DNA-BOT

Feb 2022 - DNA-BOT has now been updated to produce scripts that run on the Opentrons OT2 in APIv2. It also incorporates options for different hardware configurations by producing multiple scripts for each of the 4 steps. The Thermocycler module can now be optionally used in the step 1 clip reactions as well as the step 4 transformations. In addition, different labware inputs are available through the Graphical User Interface (GUI) or Command Line Interface (CLI).

This work was originally published in OUP Synthetic Biology

Getting Started

Users looking to implement the DNA-BOT workflow are encouraged to consult the instructions. If you are looking to contribute to this project, please raise an issue or pull request. Otherwise, feel free to reach out to geoffbaldwin.

dnabot can be run in 2 modes:

• with a graphical interface: see Running the dnabot app section in instructions. dnabot was developed using Python v3.7. Refer to requirements.txt.
• without a graphical interface: you need to specify all settings through the command line, you can see the instructions in the sections below.

Installation from conda

conda create --name <myenv>
conda activate <myenv>
conda install -c conda-forge -c brsynth dnabot

<myenv> has to be replaced by whatever meaningful name that will pleased the user.

Usage

Using the GUI

conda activate <myenv>
python -m dnabot.dnabot_app --help
python -m dnabot.dnabot_app

Using the CLI only

conda activate <myenv>
python -m dnabot.dnabot_app nogui --help
python -m dnabot.dnabot_app nogui \
    --construct_path /path/to/constructs.csv \
    --source_paths /path/to/linker_parts_coord.csv /path/to/user_parts_coord.csv \
    --output_dir /path/to/output/dir

Command line arguments

GUI mode

usage: dnabot_app.py [-h] [--default_settings_file DEFAULT_SETTINGS_FILE] {nogui} ...

DNA assembly using BASIC on OpenTrons.

positional arguments:
  {nogui}               Optional, switch to define settings from the terminal instead of the graphical interface. Type "python dnabot_app.py
                        nogui -h" for more info.

optional arguments:
  -h, --help            show this help message and exit
  --default_settings_file DEFAULT_SETTINGS_FILE
                        Optional, file providing labware IDs and parameter to be used. Default:
                        /Users/tduigou/code/dnabot/dnabot/default_settings.yaml.

No GUI mode

usage: dnabot_app.py nogui [-h] --construct_path CONSTRUCT_PATH --source_paths SOURCE_PATHS [SOURCE_PATHS ...]
                           [--etoh_well ETOH_WELL] [--soc_column SOC_COLUMN] [--output_dir OUTPUT_DIR]
                           [--template_dir TEMPLATE_DIR]

optional arguments:
  -h, --help            show this help message and exit
  --construct_path CONSTRUCT_PATH
                        File listing constructs to be implemented.
  --source_paths SOURCE_PATHS [SOURCE_PATHS ...]
                        File(s) listing parts to be used in constructs.
  --etoh_well ETOH_WELL
                        Coordinates of the well plate providing ethanol for the purification step. Default: A11
  --soc_column SOC_COLUMN
                        Coordinate of the column plate providing SOC media for the transformation step. Default: 1
  --output_dir OUTPUT_DIR
                        Output directory. Default: same directory than the one containing the 'construct_path' file
  --template_dir TEMPLATE_DIR
                        Template directory. Default: 'template_ot2_scripts' located next to the present script.

Change default values

Use the --default_settings_file argument to set different default values. This option is available either using the GUI or the CLI interface.

conda activate <myenv>
python -m dnabot.dnabot_app --default_settings_file /path/to/custom/default_settings.yaml.

The default settings file should follow the structure below (yaml file). The labware IDs to be used can be updated with the labwares section, while the parameters for the seperation step are listed in the parameters section.

labwares:

  # Pipettes #############################################
  # Opentrons P20 Single-Channel Electronic Pipette
  p20_single:
    id: p20_single_gen2

  # Opentrons P300 8-Channel Electronic Pipette
  p300_multi:
    id: p300_multi_gen2

  # Modules ###############################################
  # Opentrons magnetic module (step: purification)
  mag_deck:
    id: magdeck
    # id: magnetic module gen2  # BRS

  # Tip racks #############################################
  # Opentrons 20μL tips rack
  96_tiprack_20ul:
    id: opentrons_96_tiprack_20ul
    # id: tipone_3dprinted_96_tiprack_20ul  # BRS

  # Opentrons 300μL tips rack
  96_tiprack_300ul:
    id: opentrons_96_tiprack_300ul
    # id: tipone_yellow_3dprinted_96_tiprack_300ul  # BRS

  # Plates ################################################
  # Opentrons 4-in-1 tubes rack for 1.5 ml eppendorf tubes (steps: clip, assembly, transformation)
  24_tuberack_1500ul:
    id: e14151500starlab_24_tuberack_1500ul
    # id: opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap  # BRS

  # Clip reaction source plate (steps: clip)
  clip_source_plate:
    id: 4ti0960rig_96_wellplate_200ul
    # id: green_96_wellplate_200ul_pcr  # BRS (transparent)

  # Clip reaction plate (steps: clip, purif, assembly)
  clip_plate:
    id: 4ti0960rig_96_wellplate_200ul
    # id: black_96_wellplate_200ul_pcr  # BRS (black)

  # Mix plate (step: purification)
  mix_plate:
    id: 4ti0960rig_96_wellplate_200ul
    # id: black_96_wellplate_200ul_pcr  # BRS (black)
  
  # Final assembly plate (steps: assembly, transformation)
  final_assembly_plate:
    id: 4ti0960rig_96_wellplate_200ul
    # id: black_96_wellplate_200ul_pcr  # BRS (black)
  
  # Transformation plate with thermocycler (step: transformation)
  transfo_plate:
    id: 4ti0960rig_96_wellplate_200ul
    # id: black_96_wellplate_200ul_pcr  # BRS (black)

  # Transformation plate without thermocycler (step: transformation)
  transfo_plate_wo_thermo:
    id: 4ti0960rig_96_wellplate_200ul
    # id: green_96_wellplate_200ul_pcr  # BRS (transparent)

  # Agar plate (transformation step)
  agar_plate:
    id: 4ti0960rig_96_wellplate_200ul
    # id: thermoomnitrayfor96spots_96_wellplate_50ul  # BRS

  # Reservoir plate 21 mL 12 channels (step: purification)
  12_reservoir_21000ul:
    id: 4ti0131_12_reservoir_21000ul
    # id: citadel_12_wellplate_22000ul  # BRS

  # 96 deep well plate 2 mL wells (steps; purification, transormation)
  96_deepwellplate_2ml:
    id: 4ti0136_96_wellplate_2200ul
    # id: transparent_96_wellplate_2ml_deep  # BRS
  
  # Corning 12 Well Plate 6.9 mL Flat (step: transformation)
  12_corning_wellplate:
    id: corning_12_wellplate_6.9ml_flat
    # id: sarstedtcplatte_12_wellplate_6640ul  # BRS
  

parameters:

  # Clip reaction step ####################################
  # Keep the thermocycler lid closed at 4°C at the end of execution?
  # 1 for yes, 0 for no
  clip_keep_thermo_lid_closed:
    value: 0

  # Purification step #####################################
  # Magnetic module height (mm) - purification step
  purif_magdeck_height: 
    value: 20
    # value: 10.8  # BRS

  # Washing time (min) - purification step
  purif_wash_time:
    value: 0.5

  # Bead ratio - purification step
  purif_bead_ratio:
    value: 1.8

  # Incubation time (min) - purification step
  purif_incubation_time:
    value: 5

  # Settling time (min) - purification step
  purif_settling_time:
    value: 2
    # value: 6  # BRS

  # Drying time (min) - purification step
  purif_drying_time:
    value: 5
    # value: 15  # BRS

  # Elution time (min) - purif step
  purif_elution_time:
    value: 2
    # value: 5  # BRS
  
  # Transformation step ###################################
  # Incubation temperature
  transfo_incubation_temp:
    value: 4
    # value: 8  # BRS

  # Incubation time (min)
  transfo_incubation_time:
    value: 20
    # value: 30  # BRS

For developers

Development installation

After a git clone:

conda env create -f environment.yaml -n <dev_env>
conda develop -n <dev_env> .

You may be prompted to install conda-build in your base environment (conda install conda-build). The default conda environment name will be dnabot-dev if not specified by -n <dev-env>.

Test your installation with:

conda activate <dev_env>
python -m dnabot.dnabot_app nogui --help
python -m pytest tests

To uninstall:

conda deactivate
conda env remove -n <dev_env>

Tests

You need to install pytest if it's not done yet (conda install pytest).

conda install pytest
python -m pytest tests

Authors

The update of DNABOT to APIv2 and improvements to the front end involved the work of several people:

• Thomas Duigou - tduigou
• Geoff Baldwin - geoffbaldwin
• Gizem Buldum - gizembuldum

Initial work revising the template scripts to run in Opentrons APIv2 was done by a team of Masters students from the MRes in Systems and Synthetic Biology at Imperial College London, thanks to:

• Xin Luo
• Ruihan Bai
• Zhenhua Wu
• Lianne Wu
• Ting An Lee
• Xiangming Xu

The original code for DNABOT produced OT2 scripts that ran in APIv1 and was authored by:

• Matthew C Haines - hainesm6

License

This project is licensed under the MIT License - see the LICENSE file for details

Acknowledgments

• Marko Storch for all his help with instructions and DNA-BOT manuscript.
• Geoff Baldwin for all his help with the DNA-BOT manuscript.