• What is UNIX?

• Key Concepts

• The Shell

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).

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 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.

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

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.

Inside the pump head, the pump tubing can be removed and inspected. The tubing is 2x4mm Pharmed BPT, which has similar chemical compatibility as the silicone tubing for the rest of eVOLVER fluidics, and is rated to over 1000 hours of operating life. Learn more here:

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.

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.

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.

For more information on luer connections (and credit for the above images), see here:

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.

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.

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 Arduino Resources

SAMD21 breakout board documentation, hookup guide

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

Alumni

• Chris Mancuso

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

Buying

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

Connecting to eVOLVER

Currently eVOLVER requires a dedicated computer connected to it to run experiments. We recommend getting a Mac, which are easier to use and more reliable.

The computer needed for running experiments does not have to be new or powerful.

Upgrades

Consider implementing these to the Fynch Bio eVOLVER.

Variants

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 for CAD files and electronics parts lists for eVOLVER PCBs.

Vial Caps

Choose and 3D print vial caps for your application . For most applications choose the "universal" cap.

Other "Consumables"

See Consumables tab in spreadsheet above.

Recommended to buy several vial racks as well for ease of handling vials.

Relevant Forum Posts

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

Publications

Background, final data and experiments

Extensive documentation in the supplements, meant for reference

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:

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:

Windows

1. Go to the official to download python 3.9.

2. Follow to install python on your machine.

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

DPU Installation

1. Navigate to the

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 if you want a terminal with some quality of life enhancements. On Windows, you can use PowerShell.

7. We use 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:

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 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.

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

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)

1. Fill a large beaker with water and submerge all of the pump lines (inputs and outputs of the pumps), ensuring all of the ends are below the surface of the water.

2. Run the pumps on the Setup page to completely fill the lines with water (about 20s)

3. Place 16 vials into a rack and place the output ends of the pumps to be calibrated into the vials.

4. Select the eVOLVER on the top of the GUI home page to be calibrated

5. Navigate to the Pump Calibration page

6. Enter a name for the pump calibration

7. Select the pump configuration for the eVOLVER. In a standard setup, IN1 are the media in pumps, and E refers to the efflux pumps. If you have another pump array (for a second media input source), it is typically going to be in IN2. Click Start Pump Calibration. The standard pumps are the FAST pumps (~1 mL/s, black pump heads). If you have the slow flow rate pumps (~ 1 ml/m, pink pump heads), select the SLOW radio button for that array.

8. Select all of the vials either by clicking the button on the bottom right or clicking and dragging across the vials on the selector. Click PUMP IN (or whatever the pump array you are calibrating) to run the pumps for 10s if the FAST radio button was selected or 100s if the SLOW radio button was selected.

9. After the pumps finish pumping, measure how much water was pumped by pouring out the water from each vial into a 25 mL graduated cylinder (or something similar). Enter this number into the box for each vial. The flow rate will appear on the vial selector on the right side of the screen.

10. Repeat this process for each pump array you have selected for calibration.

11. Click the pen button to submit and save the calibration. Then click Exit after the calibration is logged.

12. Verify the calibration appears on the setup page.

About

Why are OD measurements important?

The turbidity (cloudiness) of the culture typically correlates to how many cells are in a culture. By using a simple LED and photodiode pair, we can quantify how many cells are in the population, track how the culture is growing over time, and observe the culture changing over the course of the experiment.

Why is calibration necessary?

Readings sent from the Raspberry Pi to the computer/server are the raw voltage values measured by the Arduino in the Motherboard. In other words, to make sense of what these readings are, we need a calibration file that converts this to a useable optical density measurement.

Is calibration different between different organisms?

Yes. Cells with different size and shapes scatter differently and would result in unique calibration curves. eVOLVER is set up such that one could easily use the appropriate calibration files for each experiment/organism.

Is calibration different between different temperatures?

Yes. Temperature effects the photodiode sensors that we use to measure OD. If you plan to use different temperatures we suggest doing a new calibration for each one.

Prepare the day before:

• 16 assembled glass vials:

  • Glass vials (Chemglass, CG-4902-08)

  • Vial screw caps

  • Octagon magnetic stir bars (Fisher, 14-513-57)

OD Standard Preparation

Wash cells and resuspend in 1X PBS to avoid cell growth

1. Spin down saturated cultures in 50mL or 250mL centrifuge tubes.

   1. 2500 rcf for 5 minutes should be fine for bacteria or yeast

2. Pour off media and resuspend cells in 50mL 1X PBS

3. Spin down cells in a 50mL conical tube

Find OD of Concentrated Cell Stock and Make Standards

1. Measure the OD of cell stock using a dilution series

   1. Dilute 200uL of cells into 1800uL of 1X PBS (1:10)

   2. Dilute the 1mL of the 1:10 dilution into 1mL 1X PBS (1:20)

   3. Make 1:40 and 1:80 dilutions

1. Calculate OD of concentrated cell stock

   1. Input ODs for dilution and 1X PBS blank into the spreadsheet

   2. Enter calculated OD into 'OD of Cell Stock'

Measure OD of Standards

1. Make dilutions for standards above 0.5 OD

   1. Use a 96-well plate to ease dilutions and transfer to plate reader

2. Measure ODs and calculate original OD from dilution OD

Calibrate OD using the GUI

1. On the eVOLVER app home screen, make sure you are connected to your eVOLVER (green symbol next to active eVOLVER), then choose CALIBRATIONS and then O.D.

1. Give the calibration a name (a standard suggestion is YYMMDD_YourInitials_od) and click enter.

2. Click on the white box by the S0. The display should now have an overlay with Sample 0 on top of a box with a keypad. Input the OD values of each sample.

1. Press ▷. After the eVOLVER has logged the vials in one configuration, click the forward arrow. Move vials into their new positions, as seen on the right side of the GUI.

2. Click VIEW COLLECTED DATA to monitor the smoothness of the curves as you calibrate. If a particular value is a big outlier, consider rewinding to that vial position and doing it again. Don't worry your other work will be saved!

3. Continue until all sixteen sets of values have been saved for each standard in each vial.

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

1. Mac

2. Windows

3. Linux

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!

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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

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.

Vial Cap Upgrades

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.

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

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

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

About

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

Materials

Nalgene 20L carboy

Nalgene fitting/venting closure

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

Overview

Reasons you might use command line experiment start:

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.

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.

Checklist

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

Overview

Purpose

Long have we been plagued by vial overflows. Certainly, many were our fault when we pumped too much in during setup or made unscrupulous changes to code. However, not always! And the cost was always too high for the magnitude of the mistake. Testing vials, rebuilding, and recalibration took weeks of our time.

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

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

Algorithm

1. OD is constantly monitored

2. When OD goes above the an upper OD threshold

Why

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

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.

Why is running the growth curve algorithm a good first experiment?

When setting up eVOLVER for the first time, we recommend performing a simple growth curve as a starting experiment. The growth curve algorithm functions similar to the turbidostat algorithm, except the feedback between any experimental parameters are turned OFF (or ). That way, you won't have to worry about any leaks or overflow issues from pumps misbehaving while testing the system.

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 .

Overview

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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.

Ways you might accidentally change the OD reading

1. Moving vials once experiment has started

2. Altering

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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.

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.

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

1. Download the Arduino IDE

should be fairly easy to navigate.

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

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Protocol

Page Under Construction

Design

Eagle

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

- Recommended

Overview

Reasons you might want to do this:

1. Changing - ie how often the s erver cycles

About Server Logs

We use to run and manage the logs for the . 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.

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.

Relevant Forum Posts

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.