Purpose

The min-eVOLVER was originally conceived of as a minimal apparatus to conduct phage-assisted continuous evolution (PACE). Thus it has two bioreactors (eVOLVER Smart Sleeves): one for a "host cell reservoir" and one for a "lagoon". It also has six pumps: four for bulk media in and out of the two Smart Sleeves and two for fine control over inducers into the lagoon.

However, base min-eVOLVER can be used for most continuous culture scenarios, such as turbidostat and chemostat. It can also be readily adapted to new experiments.

Differences from Main eVOLVER

Software-Hardware Architecture

The software architecture of the min-eVOLVER is similar to the , with the exceptions that:

• The Raspberry Pi server has been replaced with a server run on the connected laptop

• A single Arduino SAMD21 actuates all functions and collects vial data through the min-eVOLVER board

Maximum Power Draw

• Maximum current draw* = 2 Amps

• Power supply voltage = 12V

• Maximum power draw = 24W

*Maximum current draw was measured from a bench-top power supply with the heaters fully on, all pumps running, and stirring on. Instantaneous spikes in current could be higher when pumps and stirring start up

Overview

• Calibrations are important for the eVOLVER to match its sensor inputs with its actuator outputs

• The min-eVOLVER is not yet compatible with the normal eVOLVER GUI

• Therefore, calibrations must be done manually (ie via recording of values and fitting of lines in excel)

Questions?

Ask about calibrations in the relevant category on the .

Before Calibration

1. Complete the page

2. Gather materials (see below)

3. Start cells for OD calibration the night before

Materials

• Glass eVOLVER vials (6X)

• eVOLVER vial caps (6X)

• Temperature probe (we use a )

Temperature Calibration

About

• During temperature calibrations we will be setting the min-eVOLVER temperature to various 'raw' (uncalibrated) values

• We will then measure the actual temperatures that the min-eVOLVER vials reach using a temperature probe

• We then fit a line correlating the raw values to temperatures in degrees Celsius

Procedure

1. Make a copy of temperature_calibration.xlsx and label with the date.

   1. This can be found in /dpu/calibration/

1. Record the equilibrated temperature (in Celsius) in the space in the spread sheet for the correct vial

2. Repeat steps 3 - 6 for the other three values

3. Ideally the resulting plot will be linear, with little variation of the points from the line

Pump Calibration

1. Make a copy of pump_calibration.xlsx and label with the date.

   1. This can be found in /dpu/calibration/

2. Fill a large beaker with water and submerge all pump input and outputs in the water

1. Wait for the pump lines to fill

2. Calibrate the fast (grey) pumps

   1. Leave fluidic inputs in water and put the outputs in eVOLVER vials

1. Calibrate the slow (pink) pumps

2. Leave the inputs in water and put the outputs in 1.5mL Eppendorf tubes

   1. It may be helpful to remove the plastic Luer lock on the tube end

OD Calibration [Not for ePACE]

About

• OD calibration allows for the min-eVOLVER to accurately read ODs of the cells growing inside.

• While not completely essential with running chemostats, good OD calibrations are essential for turbidostats to give accurate results.

Procedure

1. Set the min-eVOLVER to the temperature your experiment will be at

   1. Open your temperature calibration file

   2. Change the temperature in the "Set to ( C )" field for each vial

1. Copy and paste the Standard (OD600) and Median Values for each vial into od_data.xlsx

   1. Do not include empty cells or change the formatting of od_data.xlsx

1. Copy and paste the values output from sigmoid_fit.py in evolver-min/evolver/calibrations.json for the correct vial and correct min-eVOLVER

1. Replace the values after "coefficients" (shown below)

   1. To avoid confusion, replace everything until the "raw"

Overview

Overview

The min-eVOLVER is a minimal, mini version of the main eVOLVER. It runs in the same software ecosystem with minimal changes to the code. However, the min-eVOLVER controls two eVOLVER Smart Sleeves and six pumps, making it simpler, smaller, and less costly.

Purchasing a Unit

min-eVOLVERs may not be purchased as a product at this time. Instead min-eVOLVER parts must be acquired individually and assembled.

Requirements for Construction

1. Soldering iron + solder - pumps will need to be soldered in place

2. Ability to through-hole solder

   1. Can be self-taught quickly through YouTube videos

Questions About min-eVOLVER Construction or Hardware?

Ask questions on the forum .

Parts Spreadsheet

Below is a spreadsheet containing all parts required to make a min-eVOLVER.

min-eVOLVER PCB

If you are making a min-eVOLVER Control Board PCB from scratch you have two options (as of 11/20/2024):

1. [Recommended] Order an assembled PCB from (guide below) or another manufacturer

2. Buy the parts needed from Digikey, order the from or another manufacturer, and solder the board yourself

   1. NOT recommended for anyone not already familiar with surface mount soldering.

Ordering an Assembled min-eVOLVER PCB from PCBway

1. Make an account on .

2. On the homepage, scroll down and click on PCB assembly

1. Choose the following:

   1. Turnkey, Single pieces, Both sides, fill out the quantity you want, select No "Select PCBway's PCB Order #", uncheck PCB Specifications, and click the green 'Calculate' button.

1. Download the following files from folder, upload to PCBway, and click "Submit the File Now":

   1. Gerber files:

   2. Parts List (BOM):

1. Wait for PCBway to review your order (1-2 business days) before paying

Pump PCB

Should be ordered from or another manufacturer. You can find the .zip of the gerber files . Assembly is not needed.

min-eVOLVER Case

Options:

1. We use to manufacture the cases

   1. Request a quote for the number of cases you require by emailing Jenn Hurd (jhurd@protocase.com)

   2. Mention you want to order the previously ordered name and number below:

12V Power Supply

Several 12V power supplies were tested and were selected for reliability and low noise. While most 12V power supplies should work well in the short term, long term usage may vary. Use a different power supply at your own risk.

Vial Cover

NOT AN OPTIONAL COMPONENT - Protects OD photodiodes from environmental light, which will completely change your OD calibrations. Also protects smart sleeves from spills and splashes.

3D print the latest vial cover to allow for more consistent OD readings. Choose the cheapest black FDM printing on hubs.com, shapeways.com, or a local 3D printing shop and give them the .stl file in the github repository.

eVOLVER Smart Sleeves

May be purchased from Fynch Bio. If interested in making your own see the main and follow assembly guide. This is not recommended for its added complication (you will need to machine the aluminum tube for example), but will save money.

Consumables / General

Listed in the "Consumables / General" sheet of the above spreadsheet

• Anything not listed as (Optional) is required for a full experiment.

• You need a thermometer for calibrating temperature - we suggest one.

• If you plan to run multiple min-eVOLVERs from a computer, you will need to have a USB port available for each one. You can use a USB splitter dongle to accomplish this. Example .

Questions?

Ask questions about this guide on the forum here.

Software Installation

1. Download files from GitHub repositories for the (experiment code) and (communicator with min-eVOLVER).

1. Follow the dpu installation .

   1. We will use the virtual environment made here to run the experiment

   2. Note: as of now, there is no GUI integration. Command line only.

Server Startup

1. Plug the min-eVOLVER in to your computer using micro-USB and THEN plug it in to the 12V DC power supply.

1. Make sure that you are in the server virtual environment

2. This means navigating to the server folder in a command line window and inputting commands:

   1. Mac OS:

1. This will tell you the list of min-eVOLVERs plugged in to the computer

2. Exit from the server log using control + C

3. Copy the full port address into the serial_port variable in the conf.yml

1. Start the server using python3 evolver.py

2. Observe the server for expected behavior

   1. The server cycles once every 20 seconds

Multiple min-eVOLVERs

You can connect multiple min-eVOLVERs to one computer, as long as you have enough USB ports!

1. Make a duplicate min-eVOLVER server code file with a new number ie evolver-min2

2. Plug in the new min-eVOLVER and follow the Server Startup guide

   1. The new min-eVOLVER will have a different serial_port

Test min-eV Hardware

Startup

1. Plug in the micro-USB and then the 12V power supply

2. Start the server

3. In another terminal window, enter the dpu virtual environment

Familiarize yourself with and make sure you can:

1. Send a pump command to all pumps to make sure they actuate

2. Send a command to start and stop stirring

3. Turn temperature off

test_hardware.py

1. Run a full hardware test using test_hardware.py

2. Use the command: python3 test_hardware.py

1. Check that all pumps actuate individually

2. Check that stirring turns off and on

3. Check that OD sensing works as expected (values decrease with OD LED on)

It may be helpful to have the parts page open as well, so you can identify what you need for each step.

Make Ribbon Cables

1. Measure ribbon cables and mark length (figure above A)

   1. 5" ribbon cable for pump board to min-eVOLVER PCB connection

   2. Two 4" ribbon cables for smart sleeve PCB to min-eVOLVER PCB connection

2. Cut ribbon cables to size using scissors or razor.

3. Open the connector and align to the end of the ribbon cable (figure above B)

   1. The connectors should both have the knob on the same side (figure above A, red arrows)

   2. You can apply pressure to the connector so that it stays in place as you work with it

4. Crimp on ribbon cable connectors (one on each side of cable)

   1. Be careful not to break the connectors, they're fragile!

   2. The easiest way crimp these connectors is to use a clamp or vice (figure above C), unless you have a specialized tool

Prepare case

1. Unscrew the 3 screws on the top of the case (figure below, left)

2. Unscrew the 2 screws on the bottom (figure below, right) and unscrew the right middle rubber foot (yellow arrow)

Construct Pump Board

Solder 2x8 ribbon cable connector into pump PCB, screw pumps into front of case, and solder pumps into pump PCB.

Use the below video:

Construct min-eVOLVER PCB

Solder SAMD21 Arduino

1. Place the 2x12-pin header on a workbench with the flat (non conical) side of the pins up (figure below A). Place the SAMD21 board on top of it

   1. You can also carefully plug the SAMD21 into a breadboard if you want to avoid it moving

2. Carefully solder the SAMD21 board pins.

Assemble min-eVOLVER PCB

1. Press the SAMD21 board into the min-eVOLVER PCB until you feel a click and the SAMD21 sits mostly flush

   1. Micro-USB port should be on edge of board

   2. Carefully align the pins with the sockets

OD 90 vs OD 135

1. OD90 = OD LED and photodiode at 90 degrees from eachother

2. OD135 = OD LED and photodiode at 135 degrees from eachother

3. By default, we choose to use OD90 for our vials, which is typically better at measuring higher ODs > 0.4 and is what most people want

Assemble min-eVOLVER PCB and pumps with the case

1. Place min-eVOLVER PCB on standoffs. Screw 1/4" screws into the front left and back right corners (figure A below)

2. Plug the 5" ribbon cable in to the pumps and to the ribbon cable port to the left of the SAMD21 (figure B below)

3. Close the case and replace the 5 screws and rubber foot that you had removed previously (figure C below)

Construct eVOLVER smart sleeves and screw in to case

1. If your smart sleeves came disassembled:

   1. See general smart sleeve construction guide

   2. Firmly push the OD LED and photodiodes in to the smart sleeve

Flash the min-eVOLVER with Arduino code

1. Plug the min-eVOLVER into your computer via the micro-USB cable

   1. Do not plug in the 12V power supply

2. Download the min-eVOLVER Arduino

Server Troubleshooting

My min-eVOLVER is plugged in but I can't connect to it via the server code

• If the server code says that the expected min-eVOLVER port is plugged in, check that the conf.yml file has the correct port address in it (as detailed in the ).

• As a last resort, the Arduino

The server is cycling (every ~20 seconds there are new values) but all commands are erroring

• This happens when the micro-USB is unplugged and replugged in, but the power supply is not

• Quit the server (ctrl+C), unplug the min-eVOLVER from power and micro-USB, plug the micro-USB back in, then the power supply, and restart the server

• As a last resort, the Arduino

Correct 'serial_port' but Server is Not Starting

I have found this happens when the Arduino IDE program (used for putting min-eVOLVER code on the control board) Serial Monitor is open and connected to the SAMD21. Close the Serial Monitor and you will be able to start the server.

Re-Upload the Arduino Code

Rarely, the Arduino that runs the min-eVOLVER needs to be uploaded with its software again. Follow the Arduino Software Installation and upload the min-eVOLVER Arduino onto the device.

Experiment Troubleshooting

Disconnecting During an Experiment

The most common reason for this is the computer is falling asleep. Make sure your computer is set to not fall asleep.

Parameters

OD

OD is one of the most useful and most challenging aspects of eVOLVER. However the min-eVOLVER OD system is nearly the same as the main eVOLVER. Check the main OD troubleshooting for potential causes and fixes.

Overview

This page only details differences from the general min-eVOLVER experimental protocol. You are also expected to have an understanding of PACE and how it is normally run before doing ePACE.

Implementing Controlled Host Cell Density in Reservoirs

Problem: PACE host cells overgrow

• In previous PACE experiments, host cells would increase growth rate as the experiment wore on

• This caused problems with phage replication in the lagoon and the selection plasmid breaking

• Solution: controlling host cell density in cell reservoirs

Implementing Controlled Host Cell Density in Reservoirs

• We control cell density in eVOLVER by running a turbidostat, which checks cell density and dilutes the culture if it is over a threshold.

• In PACE we remove volume from the cell reservoir and transfer it to the lagoon

  • This changes our turbidostat's volume

Chemostat and Turbidostat on the Same Vial

• We implement a "hybrid" function

• The "hybrid" function uses both a turbidostat and a chemostat on the host cell reservoir

• Turbidostat for keeping the cells from overgrowing

Vial Setup

Needles used were all 16ga

Reservoir volume = 30 mL

• Efflux needle = 3" needle in the tallest vial cap port

• Media in = 2" needle in the shortest port

• Vial to Vial = 3" needle in the second tallest port

Lagoon volume = 10 mL

• Efflux needle = 4" needle in the second tallest vial cap port

• Vial to Vial = 3" needle in the lowest port

• Inducer = 4" needle in the tallest port or 2" needle in the second lowest

Fluidic Lines

Hook up pump lines in the configuration shown below

Alter Settings in custom_script.py

1. Copy the whole ePACE template folder (/dpu/experiment/epace-template/)

2. Rename the copied folder to your experiment name

3. Change the EVOLVER_PORT to your eVOLVER's port

lower_thresh and upper thresh

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

• Format: [vial 0, vial 1]

start_time

• chemostats will not pump until this amount of hours has elapsed

• Useful to allow cells in reservoir to grow up before starting experiment

rate_config

• Format: rate_config = [reservoir, lagoon]

• In vial volumes per hour (V/h)

For the Reservoir

• Replaces volume in turbidostat that is removed via vial to vial

• Must be greater than the volume you are taking out

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

For the Lagoon

• Set based off of phage replication rate

Example Settings:

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

2. Setting to rate_config = [1, 1]

   1. 30mL media into reservoir and 10mL from reservoir into lagoon per hour

Inducer

inducer_on

• Turn inducer off to start (inducer_on = False)

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

inducer_concentration

• Times greater (X) the concentration of your inducer in its bottle compared to its final concentration in the lagoon

• Format [pump 5, pump 6]

For example:

1. Your arabinose stock is 1 M

2. The final lagoon concentration you want is 10 mM

3. Therefore 1000 mM / 10 mM = 100 X your final concentration

Optional Settings

You do not need to alter these settings

Swapping Lagoon and Reservoir Vials

If you do want to alter these variables, you also need to swap the vial locations in the turbidostat and chemostat settings of:

• lower_thresh and upper thresh

• rate_config

reservoir_vial

• Vial number of host cell reservoir

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

lagoon_vial

• Vial number of lagoon

• Only a chemostat, can have up to two inducers

Questions?

Ask about experiments in the relevant category on the .

Setup

1. 1. Use send_command.py to fill the vials to appropriate volumes

1. Use send_command.py to set to correct temperature

   1. A calibrated temperature command is in your calibration file temperature_calibration.xlsx

Running the Experiment

In a separate window from your server command line, follow the command line start .

1. If running more than one min-eVOLVER at a time: you need a separate experiment folder and command line window for each min-eVOLVER (and different port number)

2. Start the experiment using the command:

   1. python3 eVOLVER.py -i localhost:<your_min-eV_port>

Ending an Experiment

1. the experiment

2. Data is in the

About

Used to send arbitrary commands to a server outside of both the GUI and an experiment.

Useful for:

1. Controlling the

2. Sending custom commands if you have

3. Sending pump commands greater than 20 seconds if you are using the slow (~1mL/min) pumps or using

Usage

Send a Command

The <port> variable tells the program which eVOLVER to connect to

1. This is designated in the conf.yml file of the eVOLVER under 'port'

2. It is arbitrary, but we can choose something like 5555

Commands in the Server Log

When you send a command, you should see it come up in the window you're running the server in. See for more information about the eVOLVER server and commands.

Annotated Server Log as a Command Comes in

Examples

To set a parameter on all vials to one value: python3 send_command.py

To run specific pumps (where s is a number of seconds):

python3 send_command.py 5555 pump s,s,s,s,s,s

For example:

python3 send_command.py 5555 pump 0,0,0,0,5,5

To set a non-pump parameter on specific vials:

python3 send_command.py <port> <parameter> <list_of_values>

For example:

python3 send_command.py 5555 temp 30000,31000

Info

More about pumps here.

More about tubing and connectors here.

Protocol

Fit tubing to your application

General Notes

1. If you are doing ePACE in the min-eVOLVER, check the experiment for fluidic routing

2. Make sure it's easy to get both tube ends into a beaker for sterilization

3. Alternatively you can connect an additional line to short tubing for easy sterilization

To-Vial Tubing Method (Right Port of All Pumps)

1. Start with tubing that is longer than you think you will need - you can trim it later easily

2. Put eVOLVER vials with caps in the smart sleeves

3. Estimate the tubing lengths for the to-vial pump tubing

Efflux (Pumps 1 + 2 Right Port)

1. Use clear blue 1/16" ID tubing

2. Can be relatively short because it connects just to the vial

Waste (Pumps 1 + 2 Left Port)

1. Use red 1/16" ID tubing

2. Should reach from left port of bottom pumps into waste carboy

Media influx + Inducers (Pumps 3 - 6)

1. Use clear 1/16" ID tubing

2. Left pump port should be long enough to reach media / inducers easily

3. Right pump port can be relatively short because it connects just to the vial

Overview

Advantages:

• Saves time from assembling, autoclaving, and setting up new vials