Aug 06, 2024

Public workspaceOT-2 Custom Mixing with Single or Multi-Channel Pipette

DOI
dx.doi.org/10.17504/protocols.io.5jyl82686l2w/v1
  • 1CNB CSIC;
  • 2CBGP UPM-INIA
  • Ángel Goñi-Moreno: angel.goni@cnb.csic.es;
biocomp.cbgp Biocomputation Lab
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DOI: dx.doi.org/10.17504/protocols.io.5jyl82686l2w/v1
Protocol Citation: Ángel Goñi-Moreno, Ana Mariya Anhel, Paula Mugica-Galan 2024. OT-2 Custom Mixing with Single or Multi-Channel Pipette. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl82686l2w/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 25, 2024
Last Modified: August 06, 2024
Protocol Integer ID: 104076
Funders Acknowledgements:
Comunidad de Madrid
Grant ID: Y2020/TCS-6555, 2019-T1/BIO-14053
MCIN/AEI
Grant ID: CEX2020-000999-S, PID2020-117205GA-I00
European Research Council
Grant ID: 101044360
Abstract
This protocol is created to help efficiently produce high-throughput plates with different reagents using either a single or multi-channel pipette controlled by an Opentrons OT-2 robot. By running this script, users can achieve a precise layout of reagents in specified volumes across the final plate(s) using a single-channel or multi-channel pipette based on a customizable Excel input file.

The protocol uses a Python script for the Opentrons OT-2 robot in conjunction with an Excel file that contains all the necessary parameters and will be processed by the Python script.

This Excel file contains customizable variables such as the number of final plates, transfer volumes, plate types, and the type of pipette used.

This method ensures the accurate distribution of reagents based on user-defined configurations, making the high-throughput process more efficient.
This protocol has been used in our laboratory to check how different quorum sensing systems work in P. putida KT2440.

This protocol is a set of instructions or description of the LAP repository entry LAP-CustomReagentMixingMultiSinglePip-OT2-1.0.0
You can find the script and complementary information for this specific version of the protocol in this LAP entry link and GitHub Link to LAP entry documents
Guidelines
This protocol was developed using Python 3.7.1, OT App Software Version 7.0.2, and API level version 2.14 on a Linux 4.14.74 system (these are the OT-2 specifications).
In the script, several packages are used: pandas (0.25.3), openpyxl (3.1.2), math, random and numpy (1.15.1)

It has been tested with cultures from Pseudomonas putida KT2440 as part of testing quorum sensing systems with different concentrations of 3mBz to control the systems programmed in them.

The maximum number of 96-well plates per run is 9 final plates with no replicas, replacing the tiprack, using only a single-channel pipette and one falcon tube rack necessary to distribute the reagents to all the final plates.
Materials
Materials
Software
  • Python 3.7.1
  • opentrons software version 7.0.2
  • python packages: pandas (0.25.3), openpyxl (3.1.2), numpy(1.15.1), math, random, itertools, time
  • OT App version 7.0.2
  • Excel

OT-2 Labware
  • Opentrons Tip racks
  • Opentrons Pipettes
  • Opentrons Tube-racks
  • Well plates (such as Corning 96 Well Plate 360 µL Flat)
  • Reservoirs (such as a NEST 12 Well Reservoir 15 mL)
  • Opentrons HEPA filter (Optional)
Files Preparation
Files Preparation
Preparing Customized Template

Preparing the template (a .xlsx) with the specific variables for each experiment.

Attached is a template of the variable file with several sheets and a PDF file explaining each variable:
  1. GeneralVariables: These are variables mainly related to the labware to be used, optimization for the source plate, and sterility variables.
  2. PipetteVariables: These are variables related to the pipettes to be used.
  3. FinalPlatesVariables: variables related to the specifications of each final plate to be created, such as the maps associated with this final plate, the number of replicas to make and the type of pipette used to create each.
  4. MapsReagents: These are the sheets that are established in variables in FinalPlatesVariables that define the layout of reagents to transfer/distribute to each one of the final cells
  5. MapsVolumes: These are the sheets established in variables in FinalPlatesVariables that define the layout of volumes of each reagent defined in MapReagents to transfer/distribute to each of the final cells.

Download TemplateVariablesCustomMixing.xlsxTemplateVariablesCustomMixing.xlsx
Download CustomMixingInstructions_v100.pdfCustomMixingInstructions_v100.pdf

Note
The most updated Excel template can be found on the LAPrepo Repository Page

Fill the template with the corresponding values
Save it with the name VariablesCustomMixing.xlsx

Note
The file should be spelt precisely as VariablesCustomMixing.xlsx or the Python script won't work correctly.

Setting the robot
Setting the robot
Prepare the system of the robot to run the protocol

For this protocol to work, we need to transfer the file VariablesCustomMixing.xlsx to the directory /data/user_storage of the OT system we will use to perform the protocol.

Also, if we use custom labware, we must upload it to the OT App and send it to the directory /data/labware/v2/custom_definitions/custom_beta if the labware is not there yet.

Finally, we must ensure the package openpyxl is installed in the robot system.

We can do this by following the protocol Setting and Customizing OT-2 for LAP Entries with the specifications in the text above.
Protocol
Setting and Customizing OT-2 for LAP Entries
NAME
Setting and Customizing OT-2 for LAP Entries
CREATED BY
biocomp.cbgp Biocomputation Lab

Running protocol
Running protocol
Load script in OT-App

Now that we have transferred the variable files to the robot, we can import the script and run it in the selected robot

Note
This step has been developed and tested with version 7.0.2 of the OT-App.

Indications may vary from version to version of the opentrons App and the script version.

Software
Opentrons App
NAME
Windows >=10, Mac >=10 , Ubuntu >=12.04
OS
Opentrons
DEVELOPER
https://opentrons.com/ot-app/
SOURCE LINK

Load the script in the App

Protocols -> Import -> Drag Python script

This protocol version was developed when the last version of LAP-CustomReagentMixingMultiSinglePip-OT2 was 1.0.0; the script is attached.

Download ScriptMixingReagents_v100.pyScriptMixingReagents_v100.py

The name of the Python file is the user's choice; it will work with any name in the app.

Note
The last script version can be found at https://github.com/BiocomputationLab/LAPrepository/tree/main/LAPEntries. The name of the directory should be LAP-CustomReagentMixingMultiSinglePip-OT2, followed by the version.

Also, the latest version of the script can be found at https://www.laprepo.com/repository/, with the same name as on GitHub.

Software
LAP Repository
NAME
https://biocomputationlab.com/
DEVELOPER
www.laprepo.com
SOURCE LINK

Note
The App with version 7.0.2 analyzes your protocol before setting a robot to run, so the labware will not be shown before assigning the protocol to a specific robot when you import it into the App.

Select Robot to Perform Script

Click in the protocol -> Start setup -> Choose the OT where the file VariablesCustomMixing.xlsx is -> Proceed To Setto overpour to account for any pipetting errors slightlyup

After clicking on Proceed to Setup, you should obtain the labware's positions in the Labware tab and in the Liquids tab, the reagents and their corresponding volume.
If the protocol with the set variables cannot run, an error will occur during the simulation's run in the app after Start setup. Many errors are already contemplated and have a specific message that hints tothe user what could have gone wrong.

Note
The reagent volumes in the reservoirs for the multi-channel pipettes or tubes for the single-channel pipettes are precisely measured, so it is recommended to overpour slightly to account for any pipetting errors.

Note
It is recommended that you perform a labware position check.
You can do it with test plates after loading the script but before cleaning the surface to prepare the experiment layout. That way, you reduce the probability of contamination (using the test plates and labware) and pipetting errors (position check).

Run Protocol in OT
Make sure the needed calibrations are done

Pipettes, tip racks and tip length calibrations need to be done for the items used in this run
Labware position check is performed (if required)
Turn the HEPA Filter on
Optional
Clean the surface of the robot with 70% ethanol to clean and disinfect the surfaces

Note
Check the Opentrons page https://support.opentrons.com/s/article/Cleaning-your-OT-2? for more information about cleaning the OT-2 robot with the proper materials.

Set the labware and reagents as shown in the OT-App
Start Run

The procedure that the robot is going to do is mainly divided into 2 parts:
  1. Distribute reagents with single-channel if any final plate is set to be created with it.
  2. Distribute reagents with multi-channel if any final plate is set to be created with it.

Expected result
One or more plates, with the reactive-volume layout set by the user in the Excel input file.

After-running
After-running
Retrieve labware from the OT
Example
Example
We want to create three plates: 1 with a single-channel pipette and 2 with a multi-channel pipette.
The two plates filled with the multi-channel will have different concentrations of IPTG and certain bacteria. At the same time, the single-channel pipette will create a plate with varying combinations of reagents.
The multi-channel reservoir source's optimization will be low, and the reservoir will have 15 columns and 1 row.
The tip will be changed every time a reagent is changed, and the liquids will be dispensed at the top so the source reagents are not contaminated.

We will use a computer with a Windows 10 system.
Prepare variable file

Excel template that we can find Go togo to step #1 filled and saved with the name
VariablesCustomMixing.xlsx

Download VariablesCustomMixing.xlsxVariablesCustomMixing.xlsx

Upload custom labware to app
We are using a custom labware called vwrblueprintdepth105_96_wellplate_390ul that has been created with the labware creator that opentrons offers (https://labware.opentrons.com/create/)

Download vwrblueprintdepth105_96_wellplate_390ul.jsonvwrblueprintdepth105_96_wellplate_390ul.json

We upload it to the opentrons app (make sure that it is in the robot app) and the robot system as stated in the protocol in step Go togo to step #2 Setting and Customizing OT-2 for LAP Entries
Export the Excel file to the robot

Export the VariablesCustomMixing.xlsx file to the /data/user_storage directory in the robot where the script is going to be played following the instructions in Go togo to step #2 Setting and Customizing OT-2 for LAP Entries

Import the script to the robot

Download ScriptMixingReagents_v100_example1.pyScriptMixingReagents_v100_example1.py

Result of importing the Python script in the OT-App

Note
An error during the simulation of the script in the app is normal. The script is meant to work in the opentrons robot, not on your computer.

Run the protocol

ScriptMixingReagents_v100_example1.py -> Start setup -> Select the robot in which we are going to run the protocol

If we do not have any errors, the output should look similar to the following pictures



















Turn the HEPA filter on
Optional
Clean the deck
Perform calibrations if needed
Optional
Prepare all reagents and labware

The places and volumes should correspond to the ones shown in the Go to pictures.
Place everything, taking into account the notes in step Go to Notes

Start Run
.
Expected result
After the run has finished, we should obtain 3 final plates with the reagents and volumes described in the input Excel file.




Final content of C3 in PlateMulti1 located in Slot 1

Final content of A6 in PlateMulti2 located in Slot 2

Final content of A7 in PlateSingle1 located in Slot 3


Retrieve Labware from the robot
Protocol references
The Laboratory Automation Protocol (LAP) Format and Repository: A Platform for Enhancing Workflow Efficiency in Synthetic Biology (ACS Synth. Biol.) https://doi.org/10.1021/acssynbio.3c00397