Calibrations

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)

Each min-eVOLVER needs its own calibration files. These are kept inside of the server folder for each min-eVOLVER (ie in evolver-min/evolver/calibrations.json)

Questions?

Ask about calibrations in the relevant category on the forum.

Before Calibration

1. Complete the setup page

2. Gather materials (see below)

3. Start cells for OD calibration the night before

4. Calibrate temperature before OD

Temperature calibration should be done before OD calibration because the OD sensors are effected by temperature. Calibrate OD at the temperature that you intend to run experiments. (For example 37C for bacteria and 30C for yeast)

Materials

• Glass eVOLVER vials (6X)

• eVOLVER vial caps (6X)

• Digital temperature probe (we use a Fisherbrand™ Traceable™ Hi-Accuracy 0.01° Refrigerator Thermometer)

• 20mL pipette or a small graduated cylinder

• 200mL cells at OD600 > 2

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/

2. Fill eVOLVER vials with 15-20mL of water, put in stir bars, and place in aluminum sleeves.

3. Turn on the min-eVOLVER and start the server as in setup.

4. In the dpu virtual environment, send the following temperature command to the min-eVOLVER using send_command.py:

   1. python3 send_command.py <port_number> temp 31000

5. Wait for the temperature to equilibrate (using a digital temperate probe)

While waiting for equilibrations you can start calibrating pumps.

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

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

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

9. Copy and paste the values under "Copy + Paste" in evolver-min/evolver/calibrations.json for the correct min-eVOLVER

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

   2. WARNING: do not alter the format of the calibrations.json file. Doing this, (accidentally adding an extra square bracket or comma for example) could easily give errors during experiments.

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

   
3. Fill the pump lines by sending the following pump command to the min-eVOLVER using send_command.py:

   1. python3 send_command.py <port_number> pump 30,30,30,30,300,300

If you need the pumps to stop before the time is up, send the command:

python3 send_command.py <port_number> pump 0

4. Wait for the pump lines to fill

5. Calibrate the fast (grey) pumps

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

   2. Send the command:

      1. python3 send_command.py <port_number> pump 20,20,20,20,0,0

   3. Measure the resulting water output (either using a 20mL autopipette or using a small graduated cylinder)

   4. Input the values into the excel spreadsheet

   5. If you see significant variability, make sure all inputs are in water and try again

6. Calibrate the slow (pink) pumps

7. 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

   2. Make sure that the tubing is still full (send another command if not)

8. Send the command:

   1. python3 send_command.py <port_number> pump 0,0,0,0,40,40

9. Measure the resulting water output (using a 1mL pipette)

10. Input the values into the excel spreadsheet (in mL)

11. Copy and paste the values under "Copy + Paste" in evolver-min/evolver/calibrations.json for the correct min-eVOLVER

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

OD Calibration

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.

For ePACE in the min-eVOLVER, consider running calibration of the lagoon vial with 10mL volume for more accurate readings.

Procedure

Make sure you have your vial cover on! This will drastically alter calibrations if you leave it off.

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

   3. Copy and paste the command next to "Temperature command:" field. (Change to target your min-eVOLVER's port)

2. Follow the OD calibration tutorial for the main eVOLVER until it asks to begin calibration via GUI

   1. We will use this to make 8 standards, rather than 16

   2. This will be less vials to deal with

3. Heat OD standards to temperature

   1. Ideally, the vials will be stirring and continuously at the correct temperature

      1. Stirring because cells will settle otherwise

      2. At temperature because it effects the OD sensor

   2. However, the min-eVOLVER only has 2 vials, so we must find a way to make sure the vials are at temperature before reading the OD values

   3. We can either do this by

      1. Waiting for vials to come to temperature in the min-eVOLVER vials (slow)

      2. Keeping vials in an incubator until ready for reading (faster)

         1. Make sure cells are suspended before reading values either through keeping in a shaking incubator or through pipetting or swirling

4. While OD standards are heating

   1. Make a copy of od_calibration.xlsx with the date

   2. Input the optical density of your standards into the Standard (OD600) cells

5. Make sure the od_led is set to 4095

   1. Either check the server cycling log or send the command:

      1. python3 send_command.py <port_number> od_led 4095

6. Record 3 server values for each OD standard

   1. Cycle through the standards in an orderly fashion

   2. For example:

      1. Label the vials 1 - 8

      2. Start with vial 1 in smart sleeve 1 and vial 2 in smart sleeve 2

      3. Then rotate so that vial 8 is in sleeve 1 and vial 1 is in sleeve 2

      4. Repeat for all standards

After changing which standards are being read, wait for the server to cycle a couple of times before recording values. Values are averaged and the values from the previous two OD standards could still be in the mix.

Highlighted are the OD sensor values for the first and second vials respectively on a min-eVOLVER.

9. 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

   2. If you only have one min-eVOLVER you are calibrating, clear the rest of the rows of data to avoid confusion.

10. In a terminal in the dpu environment, install pandas using the command

    1. pip install pandas

11. Run the following script for fitting a sigmoid function to the curve:

    1. python3 sigmoid_fit.py

12. Evaluate the resulting curve fit in the window that pops up

Fitting curves to OD calibration data for two min-eVOLVERs. While there is some noise, standards generally follow the sigmoid function.

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

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

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