduino (hard coded)

• Follow the Arduino Software Installation

• Unplug the SAMD21 from the pressure regulator before uploading code (otherwise there might not be enough power to the SAMD21 from the USB)

• Upload modified pressure code to SAMD21

Option 2: Add a 'pres' command to the evolver conf.yml and modify conf.yml

Fluidic Lines

Connect a 8-10 psi air input into the input port (only port on its own in 1.0 design)

Connect regulated line to both sensor (below) and controlled output (white, above) as shown below:

Set Up Solenoid Control

Solenoid Bank Diagram (facing holes)

• The solenoids should be normally open and connected to the 8 psi air source. ie Vacuum when closed

• Left = 8 psi

• Right = Vacuum

• Middle = 8 different solenoids, groups of 3 are IPP controls

Connect solenoid ribbon cable to slot 5 on the fluidics box

Turn on eVOLVER server and fluidics box

Check that the solenoid bank is correctly set

Run ipp_cal.py on the server:

• Use command:

  • python3 ipp_cal.py <frequency (hz)> <seconds to run>

  • For example: python3 ipp_cal.py 1 100

Set Up IPPs

• Hook IPPs up to a pressurized input bottle for consistency

• Make sure that IPPs are operating as expected and producing the expected flow rate

Miscellaneous

"" after a very long term experiment

- another alternative is to tape the switches on

Overview

Optical density (OD) is one of the most integral parts of continuous culture. While chemostats do not innately rely on OD, it is still a useful metric for your evolution. Meanwhile, turbidostats and growth curves heavily rely on OD to function.

However, OD is the most easily disrupted parts of eVOLVER hardware. This guide serves as an initial list of things to check when troubleshooting OD.

Most Common Problems

Variability in OD Sensing Range Between Vials

2. If you already have a version of the 3D printed tube holder with defined OD LED and photodiode distances:
5. Try different ADC resistor packs

   1. eVOLVERs come with a variety of these

   2. Ask on the forum for help

6. Check that your vial construction is not variable

Noisy OD Data

1. Stir bars jumping around during OD readings

   1. Decrease stir speed
2. You are near the edge of your calibrated OD range

Incorrect OD

ie eVOLVER OD is offset from the cuvette or plate reader measured value.

1. 1. Especially because you did not wait for temperatures of the vials to equilibriate

2. Poor quality OD calibration

   2. Different temperatures require different OD calibrations

No Change in OD

1. Photodiode or IR LED not working

   2. A failed photodiode will show a raw OD value close to 64,000

   3. A failed IR LED will most likely show a raw OD value much closer to your other vials

   4. Check that they have the long (positive) lead in the correct positive terminal

2. ADC board or motherboard broken

   1. Can happen after vial overflow

Other Considerations

Pre-Calibration Considerations

• od_led value in conf.yml file (PWM value)

• • Use the splash guard that comes with your eVOLVER

  • You may need to recalibrate if you calibrated with ambient light

Experiment Considerations

• Photodiode Temperature - make sure temperature is equilibrated before calibration and before start of experiment

• eVOLVER recently turned on - causes rapid change in values

• Needles or other structures coming below 10mL line (close to OD PD etc)

• Vial volume - a volume of 10mL will totally change OD compared to 30

• Stirring with low vial volume - this will give noisy readings as the funnel created by stirring fluctuates and is more and less reflective

Non-standard changes

• Vial holder reflectiveness - ie white vs black vs metallic

Stir Bar Jumping

• by increasing space between fan and vial

Relevant Forum Posts

From eVOLVER Forum

Motherboard Replacement

The is the main printed circuit board (PCB) of the eVOLVER. It houses the PWM boards, ADC boards, and SAMD21 mini breakout boards. In the event that the motherboard is damaged, it will need to be replaced. Some damage to the motherboard will be immediately obvious.

In the example below, overflow events during an experiment caused media to leak through the Smart Vial ribbon cable ports into the vial platform, which houses all of the eVOLVER circuitry. This resulted in visible damage to the motherboard, which can be seen below.

Protocol

Unfasten Motherboard

The motherboard is fastened to the eVOLVER platform and other internal components in several ways (numbers correspond to image below):

1. Ribbon cables connecting the motherboard to the Smart Sleeves (not shown)

2. Screws in gold holes

3. 6 screws that fasten wiring from the power supplies to the motherboard

4. Cable connecting motherboard to Raspberry Pi (fastened to base of platform)

5. Cable connecting motherboard to display screen (fastened to lid of platform, obstructed by ribbon cable in image)

Remove Motherboard

1. Unplug the cables connecting the motherboard to the Raspberry Pi and display screen.

2. Remove the screws in the gold holes using a Phillips screwdriver.

3. Remove the screws holding the wiring in place using a flathead screwdriver.

4. Close the eVOLVER chassis lid half way, making sure to hold the motherboard with one hand. Unplug the ribbon cables that connect the Smart Sleeves to the motherboard, starting from the last row of vials and moving towards the first row of vials.

5. Maneuver the motherboard around the 3D printed part housing the display screen.

Transfer Parts to New Motherboard

The PWM boards, ADC boards, SAMD21 mini breakout boards and jumpers need to be transferred to the new motherboard.

Remove the ADC boards, PWM boards, and SAMD21 mini breakout boards from their slots using an Integrated Circuit (IC) puller, as seen in the image below. Pull the IC puller perpendicular to the motherboard to prevent bending or breaking the SA boards’ pins.

1. If pins are bent in the process of removing the boards, they can be straightened using tweezers. Avoid bending the pins too much; excess bending can cause the pins to snap off.

2. If pins are snapped off during this process:

   2. Alternatively, the pins can be soldered back into place onto the board, but this is much trickier to do.

   3. The board should be replaced with a new board if other options do not work

Optional: If your motherboard includes a variable resistance board, proceed to steps 3 and 4. If not, skip to step 5.

1. Remove the variable resistance board from the motherboard. The ADC boards on this board can be removed first, or the variable resistance board and ADC boards can be removed as a single part.

2. Remove the headers under the variable resistor board and transfer to the new motherboard. These can be removed with pliers.

1. Remove the yellow resistor arrays (see below). An IC puller or pliers may be helpful here. Transfer the resistor arrays to the new motherboard, making sure to insert them in the proper orientation.

2. On the side of the resistor array, there is a description of the part and a black dot, seen in the image below. This black dot indicates the ground pin of the resistor array, and should go into the slot towards the top of the motherboard.

1. Remove the jumpers to the old motherboard by pulling them straight up. Transfer the jumpers to the new motherboard, recreating the same configuration as shown below.

1. After checking for damage, transfer the variable resistance board (if applicable) and SA boards to the new motherboard (it is easiest to move these boards after transferring the resistor arrays and jumpers). Be sure to align the boards’ pins with the slots on the motherboard before pushing the boards into place.

Adding the SA parts to their slots on the motherboard can be quite tricky, especially if their pins were bent during the removal process. Some tips for this step:

• Inspect boards and use tweezers to carefully straighten pins that have been bent out of place. Bending the pins back and forth too much can cause them to break off the board, so this should be kept to a minimum.

• If pins break off an SA board, they can be soldered back into place, or the entire board can be swapped out.

• Place the SA board over its slots and check for pin alignment. Try to wedge the bent pins into place first, before pressing the board into place.

• Make sure ALL PINS enter their respective slots before pressing the SA board into place.

• The SA boards can withstand some force when being pushed into the motherboard. Press the SA boards into their slots until the boards lay flush with the slots on the motherboard.

1. Unscrew the 3D printed corners from the old motherboard and fasten them to the new motherboard. These parts act as spacers to prevent the backside of the motherboard from touching the eVOLVER platform.

Prepare New Motherboard and Plug in to eVOLVER

1. Use electrical tape to cover all exposed elements on the back of the motherboard. There are exposed pins on both the top and bottom of the motherboard.

The new motherboard is now ready to be fastened to the vial platform!

1. Align the motherboard correctly by lining up the ribbon cable ports with the openings in the platform.

2. Fasten the motherboard to the platform using the screws and a Phillips screwdriver.

3. Fasten the wiring from the power supplies to the motherboard. The new motherboard will have screws in place for where the wires should go, so those should be removed and then used to fasten the wires to the motherboard.

4. Plug in the cables to the Raspberry Pi and display screen.

5. Plug in the Smart Vial ribbon cables to the motherboard from the top of the eVOLVER.

Power Up eVOLVER

You are now ready to power up your eVOLVER!

The first time you turn on the eVOLVER after replacing the motherboard, we recommend opening the platform and keeping an eye out for a few things:

• Be sure to turn on the 5V power supply first, wait 10 seconds, then turn on the 12V power supply.

• If you notice smoke or the smell of something burning, immediately turn off both power supplies. If this occurs, inspect the motherboard and the SA boards for melting or burn spots.

• The red light on the Controls Raspberry Pi should turn on steady, and the green light should blink on and off.

• The red and blue lights on the SAMD21 mini breakout boards should turn on steady.

The 12V power supply powers the heating and stirring elements in the Smart Vials. If both of these elements are not properly functioning and you have inspected the internal circuitry for damage (see for more information), the next element to check is the 12V power supply.

Troubleshooting

• measuring w multimeter?

Replacing a 12V Power Supply

If you have found that the 12V power supply is not functioning, you can replace it following the steps below.

1. The 12V power supply is fastened to the bottom of the vial platform with 4 hexagonal bolts. Use an Allen wrench to remove these bolts.

1. Unscrew the wiring that connects the motherboard to the 12V power supply using a flathead screwdriver. This is easiest to do when the power supply has been unfastened from the case so that it can be turned on its side.

1. Attach the wiring to the replacement 12V power supply, making sure that the wires are in the correct configuration, as shown below.

What's in the box

The fluidics box can be opened by removing the 8 phillips head screws on the sides. Inside the box is the fluidics board (with a SAMD21 and three PWM boards) with RS485 and 5V connections for the SAMD21, and a 12V power supply beneath the board. Should the need arise to update the arduino code on the SAMD21, you'll need to open the box to get to it.

The connections to the pumps are numbered according to their address, i.e. cable 1 connects to pumps 1-8, cable 2 connects to pumps 9-16, etc.

Troubleshooting and repair

Not many things can go wrong inside the box, but we have dealt with a situation where the 5V power connection was faulty and led to malfunction. This part can be replaced by opening the box and replacing the faulty connector.

Modifications

Adding another pump array

The existing pump pox can control up to 16 additional pumps (or any other 12V valve, actuator, etc) with no modifications. Typically this would mean using connections 5 and 6 on the back of the box, which correspond to indices 33-48.

Modifying fluidics code

Daisy-chaining multiple boxes

Two RS485 ports (the telephone jack-looking ports) are available on the back of the fluidics box so that multiple boxes can be linked together on the same RS485 "line". All SAMD21 microcontrollers are receiving the same serial data from the Raspberry Pi, and only responding to commands when the appropriate address for that SAMD21 is in the command, like "temp" or "stir". Additional fluidics boxes can have the same "pump" address with higher pump indices (i.e. pumps 49-96 for a second box), so long as you change the number of expected pump values on the conf.yml file. You will also need to modify the SAMD21 arduino code accordingly. You can also have a different address for the other fluidics box, by adding another parameter in the conf.yml and modifying the arduino code accordingly.

Guide

1. Check the server software for more information

2. Monitor the server log file

   1. Guide

3. Any time you are troubleshooting the server, make sure to save your calibrations.json file in a secure location before going further

4. If the log file is not updating (via evolver tail -f)

   1. If you have manually edited the conf.yml file, most likely you have made an error somewhere there

      1. This could be formatting or a typo

      2. Copy a conf.yml that you know works (for example to )

         1. Using the server update as a reference

         2. ONLY copy the conf.yml to home/pi/evolver/evolver/

         3. Otherwise you will overwrite your calibration files

      3. Restart the server

         1. Stop following the server using ctrl+C

         2. Type restart evolver

         3. evolver tail -f

      4. The server log should now be updating each cycle

   2. If not, and you have not recently updated the evolver.py or other python files,

Relevant Forum Posts

1. Smart Sleeve

2. Motherboard

   1. SAMD21 Arduino Microcontrollers

   2. Characterized SA Boards

      1. PWM Board

      2. ADC Board

3. Auxiliary Board

General Overview

The eVOLVER hardware framework contains three levels of organization: (1) programmable sensors and actuators (e.g. Smart Sleeve components, pumps/fluidic control elements) (2) a Motherboard and microcontrollers, and (3) a Raspberry Pi. At the level of individual culture vessels, Smart Sleeves enable individual control over several experimental parameters in the culture.

Specifically, each sleeve contains sensors and actuators (e.g. heaters, LEDs, thermometer/thermistor) that measure and adjust aspects of the culture environment of a glass vial housed within. At the next level of organization, the Motherboard, Arduino microcontrollers, and other core electronic boards form a robust hardware infrastructure that communicates internally and coordinates activity of each individual Smart Sleeve to control each experimental parameter.

At the final level of organization, a Raspberry Pi forms a link to the outside world by relaying information and commands to and from a computer/server, permitting the same computer/server to run many eVOLVER devices across a network. Layered on top of the hardware framework, control software enables programmable feedback between parameters and orchestrates experiments at an abstract level, providing an easy method of customization that is shareable with other users. Below we present the core hardware framework as well as the particular configuration enabling the experiments described in this study.

Smart Sleeve

The Smart Sleeve houses each culture vial and contains all the necessary sensors and actuators to regulate desired culture parameters. An aluminum sleeve surrounds the vial, which is mounted into a 3D printed part that houses a printed circuit board (PCB) called the Component Mount Board (CMB). The CMB contains a variety of different sensors and actuators needed to regulate culture conditions. As the most flexible component in the eVOLVER Framework, the Smart Sleeve can be re-designed to regulate other culture conditions based on the user's end application.

Motherboard

The Motherboard is responsible for housing custom PCBs and Arduino microcontollers that control user defined culture conditions by routing signals to and from an array of Smart Sleeves. Custom PCBs are plugged into SA (sensor/actuator) slots, of which there are 8. Using the routing system, these custom PCBs can collect data from CMB sensors or deliver power to CMB actuators. For example, in the base eVOLVER setup, there is a SA slot dedicated to individually powering heaters across all 16 Smart Sleeves.

SAMD21 Arduino

On the Motherboard are 4 SAMD21 Arduinos, each of which is responsible for regulating a single culture parameter. To do so, each SAMD21 board is electrically connected to 2 SA slots since many culture conditions require both a sensor and actuator. To regulate temperature, for example, a single SAMD21 will leverage its SA slot pair to sense the temperature for each Smart Sleeve and provide the necessary power to each Smart Sleeve heater to maintain the user-defined temperature for each culture vial. This modular design enables functional parallelization of culture parameters, providing a high degree of flexibility to users when designing hardware for experiments.

Characterized SA Boards

FynchBio has designed and characterized the PWM Board and ADC Board, two PCBs that plug into Motherboard's SA slots. These boards are designed to power actuators and collect sensor data for existing CMB components and should be sufficient to handle custom reconfigurations.

PWM Board

The PWM Board is designed to power 16 actuators, in parallel, by amplifying the SAMD21 voltage ouptut using the Motherboard's voltage source. The PWM Board leverages pulse-width modulation (PWM) signals to power actuators, allowing digital signals to better approximate analog signals. This is essential because analog signals provide precise control of actuators, rather than simple ON/OFF dynamics with digital signals.

ADC Board

The ADC board is a 16-1 de-multiplexer that collects data from 16 sensors and filters it before sending it to its cognate SAMD21 Arduino. Specifically, the ADC Board reads the output voltage from a basic voltage divider circuit integrated with the sensor on the CMB. After analog signals are collected, they are filtered to remove noise, sent to the SAMD21 Arduino through an input pin, and finally converted to a digital signal for downstream data processing.

Auxillary Board

Akin to the Motherboard, the Auxillary Board is a custom PCB designed to control a peristalitc pump array to handle fluidic functions. This includes removing waste and adding nutrients/chemicals to cultures. The Auxillary Board contains a single SAMD21 Arduino, which connects to 3 PWM Boards to actuate 48 peristaltic

Description

The programmable Smart Sleeve is the foundational unit on which the eVOLVER is built (Fig. 2 and Supplementary Fig. 1). The Smart Sleeve is comprised of all the sensors and actuators required to control the culture conditions inside a 40 mL borosilicate glass vial. At the core is an aluminum sleeve, which surrounds the vial and is used to control temperature via two resistive heaters and a thermistor integrated within. Near the base of the vial sits a 3D printed part that houses and aligns the optical density LED and photodiode. Below that sits a fan motor equipped with magnets to rotate a stir bar within the vial. The Smart Sleeve represents one of the most easily customized features of the eVOLVER: by changing which sensors and actuators are used and their layout, the user may develop culture vessels that fit their experimental needs. For a detailed description of the sensors and actuators used to control stirring, temperature, and optical density in Smart Sleeves featured in this study, as well as strategies for modifying the Smart Sleeve to fit experimental needs, refer to Supplementary Note 4. Liquid handling is also controlled at the level of the individual culture vessel, yet these components are housed in a separate fluidic module, described in Supplementary Note 5.

The sensors and actuators on each sleeve are integrated in a small printed circuit board (PCB), termed the Vial Board (VB). We designed the VB such that we can easily solder electrical connections and efficiently manage/package wiring from the sensors. The VB is a very simple PCB, containing only a few resistors, and is straightforward to redesign and inexpensive to manufacture, if needed. The simplicity in the VB leads to robustness in the system. For example, any accidental overflow and spillage from the vials (e.g. from clogged fluid lines or user error) should minimally impact the rest of the system, as critical components are located at the Motherboard rather than the sleeve itself. Ribbon cables provide a modular way to connect the integrated Smart Sleeves to the Motherboard.

The VB is designed to rest atop a 3D printed piece, which houses optical density and temperature components (see Supplementary Note 4). The printed part can be fabricated with Nature Biotechnology: doi:10.1038/nbt.4151 5 any commercial or DIY 3D printer, readily available at almost any university or hacker space, and customized to the requirements of the user. For example, if a user wanted to change the mode of optical density detection between scattering and absorption, they could redesign the 3D printed part housing the LED-diode pair such that it would have the correct offset angle for the desired mode of measurement.

Vial

Vial Holder

Vial Sleeve

Vial Cap

Vial Board

Standard components

Tubing

The standard eVOLVER package includes 1/16" ID (1/8" OD) soft silicone tubing for all fluid handling operations. For exact specifications, see the , "Fluidics" tab.

Silicone is the tubing of choice for eVOLVER because of its flexibility, durability, cost, and resistance to beach and ethanol. It is also autoclavable, although 30 minutes of 10% bleach sterilization is sufficient to completely sterilize the lines.

Silicone tubing is highly permeable to gasses, including water vapor. If you leave media in the tubing for >1 week, it will likely dry out and cause an obstruction (see ).

Connectors

The standard eVOLVER package includes 1/16" barb to luer-lok connectors for all pump-to-vial connections. For exact specifications, see the , "Fluidics" tab.

The barb side of the connector is rated for the ID of tubing it is mean to be used with, i.e. 1/16" barb for 1/16" ID tubing, 1/8" barb for 1/8" ID tubing, etc. Barbs are designed to get a tight seal around their widest diameter, with the max pressure and resistance to slippage depending on the softness of the tubing and style of barb (40 psi for standard eVOLVER connectors).

Luer connectors work by pressing a "male" plug into a "female" socket, both tapered at 6%. Because of the smooth surfaces and large contact area, this creates an effective seal without tools, held in place by friction. Syringes and medical equipment commonly make use of luer connections.

To hold luer connections in place more securely, luer-lok connectors use a screw-like "skirt" to prevent luer connectors from simply being pulled apart. eVOLVER tubing connections are luer-lok, allowing for easy but relatively secure connections between most components.

However, luer-lok connections have some common failure modes. Because the threads of the skirt are so coarse, it only takes a 1/4 turn to break the seal at the plug-socket interface, leading to a leak (if positive pressure) or air bubble source (if negative pressure). Loose luer connections easily become disconnected, leading to spills (the major source of electronics failure) and of course, failed experiments. One should always ensure luer connections are snug (hand tight), and tubing free from torsion that might unscrew the luer-lok.

Additionally, because the male connector comes in direct contact with fluid, it becomes your most likely contamination source. Be careful not to bump the tips of the male connectors against contaminated surfaces when setting up your experiment. Doing a final wash with 70% ethanol after bleach sterilizing can prevent some contamination from this route, depending on your media and organism.

Swapping and modifying

Any soft tubing (durometer 40-70A) works well with the barbed connectors. Vinyl (PVC) or tygon tubing might be a suitable alternative for decreased permeability. Opaque tubing may make sense for light-sensitive media or antibiotic feeds that have extended time exposed to light in the tubing due to low flow rates, at the cost of not being able to observe media in your line. Remember that 1/16" ID tubing contains about 0.75 mL per foot.

For eVOLVER to be more than a 16-well plate reader, there needs to be control of liquid flow through each vial. The most common hardware for this application is the peristaltic pump, used in continuous culture for almost a century. Novel aspects of eVOLVER include the automated, individual control of these pumps, and modularity for different types and numbers of pumps. This is enabled by an auxiliary fluidic board, separate from the motherboard, that controls up to 48 fluidic actuators. For typical eVOLVER use, this is coupled with 32 pumps (16 for influx, 16 for efflux), 64 lines of tubing (to and from each pump), and 128 barbed connectors (for both ends of each line of tubing). This section of the Wiki will go into detail of how each component works, and options for repair, troubleshooting, swapping, and modifying them in the eVOLVER fluidic hardware framework.

Version D

Pin Map

Differences to Version C bolded

1. Fan

2. Unused; linked to fan for future fan tachometer

3. Heating resistor

4. Thermistor

5. OD IR LED

6. OD 135 photodiode

7. OD 90 photodiode / Spare A

8. Spare B

Version C

Pin Map

Differences to Version D bolded

1. Fan

2. Unused; linked to fan for future fan tachometer

3. Heating resistor

4. Thermistor

5. OD IR LED

6. OD 90 photodiode

7. OD 135 photodiode / Spare A

8. Spare B

Standard components

The standard eVOLVER peristaltic pumps (since ~2020) sourced by Fynch Bio are selected specifically to eVOLVER use. Previous pumps used with eVOLVER during its early development tended to wear out quickly and have variable flow rates, primarily due to the mechanism for rotating the rollers against the tubing to drive pump flow. The motor shaft directly turned the rollers through friction, which led to slippage and abrasion over time. The current pumps do not have drirect contact between the motor shaft and rollers, instead driving roller rotation through a gear reduction system.

This gear system drives a roller casing with fixed roller axles contained in the pump head. In this way, the pump action does not rely on friction and thus much more accurate and durable than previous pumps.

Troubleshooting and repair

Because the current pump style has been introduced so recently, very few of them have failed from wear and tear. Based on our experience with other pump styles, the most likely failure mode will be collapse or tear of the tubing inside the pump head, or shearing of the tubing on the barb outside the head. This can be solved by replacing the pump head (contact Fynch Bio) or replacing the tubing.

The induction motor inside the pump is very unlikely to fail through normal use. If there is a liquid spill on the pump array and some pumps are not spinning, the most likely cause is corroded contacts with the pump array PCB. While the pumps themselves will be fine, they should be removed and replaced to a new PCB, ordered from Fynch or PCBWay. This will require removing solder, which can be tricky.

Swapping and modifying

The pump heads can be rotated 90 degrees in any direction, should your bench setup require changing the direction your pumps face.

The low-flow pumps available through Fynch use an additional gear reduction box to reduce the speed of pump action by a factor of 45. This is useful for addition of inducers, antibiotics, or other media feeds that require a small and precise bolus.

Plenty of other peristaltic pumps are suitable for eVOLVER, so long as they operate at 12V.

The flow rate of eVOLVER pumps (and any pump with an induction motor) is somewhat modifiable through PWM modulation. This effectively reduces the voltage going to the pump, which will slow its rotation, down to the point where the coils can't generate enough torque to consistently turn the shaft. This happens about halfway through the PWM range (around 2000), where you can get the pumps to run at about 1/3 speed. This is rarely necessary for experiments, so it has not been optimized, but is a potential feature for users to explore.

Description

The eVOLVER platform features tunable and independent stir rate control across culture vials. Stirring in eVOLVER is actuated by 12V brushless DC motors with attached neodymium magnets. The fastened magnets spin a stir bar (20 mm x 3 mm, PTFE coated) within an autoclaved glass vial (28 mm x 95 mm, borosilicate). The stirring module utilizes a single SA slot on the Motherboard; in the particular configuration described in this study, we utilized SA slot 1 (Supplementary Fig. 4). The two leads of the motor (12V & GND) are connected to a screw terminal on the component mount board, from which a ribbon cable connects the smart sleeve to the Motherboard. The PWM board (plugged into the SA slot) can control each motor independently to achieve different stir rates across eVOLVER vials. Briefly, the 16-channel PWM board amplifies a 3.3V signal from the Arduino microcontroller to a 12V signal to actuate Nature Biotechnology: doi:10.1038/nbt.4151 12 the motor. Arduino 1, which manages SA slot 1, was programmed to take in serial inputs from the Raspberry Pi and translate the serial values to different stir rates, determined by pulsing the motor ON and OFF at different ratios (Supplementary Fig. 4).

Relevant Forum Posts

Description

In contrast to current approaches in which all culture vessels are housed in a single incubator7,26, we developed a module for individually controlling the temperature of each Smart Sleeve in the eVOLVER. This not only allows the cultures to be maintained at distinct temperatures, but also reduces thermal mass, permitting dynamic temperature profiles. For the configuration described in this study, the temperature module utilizes SA slots 2 and 3 on the Motherboard (Supplementary Fig. 5). Typically, there are three main components to temperature control: (1) a thermometer, (2) a heater, and (3) a feedback controller. In our setup, the thermometer and the heater are integrated in the Smart Sleeve while the feedback controller is located on the Motherboard. Specifically, the temperature is measured by a 500 μm thick temperature-sensitive resistor, or thermistor (Semitec, 103JT-025). The sensor is integrated into the sleeve between the 3D printed part and the aluminum tube, and the thermistor is soldered onto the component mount board (CMB) after assembly. The aluminum tube enables even heat distribution/dissipation and shields the culture from ambient light (important for other measurements/parameters). Two heating resistors (20 Ohm 15 W, thick film) are screwed onto the aluminum tube for better contact and connected to the CMB via soldering. In our setup, the four leads, 2 from heating resistors and 2 from thermistor, are connected via a ribbon cable to the Motherboard and routed to SA slots 2 and 3, respectively. In slot 2, a 16-channel PWM board amplifies a 3.3V signal from the Arduino microcontroller to a 12V signal to actuate the heating resistors. Slot 3 contains a 16-channel ADC board, which reads the voltage difference across a 10 kilo Ohm resistor, and is responsible for analog filtering and demultiplexing the signal from the thermistor. These slots are connected to and are programmatically controlled by Arduino. Briefly, the Arduino code interprets serial inputs from the Raspberry Pi, updates the set point on the PID controller, and responds with the current measured temperature. Temperature settings can be updated as frequently as every 30 seconds. To determine how much to turn the resistive heaters on, the Arduino is programmed with a simple PID control algorithm. The PID controller can be easily tuned via software to obtain the desired overshoot and time delays. The Arduino then controls a PWM board (on SA slot 3) to interface with the resistive heaters and get the desired heat output. Calibration of the temperature measurement in the sleeve was performed by comparing the temperature of water measured in the vial using a thermocouple to the values returned by the thermistor (Supplementary Fig. 6). The dynamics of heating were determined by tracking temperature during a programmed step function, again comparing thermocouple and thermistor readings; the thermistor measures the temperature of the sleeve, while the thermocouple measured the actual water temperature. At room temperature (23°C for this experiment), a single culture (20 mL) can reach a temperature of 42°C in roughly half an hour with the current hardware setup (Supplementary Fig. 6). During an experiment, the transient offset between the recorded temperature and actual temperature may vary due to ambient temperature and volume of liquid. At steady state, the temperature can be maintained to +/- 0.1°C, with properly tuned PID constants. Max temperature and rate of temperature ramp can be changed with different power sources (e.g. 24V power source could reach temperatures >55°C). It should also be noted that at different temperatures, the optical density readings are affected accordingly. This effect was measured in both yeast cultures and evaporated milk (Supplementary Fig. 6). See the next supplementary note for more detail.

The eVOLVER uses SAMD21 mini breakout boards that can be programmed using the Arduino IDE. These microcontroller breakout boards allow the eVOLVER to read analog signals from sensors (OD, temperature, etc.) and actuate pumps, stir, LEDs, and any other culture component that is needed.

General Information

As a quick recap, the Motherboard is designed to collect and send data/power to all 16 Smart Sleeves. The Motherboard is essentially a large PCB used to route signals via a network of electrical traces. Each SA slot (8 total) posses two electrical connections (positive and negative leads) to each Smart Sleeve CMB (8*2=16 connections per CMB). These two leads per SA slot are then used to make the physical connections to a sensor/actuator. In total, this results in 2 (leads per sensor/actuator) * 8 (SA slots) * 16 (number of Smart Sleeves) = 256 total traces. This is important to keep in mind when modifying the Smart Sleeve or troubleshooting when problems arise.

Lastly, each SA slot acts as a de-multiplexer/multiplexer for bidirectional relay of signals with its cognate SAMD21 Arduino.

TODO: Include a figure to highight transfer of data including voltage divider

Tuning Sensor Behavior

As mentioned earlier, the Motherboard makes use of voltage divider circuits to acquire sensory data on cultures. Briefly, a voltage divider circuit works by connecting two resistors in series with Vin, a voltage source, to produce Vout, a set fraction of Vin (Fig 1.). The value of Vout is based on the the resistance values of the two resitors and the value of Vin, which can be modeled as:

This principle is used by the Motherboard to implement sensory circuits with components that transduce environmental signals into electrical signals (i.e. thermistors, photodiode, phototransistors, etc). This signal is read via Vout, the voltage across the resistor, which is proportional to the sensor's electrical readout (Fig 2.).

Based on experimental conditions, it may be of interest to tune how these sensory circuits behave to acquire optimal data. The Motherboard offers two primary ways to modify sensory circuits: 1) swap R1 to alter sensor sensitivity and 2) swap Vin to alter the circuit's dynamic range.

Tuning Sensor Sensitivity

To streamline sensitivity tuning, the Motherboard uses 9-pin resistor packs (one is reserved for ground), which connect to the left-and-right to each SA slots (8*2=16 parallel resistors). As a user, all you need to do is remove the two current resistor packs and replace them with packs with desired resitance values. By taking a look at the raw sensory data coming off the eVOLVER, you can assess if the sensitivity modifications are adequate or not.

Description

A key development in microfluidics was the design and fabrication of devices containing integrated (pneumatic) valves that could allow for complex fluidic manipulations with minimal number of control elements27–29. Here, we describe (1) why adapting this technology for the macro scale is valuable for automated cell culture, (2) challenges faced when scaling to larger flow rates, and (3) a new framework for fabrication and bonding of millifluidic devices featuring integrated pneumatic valves. These devices offer a scalable solution to challenges faced by traditional fluidics.

Importance of Complex Fluidic Tasks

The ability to program complex fluidic tasks could enable entirely new manipulations in automated cell culture applications (see Fig. 5, Supplementary Notes 15 - 17). For example, when growing undomesticated microbes, biofilm may form in the efflux fluidic lines and vials in as little as in 12 hours (e.g. see Fig. 5). The ability to programmatically bypass the vial in order to clean the fluidic lines with a bleach solution, and passage the culture from one vial to the next as a preventative measure, would be critical for long-term continuous growth of these microbes. However, complex fluidic tasks like vial-to-vial transfer, cleaning protocols, and mixed media inputs are extremely difficult with traditional fluidic systems used by current devices. In electronics, custom circuits can be readily created by breadboarding; however, this approach scales poorly to larger, more complex circuits because it relies on tedious manual assembly and leads to limited durability. Similarly, fluidic systems consisting of flexible tubing connecting separate control elements, like pumps and valves, can solve simple fluidic tasks. However, the number of necessary fluidic connections scales with the complexity of the desired task. For example, even with an optimal valving scheme, the ability to perform automated large volume transfers between any two culture vials in a 16-vial eVOLVER unit would require almost 300 fluidic connections and over 20 control elements. As in breadboarding, each connection would need to be routed individually by fluidic tubing and often by hand, a tedious task. Additionally, the tubing is usually fairly long, and each connection introduces dead volume, making the system less robust and impractical. Instead, by creating integrated (pneumatically valved) schematics, we sought to make a millifluidic equivalent of a printed circuit board; the complex fluidic connections are now integrated in a small device that is computer designed, manufacturable, and much easier to reproduce. With the flexibility of CAD, one would be able to customize a fluidic device to fit their particular experimental needs.

Fluidic Scaling Problem

The cost of control elements and assembly time of bioreactor units can prove to be a significant burden as one scales fluidic inputs and outputs for high-throughput operations. As previously described, most designs rely on a pinch valve or a peristaltic pump to separately control each of the media sources and another to control waste. For example, a single vial turbidostat unit with 4 different media inputs would require 5 pumps. A hypothetical 48-vial unit with the same capabilities would therefore require 240 pumps, at a cost of $7,000 to $10,000. The key problem is that the number of control elements increases linearly with the number of vials. Our pneumatically-valved devices can leverage concepts developed in microfluidics in order to scale throughput by multiplexing and demultiplexing inputs and outputs27,30. In this scheme, the number of controllable vials scales exponentially to the number of control 23 elements, needing only 30 elements to route up to 16 different fluidic inputs to 48 vials. We project the costs of this new hypothetical 48 vial fluidic schematic to be $1,000 to $2,000, roughly a 90% decrease in cost in comparison to current systems.

Desired Characteristics and Properties of Integrated Millifluidic Devices

Though inspired by microfluidic technologies, integrated millifluidic devices for continuous culture have drastically different design requirements. The following is a list of critical requirements for this technology in the context of continuous culture:

• Devices need to be on the decimeter scale. Indicated in the nomenclature, microfluidic devices operate on the nano to micro liter per second flow rate. In contrast, continuous culture in eVOLVER requires flow rates of roughly ~1 mL per second, a 1000-fold increase. As such, in order to increase flow rate with an appropriate safety factor, the flow channels and device needs to be at least 10-fold larger than typical microfluidic devices.

• New prototyping framework is necessary for larger devices. Traditional microfluidic prototyping techniques rely on standards from the microelectronics industry, namely patterning photoresist on silicon wafers29,51. Typically, 100 mm (4 inch) circular wafers are the largest size the machinery can pattern designs on, which is still too small for complex millifluidic devices. New fabrication framework is necessary to prototype devices for continuous culture.

• Design cycle must be fast and repeatable. The success of microfluidics in the laboratory is, in large part, attributed to rapid and reliable design cycles enabling iteration and testing of prototypes. New design frameworks for millifluidic devices must be equally rapid and reliable.

• Device must be transparent. Fluidic systems designed for long-term continuous culture of microbes will be prone to biofilm formation. The ability to monitor flow through the device is critical for debugging experimental issues.

• Device needs to interface with pumps, filters, and tubing. Fluidics for laboratory continuous culture typically interface with syringe pumps, pressurized fluids, sterile filters and peristaltic pumps. A robust way to interface dozens of connections between the device and other fluidic elements (e.g. vials, filters) is critical and nontrivial.

• Device must be resistant to 10% bleach and 70% ethanol. Sterilization of the device is necessary prior to any experimentation. Fluidic materials and fabrication must be resilient to these chemicals for weeks of continuous usage.

Characteristics of Pneumatic Valves for Millifluidic Devices

Our pneumatically-valved millifluidic devices enable customizable, programmable routing of liquid at volumetric flow rates of ~ 1 mL/sec. The valves pinch off fluid flow on the flow layer when 10 psi is applied to the control layer and enable flow when vacuum is applied (Supplementary Fig. 11). Improvement of device bonding will enable application of pressures above 10 psi, necessary with higher flow rates.

Coordinating New Fluidic Experimental Parameters

Fluidic tasks in eVOLVER are enabled by the sequential actuation of valves in a specific fluidic network encoded in the integrated millifluidic device. We demonstrate these fluidic manipulations in a series of experiments (see Fig. 5, Supplementary Notes 15-17). Each experiment utilizes different devices as required to meet the experimental needs. The architectures for most functions are modular (e.g. multiplexer, vial-to-vial router) and can be combined in order to achieve more complex functionalities (Supplementary Fig. 11). For example, simple single media input turbidostat function utilizes multiplexer and demultiplexer modules. The demultiplexer routes the media source to the correct vial and the multiplexer routes the efflux from vial to waste. The same multiplexer and demultiplexer modules are Nature Biotechnology: doi:10.1038/nbt.4151 25 reused in all Fig. 5 applications, but different multiplexed media selectors and vial-to-vial routers are included as needed in different experiments. Software routines to control the control elements (valves and pumps) are also divided into commonly repeated functions, usually in a similar manner to how fluidic modules were divided. The code for each fluidic function is preloaded into the Arduino to coordinate tasks between each fluidic module. For example, a simple dilution event would first actuate valves in the media multiplexer to select media, then actuate a syringe pump for metering the desired volume, followed by valves in the vial demultiplexer and multiplexer to route media into the vial and remove efflux. By loading the routine for abstract functions (e.g. dilute, clean, vial-to-vial transfer) into the Arduino, robust communication can be ensured, with rapid transition between sub-tasks and no skipped steps (which could cause incorrect media routing, mis-priming of the syringe pump, or leaks and other device failures).

eVOLVER uses Raspberry Pi for interfacing with the Arduino microcontrollers and for processing user and experimental commands. Hardware and software configurations and calibrations are saved on the RPi.

We provide a pre-configured RPi image that is configured to run all eVOLVER software.

RPi image for eVOLVER

General RPi information

Raspberry Pi Imager - Use this to upload the disk image linked above to the RPi SD card. We have a guide for this process here.

Guide for uploading a disk image to the RPi SD card via command line - Only use this if you are comfortable working on a terminal and the upload tool above isn't working for you.

Setting up a new RPi