TITLE: OT-2 Media dispensing and culture inoculation protocol
AUTHORS: Ana Mariya Anhel, Lorea Alejaldre, Ángel Goñi-Moreno

[DESCRIPTION]
This protocol is meant to distribute reactive(s) into well plates with a single channel pipette, and then transfer culture samples from a source plate to these plates with a multi channel pipette.

This protocol uses a python script for an Opentrons 2 robot and a .csv file containing the required variables to set the number of samples, volumes of transfer, type of plates, etc... 
In our laboratory, this protocol has been used as part of the "High-throughput workflow for the genotypic characterization of transposon library variants" also available in protocols.io to prepare cultures plates to perform a subsequent counter-selection 



[BEFORE_START]
Note that the source and final 96-well plates will follow the same order (If a row of the source plates has empty wells the tips for the multichannel will aspirate in that well)

[GUIDELINES]
This protocol was developed with python 3.9.7, OT App Software Version 6.1.0 and API level version 2.13. It has been tested with cultures from Pseudomonas putida KT2440 as part of the High-throughput workflow for the genotypic characterization of transposon library variants. LB and M9-citrate media with either kanamycin, gentamicin, streptomycin or ampicillin haven been successfully run.


The maximum number of 96-well plates per run is 3 source plates to 3 final plates (using 4 tip racks and 1 tube rack).

[STEPS]
SECTION: Files Preparation
1. Preparing Customized Template

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

Here we attach one excel with several sheets:
Template to use in protocol
Explanation of each variable
Several examples

  

SECTION: Run Protocol
5. Setting Labware
SECTION: After-Running
8. Retrieve labware from the OT
SECTION: Files Preparation
2. Transfering csv to Robot


Transfer the Variables-AntibioticPlatesCreation-OT.csv to the directory /data/user_storage of the OT system that we are going to use to perform the protocol.

 


Here we present a summary of how to transfer the files in 3 OS: Windows, Mac and Ubuntu (applicable to most Linux)


Mac/Linux
We will use the command line with scp to transfer the file Variables-PCRs-OT.csv to the OT system.

We need to perform the following line:
 

 

Windows
There are several ways to pass files from a windows to a linux (for example with a virtual machine).

Here we will use FileZilla (https://filezilla-project.org/download.php?type=client).

Go to File > Site Manager > New Site > Change Protocol to SFTP. Then introduce in Host the OT IP, change the Logon Type to key file, change the user name to root and give the directory were the ot key is. Should look something like this

  
 
Then press connect and we will have a conection between our computer and the robot.

After this connection, we should be able to move out Variables-SamplesMerging-OT.csv  (in our computer) to the directory /data/user_storage in the robot.

 

 


SECTION: Files Preparation
1.1. Fill the template with the corresponding values
SECTION: Files Preparation
1.2. Store it as a comma-separated values file (csv) with the name Variables-AntibioticPlatesCreation-OT.csv
 


 



SECTION: Files Preparation
3. Adding the custom labware
 

 

SECTION: Files Preparation
3.1. Creation of .json file

The description file can be obtained by describing the labware dimensions in https://labware.opentrons.com/create/
SECTION: Files Preparation
3.2. Creating description folder

We need to create for our custom labware a folder with the API name containing the description file (.json) called 1.json
SECTION: Files Preparation
3.3. Transfer the description folder to the OT robot

We need to transfer the directory or directories created in Step 3.2

For that we need to introduce the following command (in Linux), for Windows process   
 

 

SECTION: Files Preparation
4. Simulating and getting the output (user instructions)


For more information in how to setup the ssh visit the official OT support blog (https://support.opentrons.com/s/article/Setting-up-SSH-access-to-your-OT-2)
SECTION: Files Preparation
4.1. Passing python script

To make sure that the protocol will work with the variables we have introduced we need to run the python script in the robot with the OT simulator.

The last script version can be found at https://github.com/Biocomputation-CBGP/OT2/tree/main/AntibioticPlatesGeneration (name of this file is user's choice)

For that we can   but instead of passing the variables file, we pass the python script. In this case we can transfer it to the directory /data/user_storage or we can directly pass it to the directory /root (recommended)


 

SECTION: Files Preparation
4.2. Simulating

We should connect to the robot via ssh
 


Then we would move to the directory where the python script is with the cd command

Finally we are going to perform the simulation of the opentrons

 

 

SECTION: Files Preparation
4.3. In case that you want to see the instruction file before the next step, you can see the instruction file with cat
 

SECTION: Files Preparation
4.4. Retriving output file (user instructions)
To retrive the output from the OT system you can   , replicate it but transfering the file from the OT to your computer

In this file we have 3 or more sections:

(Optional) Warnings
General Information: pieces of information that are extracted from variables in the variables file and are only remainders
Deck Labware Position: this information will be provided by the OT as well
15mL Falcon Reactive Positions: for each Falcon tuberack that is needed (with 15 positions) we have a table with the reactives and their respective volumes

 

In the case that something went wrong, the variables are not consistents or there is not enough deck place to perform the experiments the instruction file will be sorter and will have a single section, Errors.

SECTION: Run Protocol
6. Load script in OT-App

Now that we have made sure that the protocol is going to work we import the python script in the OT-App.
 

SECTION: Run Protocol
7. Run Protocol in OT
SECTION: Run Protocol
5.1. Wipe the surface of the deck with 70% ethanol to clean and desinfect the surfaces
 

SECTION: Run Protocol
5.2. Set the respective labwares in the slots as the OT App and the instruction file set

 

SECTION: Run Protocol
5.3. Set the different reactives in their respective falcon tuberack positions, as stated in the instruction file

 

SECTION: Run Protocol
6.1. Load the script

Protocols > Import > Drag the python script
 

 

SECTION: Run Protocol
6.2. Select Robot to Perform Script

Click in the protocol > Run Protocol > Choose the OT where the file Variables-AntibioticPlatesCreation-OT.csv is > Proceed To Setup

After clicking in Proceed to Setup you should obtain the setup of the labware as instructed in the Deck Labware Positions section of the instructions file.

The Labware Setup tab should look similar to the following image

  
 


SECTION: Run Protocol
7.1. Make sure the needed calibrations are done
SECTION: Run Protocol
7.2. Labware position check is performed (if needed)
SECTION: Run Protocol
7.3. Start Run

The procedure that the robot is going to do is mainly divided in 2 parts:

Distributing of antibiotic or reactives in respective plates
Transfering samples to corresponding plates

 

 


 

SECTION: Example
9. We want to transfer samples from 3 source plates to different antibiotic plates, one per antibiotic and source plate (Example 2 in Template_Variables_PlateGeneration.xlsx)

We will use a computer with an Ubuntu system connected via USB to the robot
SECTION: Example
9.1. Excel temple filled and exported to csv with the name Variables-AntibioticPlatesCreation-OT.csv

  

SECTION: Example
9.2. Export the csv to /data/user_storage

  
SECTION: Example
9.3. We have never used a 96-well tissue plate labware with the API name vwrblueprintdepth105_96_wellplate_390ul, so we must pass the directory regarding this labware to the robot

  
SECTION: Example
9.4. Pass the scipt that I have downloaded from https://github.com/Biocomputation-CBGP/OT2/tree/main/AntibioticPlatesGeneration (I named it AntibioticPlateGeneration-example.py), connect to OT computer, simulate, exit OT system, store the output in instructions_AntibioticPlateGeneration_Example2.txt and import it to the computer

  

  

SECTION: Example
9.5. Prepare all reactives and labwares

Initial Labware Setup and Coldblocks setup should look like the following picture

  
  

SECTION: Example
9.6. Load to OT-App the AntibioticPlateGeneration-example.py > Run > Select robot in which we are going to run the protocol

The window and tabs of the OT-App should look like the following pictures
 
  
  

SECTION: Example
9.7.  Turn HEPA module on and desinfect OT-2
SECTION: Example
9.8. Load protocol labware as instructed in Step 9.6
SECTION: Example
9.9. Run protocol

The final results should look like the following pictures

 
   
SECTION: Example
9.10. Retrieve labwares from the OT