Jan 16, 2024
Version 1
OT-2 Modular Cloning Construct Assembly V.1
- Ana Mariya Anhel1,
- Laura Cutugno1,
- Ángel Goñi-Moreno1
- 1Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM)-Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA/CSIC), Madrid, Spain
Protocol Citation: Ana Mariya Anhel, Laura Cutugno, Ángel Goñi-Moreno 2024. OT-2 Modular Cloning Construct Assembly . protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8p82rg2w/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: December 14, 2023
Last Modified: January 17, 2024
Protocol Integer ID: 92328
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 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
Guidelines
This protocol was developed with python 3.7.1, OT App Software Version 6.3.1 and API level version 2.14 in 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)
This protocol has been tested by assembling level 1 and level 2 constructs with parts of the golden standard database
Materials
Software
- Python 3.7.1
- opentrons software version 6.3.1
- python packages: pandas (0.25.3), numpy (1.15.1), openpyxl (3.1.2), math, random
- OT App
- Excel
OT-2 Labware
- Opentrons Tip racks
Equipment
Opentrons 96 Tip Rack 300 µL
NAME
Tip rack
TYPE
Opentrons
BRAND
-
SKU
LINK
Equipment
Opentrons 96 Tip Rack 20 µL
NAME
Tip rack
TYPE
Opentrons
BRAND
-
SKU
LINK
- PCR skirted plate
Equipment
PCR 96-plate low profile Thermo Scientific
NAME
PCR Plate, 96-well, low profile
TYPE
Thermo Scientific
BRAND
AB0800R
SKU
LINK
- Opentrons Eppendord Tube Rack
Equipment
Opentrons 24 Tube Rack with Eppendorf 1.5 mL Safe-Lock Snapcap
NAME
Tube Rack
TYPE
Opentrons
BRAND
opentrons_24_tuberack_eppendorf_1.5ml_safelock_sna
SKU
LINK
- Tube Rack Eppendorf for Heater-Shaker + 24 eppendorf tube holder
TubeHolder_Eppendorfs_HS_OT2.stl Equipment
24 Eppendorf Tube holder
NAME
Tube holder
TYPE
Opentrons
BRAND
999-00030
SKU
LINK
- 1.5mL eppendorfs (without the cap) + 4ºC cold-block with adaptor (file attach)
Equipment
BRAND™ Centrifuge Tube Mini-Cooler
NAME
ColdBlock
TYPE
BRAND
BRAND
10141921
SKU
LINK
adaptor_OT_coldblock.stl Reactives:
- Water: MilliQ water
- T4 Ligase and Ligase Buffer: T4 DNA Ligase, 100uPromegaCatalog #M1801
- Restriction Enzyme: BpiI (BbsI) (10 U/µL)Thermo FisherCatalog #ER1012 BsaI-HFv2New England BiolabsCatalog # R3733S
Equipment:
Equipment
OT-2
NAME
Liquid handler
TYPE
Opentrons
BRAND
OT-2
SKU
Equipment
Single Channel Electronic Pipette (GEN2) 300uL
NAME
Opentrons Pipette
TYPE
Opentrons
BRAND
-
SKU
LINK
Equipment
Single Channel Electronic Pipette (GEN2) 20uL
NAME
Opentrons Pipette
TYPE
Opentrons
BRAND
-
SKU
LINK
Equipment
Opentrons Thermocycler Module
NAME
Thermocycler
TYPE
Opentons
BRAND
999-00174
SKU
LINK
Equipment
Opentrons Heater-Shaker Module
NAME
Heater-Shaker
TYPE
Opentrons
BRAND
999-00157
SKU
LINK
Safety warnings
If you are using the heater-shaker take in account that there is a limit of RPM that it can shake before the liquid of the eppendorfs get out. As well, take in account that there are constrictions that could prevent some labware to be placed.
This speed depends on the liquid consistency and volume
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
Files Preparation
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
- TemperatureProfile (Optional): a profile that will be performed in the thermocycler if set as True in the ModuleVariables sheet
Template-VariablesMoCloAssembly.xlsx 
MoCloAssemblyInstructions.pdf Fill the template with the corresponding values
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 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 fileVariablesMoCloAssembly.xlsx to the OT system.
We need to perform the following command
Command
File passing from our computer to robot's linux (OT raspberry)
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
Command
Transferring files to OT (MacOS 13 and 14)
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).
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
Creation of .json file
The description file can be obtained by describing the labware dimensions at https://labware.opentrons.com/create/
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
Expected result
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
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.
Command
Transferring the custom labware to OT (Linux)
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
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 protocol
If 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
Connect to the robot
go to step #2 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
Command
Connect to Linux based OT via ssh
In Windows you can do this command in Windows Powershell
Expected result
If the connection has been successful you should obtain a screen similar to the following image
Drawing obtained when entering an OT-2 system
Install the package
Once inside the robot's system, you need to run the following command
Command
Install openpyxl package (Linux 4.14.74-v7)
Note
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
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 7.0.2
Indications may vary from version to version
Software
Opentrons App
NAME
Windows >=10, Mac >=10 , Ubuntu >=12.04
OS
Opentrons
DEVELOPER
SOURCE LINK
Load the script in the App
Protocols -> Import -> Drag Python script
Note
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.
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
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
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.
Expected result
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
Expected result
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
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 needed)
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 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
Expected result
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
After-Running
Retrieve labware from the OT
Importing map from robot
To retrieve the file we can go to step #2 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
Command
Transferring files from OT to computer (Linux, macOS)
Expected result
The map(s) contains a table for each final plate in the run
Each 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
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
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.
Prepare variable file
Excel temple filled and saved with the name VariablesMoCloAssembly.xlsx
VariablesMoCloAssembly.xlsx Export the variable file to 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
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.json3KB Upload it to the opentrons app and make sure it is loaded in it
List of custom labware recorded in the Opentrons App
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 Command
Transferring the used custom labware to OT (Linux)
Import the script that we have downloaded from the step go to step #5.1 (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
Run the protocol in the robot that we have transferred the Excel file
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
Clean the platform of the robot that we are going to perform the protocol
Prepare all reagents and labware in the places as the App is showing taking into account the notes in step go to step #5.2 Notes
Start run
Expected result
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
Retrieve labwares from the OT
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 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
Golden Standard: a complete standard, portable, and interoperative MoClo tool for model and non-model proteobacteria (Nucleic Acids Research) https://doi.org/10.1093/nar/gkad758
Citations
Step 9
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. Golden Standard: a complete standard, portable, and interoperative MoClo tool for model and non-model proteobacteria.
https://doi.org/10.1093/nar/gkad758