End-to-end workflow

PUDU automates three sequential lab stages. Each stage reads the output of the previous one, creating a traceable chain from DNA design to plated colonies.

SBOL JSON design
     │
     ▼
┌─────────────┐     opentrons_simulate
│  Assembly   │ ──────────────────────▶  transformation_input.json
└─────────────┘
     │
     ▼
┌──────────────────┐  opentrons_simulate
│  Transformation  │ ──────────────────▶  plating_input.json
└──────────────────┘
     │
     ▼
┌─────────┐  opentrons_simulate
│ Plating │ ──────────────────────────▶  plating_layout.json / .xlsx
└─────────┘

Stage 1 — Assembly

Golden Gate DNA assembly in a thermocycler. Parts and backbone are loaded onto a temperature module (4 °C), combined in a thermocycler plate, and cycled 75 times between 42 °C (digest) and 16 °C (ligate).

Generate and simulate the protocol:

python -m pudu.generate_protocol \
    assembly_input.json \
    -o assembly_protocol.py \
    --protocol-type assembly

opentrons_simulate assembly_protocol.py

This writes transformation_input.json mapping each assembled product URI to its thermocycler well location.

Stage 2 — Transformation

Heat-shock transformation of assembled plasmids into competent bacteria. DNA is transferred from the assembly plate (or temp module) into the thermocycler plate alongside competent cells, heat-shocked, and incubated with recovery media.

Generate and simulate:

python -m pudu.generate_protocol \
    transformation_spec.json \
    -o transformation_protocol.py \
    --protocol-type transformation \
    --plasmid-locations transformation_input.json

opentrons_simulate transformation_protocol.py

This writes plating_input.json mapping each thermocycler well to its transformed construct.

Stage 3 — Plating

Serial dilution and spot-plating onto agar. Bacteria from the thermocycler plate are diluted (up to 2×) and spotted onto agar plates with replicates.

Generate and simulate:

python -m pudu.generate_protocol \
    plating_input.json \
    -o plating_protocol.py \
    --protocol-type plating

opentrons_simulate plating_protocol.py

This writes plating_layout.json and plating_layout.xlsx — a coloured grid showing which agar well receives which construct at which dilution ratio.

Manual bench protocols

If you don’t have access to an OT-2, PUDU can generate human-readable Markdown guides for each stage:

python scripts/manual/generate_manual_assembly_protocol.py \
    --input  assembly_input.json \
    --output scripts/manual/manual_assembly_protocol.md

python scripts/manual/generate_manual_transformation_protocol.py \
    --input  transformation_spec.json \
    --output scripts/manual/manual_transformation_protocol.md

python scripts/manual/generate_manual_plating_protocol.py \
    --input  plating_input.json \
    --output scripts/manual/manual_plating_protocol.md

Input file formats

See pudu.generate_protocol for the full JSON schemas for each input type and the advanced parameters file.

Calibration

Before running fluorescence or OD600 measurements, calibrate your plate reader using the iGEM 2022 protocol. Two calibration classes are provided:

pudu.calibration.GFPODCalibration — fluorescein + microspheres (GFP + OD600)
pudu.calibration.RGBODCalibration — fluorescein + sulforhodamine + cascade blue + microspheres

Example script: scripts/automated_ot2/run_iGEM_rgb_od.py