AssemblyTron is a package for automating DNA assembly with an Opentrons liquid handling robot. DNA-Assembly line implements combinatorial DNA assemblies based on designs created with j5 (Hillson, N.J., Rosengarten, R.D., and Keasling J.D. (2012) j5 DNA Assembly Design Automation Software. ACS Synthetic Biology 1 (1), 14-21. DOI: 10.1021/sb2000116), currently accessibe via: https://public-diva.jbei.org/.
This vignette is intended to guide a novice user through installation and use of AssemblyTron to build DNA constructs via Golden Gate assembly in the Opentrons OT-2 robot. We assume that the user has a folder/directory containing the results of a j5 combinatorial assembly design on the computer connected to their OT-2 robot. AssemblyTron then operates on the files within this folder to generate specific assembly instructions for the user and the OT-2 robot which are saved in this folder.
Technical documentation is provided in html format in the AssemblyTron/docs folder and hosted at: https://assemblytron.readthedocs.io/en/latest/AssemblyTron.html
! pip install AssemblyTron
Collecting AssemblyTron==0.0.5 Using cached AssemblyTron-0.0.5-py3-none-any.whl (479 kB) Requirement already satisfied: numpy in c:\users\opentrons\anaconda3\lib\site-packages (from AssemblyTron==0.0.5) (1.20.1) Requirement already satisfied: pandas in c:\users\opentrons\anaconda3\lib\site-packages (from AssemblyTron==0.0.5) (1.2.4) Collecting datetime Using cached DateTime-4.5-py2.py3-none-any.whl (52 kB) Requirement already satisfied: pytz in c:\users\opentrons\anaconda3\lib\site-packages (from datetime->AssemblyTron==0.0.5) (2021.1) Requirement already satisfied: zope.interface in c:\users\opentrons\anaconda3\lib\site-packages (from datetime->AssemblyTron==0.0.5) (5.3.0) Requirement already satisfied: python-dateutil>=2.7.3 in c:\users\opentrons\anaconda3\lib\site-packages (from pandas->AssemblyTron==0.0.5) (2.8.1) Requirement already satisfied: six>=1.5 in c:\users\opentrons\anaconda3\lib\site-packages (from python-dateutil>=2.7.3->pandas->AssemblyTron==0.0.5) (1.15.0) Requirement already satisfied: setuptools in c:\users\opentrons\anaconda3\lib\site-packages (from zope.interface->datetime->AssemblyTron==0.0.5) (52.0.0.post20210125) Installing collected packages: datetime, AssemblyTron Successfully installed AssemblyTron-0.0.5 datetime-4.5
Navigate to your local ~/site-packages directory to access AssemblyLine. Click on the DNA-AssemblyLine folder, and open the software directory.
! pip show AssemblyTron
Name: AssemblyTron Version: 0.0.5 Summary: A package for automating DNA assembly with an Opentrons liquid handling robot Home-page: https://github.com/PlantSynBioLab/opentrons Author: John Bryant Author-email: jbryant2@vt.edu License: Apache License 2.0 Location: c:\users\opentrons\anaconda3\lib\site-packages Requires: numpy, datetime, pandas Required-by:
! cd c:\users\opentrons\anaconda3\lib\site-packages
Run the pathswriter module in order to record your working directory and enable protocol setup scripts to navigate accurately. The pathswriter.py must be run in the AssemblyLine working directory so that the correct path is generated, and the paths.csv file is saved in the proper location.
! python -m AssemblyTron.pathswriter
Since AssemblyTron has an R-script integrated, the pathswriter module will prompt you to select the folder where 'Rsript.exe' file is located with a tkinter file explorer window. This can be found in the folder where R was downloaded, and the AssemblyTron prompt offers an example of where to look. Of course this will vary between machines.
Rscript.exe is often in a hidden folder, so be sure to show hidden files and folders on the file explorer.
Select confirm to create the paths.csv file.
Once paths.csv has been created, there are five options for protocols.
The first option is a homology dependent assembly with up to 24 combined primers and templates.
! python -m AssemblyTron.Cloning.Setup_seppcr_gradient_24
The second option is a homology dependent assembly with up to 96 combined primers and templates.
! python -m AssemblyTron.Cloning.Setup_seppcr_gradient_96
The third option is a Golden Gate assembly with a destination plasmid with restriction sites. The destination plasmid is typically the backbone fragment and does not require PCR amplification.
! python -m AssemblyTron.Golden_Gate.Setup_digests_gradient
The fourth option is a Golden Gate assembly with 24 combined primers and templates.
! python -m AssemblyTron.Golden_Gate.Setup_nodigests_seppcr_gradient_24
The fifth option is a Golden Gate assembly with 96 combined primers and templates.
! python -m AssemblyTron.Golden_Gate.Setup_nodigests_seppcr_gradient_96
This vignette will go through the workflow of option five, which is a Golden Gate assembly with 96 possible primers and templates. After running the module, a tkinter file explorer window will pop up. This is for the user to navigate to the j5 combinatorial assembly design folder saved on the computer attached to the OT-2.
Once the folder containing the assembly design file is selected, select confirm to proceed.
AssemblyTron will run the R script for parsing the combinatorial design file at this point. No additonal actions are required.
A reagents setup text file window will automatically appear. This specifies where to place primers, templates, labware, etc. For the 96 well dilution script, it specifies the deck slot and tube rack position where the template belongs as well, since the deck has up to four slots with 24-tube racks for stock primers and templates. The absolute path, date, and time are also included at the top of this file.
We will return to this file when we are ready to insert stock primers and templates into the OT-2 deck. Close for now, and follow the instructuion when finished with the setup script.
Next, another window will appear for specifying parameters of the assembly. All of the volume and concentration reaction parameters are autofilled with default values, however the user can change these as necessary. This window also displays the DNA templates that will be used for the assembly, allowing the user to retrieve stocks of these and input their concentrations in ng/µL, at this time. Finally, there are two slots for extra parameters in case the user wants to modify the source code, this can be done without having to modify this user interface. These extra parameters are 0 by default.
Once the parameter are set, click confirm.
Next, a new window will appear where the user can choose which steps of the assembly to perform. Often, it will be necessary to skip to later steps of the workflow or only perform the first steps. So this tool gives the user the ability to only run the parts of the script that they need, again without having to modify source code. Once the desired protocol steps are selected, click confirm.
Next, a reaction setup text file will pop up to specify the tube configuration that needs to be used on the thermocycler. This is similar to the reagent_setup text file. Both are saved in the Golden Gate folder with the current date.
Close this for now, and re-open after completing these setup steps. The protocol is now complete and ready to be loaded onto the OT-2.
Run the file transfer script on bash to move CSV files onto the Opentrons OT-2's internal computer for the run. The paths within this script will need to be modified to match the AssemblyLine working directory (found using pip show DNA-AssemblyLine, assuming you have installed AssemblyTron using pip). This script is called Copy_Cloning.bat and is in the src directory of AssemblyTron.
It is necessary to coordinate with your computer systems administrator and Opentrons tech support to set a a wired static ip address for your OT-2. Opentrons will provide a protocol script for setting this up, and keys will need to be set up for automated ssh access to the robot.
Do not set the IP address as either your computer or OT-2, and do not change the subnet mask. The static IP address needs to be reachable from the computer with the Opentrons App (aka selected from the proper subnet). The network needs to be specially set up to account for the OT-2 having the static IP address. Otherwise, the OT-2 could conflict with another device on the network.
There is a python script for setting up the wired static IP address in the ~/src/AssemblyTron/ directory called static_wired_IP_address.py (this script was obtained from Opentrons).
Power on the OT-2 and start the Run app. Select the protocol tab on the left side of the window. Click on choose file. Navigate to the AssemblyTron working directory (found using pip show AssemblyTron), then navigate to the Golden_Gate/date+time_GoldenGate directory you just generated.
First, run 1_Update_Input.py to ensure the robot calls the correct Input.csv file. Failure to complete this step could result in errors and failure. This file is run on the command line witht the command:
! python -m AssemblyTron.Golden_Gate.{insert dated folder}.1_Update_Input.py
or if your current working directory is the new directory you just generated:
!python -m 1_Update_Input.py
Next, run the protocol 2_dilution_96.py. Select this file and drag to protocol upload are on the OT-2 run app, or open directly through the run app.
The opentrons app will then read this protocol script and show the deck setup for the initial primer and template dilution step.
Now open the reagent_setup.txt file and follow the slot and tube position designations to place primer and template stock tubes in the correct locations.
Review the protocol steps and run the dilution script.
At the conclusion of this run, return to the protocol tab and close the dilution script. Restart the OT-2 to avoid system freezing or crashing. Select CHOOSE FILE... again and open the 2_date+time_GoldenGate.py script. Follow the layout shown on the OT-2 for rearranging the OT-2 deck, set up reagents with reagent_setup.txt, set up PCR tubes with reaction_setup.txt, and review the protocol steps, then select START RUN.
Follow prompts throughout the protocol run. For example, after PCRs are mixed, the protocol pauses and the user is prompted to move PCR strips to a gradient thermocycler. Optimized annealing and extension parameter are provided to set up the gradient thermocycler.
The user will also be prompted to refill tipracks, remove enzymes, etc.
At the conclusion of the run, retrieve assemblies for transformation. To power down, return all reagents to the freezer, power off the modules and OT-2, and clean the OT-2 deck.