OT-2 Modular Cloning Construct Assembly

Ana Mariya Anhel, Ángel Goñi-Moreno, Laura Cutugno

Published: 2024-01-17 DOI: 10.17504/protocols.io.5jyl8p82rg2w/v1

Abstract

This protocol is meant to create modular cloning constructs from different parts into a final plate and, optionally, perform the temperature profile needed to the assembly of the constructs.

The output of running this script will be the final plate(s) with the constructs and the mix needed to perform that assembly, and the corresponding map(s) with the names of the constructs in their corrresponding well which will be given by the user in the input file.

This protocol uses a python script for an Opentrons 2 robot and an excel 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 to perform plasmids using the Golden Standard modular cloning of levels 1 and level 2

This protocol is a set of instructions or description of the LAP repository entry LAP-MoCloAssembly-OT2-1.0.1

Before start

Being a one-pot reaction in this protocol only 1 restriction enzyme is used so one type of level, in the case of the golden standard database, can be produced in only 1 run

Steps

Files Preparation

Preparing Customized Template

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

Here there is attached a template of the variable file with several sheets and a PDF file explaining each variable:

GeneralVariables: variables related mainly to the labware that is going to be used
PerPlateVariables: variables related to the specifications of each source plate
PipetteVariables: variables related to the pipettes that are going to be used
ReactionVariables: variables that will determine the final mix of the wells
ModuleVariables: variables related to the modules used in the protocol, the thermocycler and the heater-shaker
Combinations: set of combinations that are going to be created in the final wells, one combination per row and one DNA part per cell
Map DNA Parts Sheet(s): sheet(s) with the names of each DNA part that can be used to create final assemblies denoted in the combinations sheet. The sheet(s) need to have also dthe name of the rows and columns of the plate and the wells that does not have any sample need to be left empty --> not included in the template but needed to be included and have the same names as established in the variable Name Map DNA Parts from the PerPlateVariables Sheet Name Map DNA Parts from the PerPlateVariables Sheet
TemperatureProfile (Optional): a profile that will be performed in the thermocycler if set as True in the ModuleVariables sheet

Template-VariablesMoCloAssembly.xlsx

MoCloAssemblyInstructions.pdf

1.1.

Fill the template with the corresponding values

1.2.

Store it with the name VariablesMoCloAssembly.xlsx

Note

The file should be spelt exactly VariablesMoCloAssembly.xlsx or the Python script won't work correctly

Transferring file to Robot

Transfer the VariablesMoCloAssembly.xlsx to the directory /data/user_storage of the OT robot

that we are going to use to perform the protocol.

Note

Before transferring any file to the OT, we need to know the IP of the robot .This can be obtained in the Networking section of the Device that is going to be used.To obtain this info go to OT-App -> Devices -> Chosen Robot (three dots) -> Robot Settings -> Networking In this tab, you can see 2 types of IP; one is shown if both the robot and you are connected to the same WiFi, and the other is shown if the computer and the robot are connected via USB. Both connections can be used for this step.

Note

To connect to the robot an OT-key should have been previously generated, and it is done with the ssh-keygen command and transferring the public key to the OT. For more information about how to generate and set the connection between your computer and the Opentrons robot, visit For more information about how to generate and set the connection between your computer and the Opentrons robot, visit https://support.opentrons.com/s/article/Setting-up-SSH-access-to-your-OT-2

Here, we present a summary of how to transfer the files in 3 Operative Systems: Windows, MacOS and Linux

MacOS/Linux

We will use the command line with scp to transfer the file VariablesMoCloAssembly.xlsx to the OT system.

We need to perform the following command

#Passing Files to OT 
scp -i [ot_key] [file] root@[IP_OT]:/data/user_storage

Note

You could face difficulties transferring files in MacOS Ventura (13) and Sonoma (14). These problems can be solved by adding the argument -O (uppercase o) to the command #Transferring files to OT (MacOS 13 and 14)scp -Oi [ot_keypath] [file path] root@[OP_robot]:/data/user_storage

Windows

There are several ways to send files from a Windows to a Linux (for example, with a virtual machine or Windows Powershell in the latest versions of Windows).

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 User to root and give the directory where the ot key is. It should look something like this

Example of setting the FileZilla to transfer files from Windows (our computer) to Linux (OT)

Then press Connect , and we will have a connection between our computer and the robot.

After this connection, we should be able to move the file VariablesMoCloAssembly.xlsx (in our computer) to the directory /data/user_storage in the robot.

This method can be used as well in some Linux and MacOS

Note

Take into account that the IP of the robot could change, so it is possible that it will be needed to change the host in these connections from time to time.

Adding the custom labware

There is only a need to do this step when the labware that you are using is not OT official or is not included in the OT app

3.1.

Creation of .json file

The description file can be obtained by describing the labware dimensions at https://labware.opentrons.com/create/

3.2.

Uploading files to the OT App

In the OT app, we need to perform the following route: Labware -> Import -> Choose File -> Select file we have created in step 3.1

Citation

After uploading the labware you should be able to see the new labware in the Labware tab of the OT App, all custom labware can be found more easily in the category Custom Labware

3.3.

Transfer labware files to the robot

If you are using the entry LAP-MoCloAssembly-OT2-1.0.0 or LAP-MoCloAssembly-OT2-1.0.1 with custom labware, an additional step is needed, which is transferring a folder with the custom labware

We need to create for our custom labware a folder with the API name containing the description file (.json) called 1.json and then transfer that folder to the robot's folder /data/labware/v2/custom_definitions/custom_beta in a similar way as in the Step 2 but with the difference that is a directory that needs to be transferred and not a file.

#Transferring the custom labware to OT (Linux)
scp -i [ot_key] -r [directory_custom_labware] root@[IP_OT]:/data/labware/v2/custom_definitions/custom_beta

Note

We do not need to execute this part every time the protocol is used, only when that labware is not included in the OT official labware and these directories are not in the robot

Prepare RobotOS

Install needed packages

This script needs the package openpyxl , which is not installed by default in the OT-2 robots

Note

This step is only needed if the package is not installed in the robot, not every run of the protocolIf the package is not installed, an error will appear when running the script in the robot. While simulating the script in the app, this error will appear, but you can ignore it

4.1.

Connect to the robot

to find the IP of the robot in which you want to run the script

To connect to the robot, you can do it via ssh with the following command

#Connect to Linux based OT via ssh 
ssh -i [path ot_key] root@[Robot_IP]
```In Windows you can do this command in Windows Powershell



<Note title="Citation" type="success" ><span>If the connection has been successful you should obtain a screen similar to the following image</span><img src="https://static.yanyin.tech/literature_test/protocol_io_true/protocols.io.5jyl8p82rg2w/pi66bxsu77.jpg" alt="Drawing obtained when entering an OT-2 system" loading="lazy" title="Drawing obtained when entering an OT-2 system"/></Note>

4.2.

Install the package

Once inside the robot's system, you need to run the following command

#Install openpyxl package (Linux 4.14.74-v7)
pip install openpyxl

Note

For more information about installing packages in the opentrons robots, check the following Opentrons page: For more information about installing packages in the opentrons robots, check the following Opentrons page: https://support.opentrons.com/s/article/Using-Python-packages-in-Python-API-protocols

Running Protocol

Load script in OT-App

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

Note

This whole step has been developed with version 6.3.1 of the OT-App and has been tested until version 7.0.2Indications may vary from version to version

Software

Value	Label
Opentrons App	NAME
Windows >=10, Mac >=10	OS_NAME
, Ubuntu >=12.04	OS_VERSION
https://opentrons.com/ot-app/	REPOSITORY
Opentrons	DEVELOPER

5.1.

Load the script in the App

Protocols -> Import -> Drag Python script

Note

The last script version can be found at The last script version can be found at https://github.com/BiocomputationLab/LAPrepository/tree/main/LAPEntries (the name of this file is the user's choice). The name of the directory should be LAP-MoCloAssembly-OT2 followed by the version. (the name of this file is the user's choice). The name of the directory should be LAP-MoCloAssembly-OT2 followed by the version.As well we can find the latest version of the script at As well we can find the latest version of the script at https://www.laprepo.cbgp.upm.es/repository/ with the same name as in GitHub with the same name as in GitHub

Software

Value	Label
LAP Repository	NAME
www.laprepo.com	REPOSITORY
https://biocomputationlab.com/	DEVELOPER

Note

The App with version 6.3.1 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

5.2.

Select Robot to Perform Script

Click in the protocol -> Start setup -> Choose the OT where the file VariablesMoCloAssembly.xlsx is -> Proceed To Setup

After clicking on Proceed to Setup, you should obtain, if there is no error, the positions of the labware in the Labware tab and the reagents, with their corresponding volume in the Liquids tab.

In case the protocol with the set variables cannot run, an error will be raised by the app. Many errors are contemplated already and have a specific message that will give the user a hint of what could have gone wrong.

Citation

A labware setup should look like the following image, where you can find the initial and final plates, the Eppendorf labware to store the reagents, the corresponding tips and, if included, the location of the heater-shaker(s) and thermocycler. The latter will always have the exact location in the OT-2

Labware Set-up example of a MoClo assembly protocol where both modules are set as True

Labware Set-up example of a MoClo assembly Protocol where both modules are set as False

Citation

A liquid setup should look like the following images, where you can find the samples in the initial plates and the different reagents in the Eppendorf labware with their respective volume. You have the reagent labware(s) where you can find the volumes and positions of the enzymes, ligases, water, etc. The you have the DNA plate(s) where you can find the different parts you have inserted in the input variables file and their needed volume

Liquid Set-up Example of a MoClo assembly Protocol where both modules are set as True

Liquid Set-up Example of a MoClo assembly Protocol where both modules are set as False

Note

Both the volume of the reagents and the DNA parts are exactly what is needed, so it is suggested to pour always more to take in account the error of pipetting

Note

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

Run Protocol in OT

6.1.

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

6.2.

Labware position check is performed (if needed)

6.3.

Clean the surface of the robot with 70% ethanol to clean and disinfect the surfaces

Note

Check the Opentrons page 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. for more information about cleaning the OT-2 robot with the proper materials.

6.4.

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

6.5.

Start Run

The procedure that the robot is going to do is mainly divided into 9 parts:

(Optional) The block temperature from the thermocycler reaches a temperature set by the user in case that variable is not empty
Distribute the needed water to each final well (the volume of water is calculated by the OT-2 according to the volume set in the variable Volume Final Each Reaction (uL) taking in account the volume of the other reagents and the number of DNA parts that is going to that specific well)
Creation of mix(es) transferring ligases, buffer, serum and restriction enzyme to new tube(s)
Mixing with either a pipette or heater-shaker
Distribute mix to final plate(s)
Distribute DNA parts to the corresponding wells (as many transferrings as set in the combinations sheet)
Generate identity maps to be exported
(Optional) Pause the program for user to put lids on the plate located in the thermocycler (if in the input variable)
(Optional) Temperature profile with thermocycler module
(Optional) Block of thermocycler stay to the set temperature

Citation

One or more plates where there is a mix between the different DNA parts and the mix of reagents needed to perform the assembly process, each combination will be in one well.A sheet for every final plate will be created as well in an Excel file with the given name in the sheet GeneralVariables in the variable "Name File Final Constructs" followed by the extension .xlsx in the folder /data/user_storage of the robot where we run the script.

After-Running

Retrieve labware from the OT

Importing map from robot

To retrieve the file we can and reproduce it by transferring the files to the computer.

They will be in the directory /data/user_storage . It will be a file with an extension .xlsx and have the name provided in the input variable file

#Transferring files from OT to computer (Linux, macOS)
scp -i [path_ot_key] root@[IP_robot]:/data/user_storage/[name_map].xlsx [final_path_computer]

Citation

The map(s) contains a table for each final plate in the runEach table will have the name given in the Combinations sheet of the input variable which has to be unique so there is no confusion on the placement of the different combinactions

Example

We want to make 47 constructs that are all level 1 from 38 parts, some created in the lab, some from the golden standard database.

Citation

Blázquez B, León DS, Torres-Bacete J, Gómez-Luengo Á, Kniewel R, Martínez I, Sordon S, Wilczak A, Salgado S, Huszcza E, Popłoński J, Prieto A, Nogales J 2023 Golden Standard: a complete standard, portable, and interoperative MoClo tool for model and non-model proteobacteria. https://doi.org/10.1093/nar/gkad758

We are going to use the heater-shaker module to mix the assembly mix (enzyme, ligase, serum and buffer) and use a thermocycler module to perform the construct assembly with a provided temperature program.

9.1.

Prepare variable file

Excel temple filled and saved with the name VariablesMoCloAssembly.xlsx

VariablesMoCloAssembly.xlsx

9.10.

Retrieve labwares from the OT

9.11.

Retrieve the final map(s) file from the robot where we run the protocol. In this case, they will be called final_construct_map-example.xlsx (name that is stated in the variable file in the variable Name File Final Constructs located in the GeneralVariablesSheet )

Command line window with the transfer command of the final file with the map(s) from the OT to our computer

final_construct_map-example.xlsx

9.2.

Export the variable file to the /data/user_storage folder in the robot the /data/user_storage folder in the robot

Command line window with scp commands to transfer the variables .xlsx from our computer to the OT-2

9.3.

Upload custom labware to app

We are using a custom labware called 3dprinted_opentrons_shaker_1.5mleppendorf that has been created with a 3D printer and its file with the labware creator that opentrons offers (https://labware.opentrons.com/create/)

3dprinted_opentrons_shaker_1.5mleppendorf.json

Upload it to the opentrons app and make sure it is loaded in it

List of custom labware recorded in the Opentrons App

9.4.

Because we are using version 1.0.1 of the script in this example, we will transfer the directory of the labware as well (here we have attached a zip, but it is the folder that must be transferred, not the zip)

3dprinted_opentrons_shaker_1.5mleppendorf.zip

#Transferring the used custom labware to OT (Linux)
scp -i [ot_key] -r 3dprinted_opentrons_shaker_1.5mleppendorf root@[IP_OT]:/data/labware/v2/custom_definitions/custom_beta

9.5.

Import the script that we have downloaded from the step (I named it Example-MoClo . py ) to the OT-App

Example-MoClo.py

The result of importing the Python script into the OT-App

As we can see, we have an error, but that is programmed because the script is meant to work in the robot but not in your computer

9.6.

Run the protocol in the robot that we have transferred the Excel file

Example-MoClo.py Example-MoClo.py -> Start setup -> Select 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

Labware and liquid set-up layout that corresponds to the run of the example variable input file

Volumes of the different reagents needed to perform the protocol that corresponds to the run of the example variable input file

Positions and volumes of different DNA parts. The volumes are calculated but the positions are the ones set in the input file

9.7.

Clean the platform of the robot that we are going to perform the protocol

9.8.

Prepare all reagents and labware in the places as the App is showing taking into account the notes in step

9.9.

Start run

Citation

Example of the content of a well, in this case A1 in the labware Final Plate 1 with Combinations Slot 7

Here, we will obtain the modular cloning mix and the diffreent DNA parts that are set in the input file. These positions are seen in the image by the grey wells, and we can see the info of the plate and the media in the plate on slot 7 in this case

A dynamic DNA tetrahedron framework for active targeting

An MSstats workflow for detecting differentially abundant proteins in large-scale data-independent acquisition mass spectrometry experiments with FragPipe processing

OT-2 Modular Cloning Construct Assembly

Abstract

Before start

Steps

Files Preparation

Prepare RobotOS

Running Protocol

After-Running

Example

推荐阅读