Part 1: Protocol Script

Part 1: Opentrons Protocol — T-Rex QR Code

Python protocol for the Opentrons OT-2 that deposits fluorescent bacteria to reproduce the T-Rex QR code on a black agar plate. Black pixels in the 33×33 grid are mapped to plate coordinates; one color (Green) is used for the monochrome design.

Protocol Code Block 1 — Main Protocol

Copy this into the first code block of the HTGAA26 Opentrons Colab notebook (after the prerequisite setup):

from opentrons import types

metadata = {
    'author': 'James Utley',
    'protocolName': 'T-Rex QR Code Bio-Art',
    'description': 'Deposits fluorescent E. coli to create a scannable QR code with T-Rex on black agar',
    'source': 'HTGAA 2026 Opentrons Lab',
    'apiLevel': '2.20'
}

##############################################################################
###   Robot deck setup constants - don't change these
##############################################################################

TIP_RACK_DECK_SLOT = 9
COLORS_DECK_SLOT = 6
AGAR_DECK_SLOT = 5
PIPETTE_STARTING_TIP_WELL = 'A1'

well_colors = {
    'A1': 'Red',
    'B1': 'Green',
    'C1': 'Orange'
}

# 33x33 QR code pixel grid (1 = black/deposit, 0 = white/skip)
# Extracted from T-Rex QR code image
QR_PIXELS = [
    [0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0],
    [0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0],
    [0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0],
    [0, 0, 0, 1, 1, 1, 1, 1, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 1, 1, 1, 1, 1, 0, 0, 0],
    [0, 0, 0, 1, 0, 1, 0, 1, 0, 0, 0, 1, 0, 1, 0, 0, 0, 0, 1, 1, 0, 1, 0, 0, 0, 1, 0, 1, 0, 1, 0, 0, 0],
    [0, 0, 0, 1, 1, 1, 1, 1, 0, 0, 1, 1, 0, 0, 1, 1, 0, 0, 1, 1, 0, 1, 0, 0, 0, 1, 1, 1, 1, 1, 0, 0, 0],
    [0, 0, 0, 1, 0, 1, 0, 1, 0, 0, 0, 0, 0, 1, 0, 0, 0, 0, 0, 1, 0, 0, 0, 0, 0, 1, 0, 1, 0, 1, 0, 0, 0],
    [0, 0, 0, 1, 1, 1, 1, 1, 0, 0, 1, 0, 0, 1, 0, 0, 1, 0, 0, 1, 0, 0, 1, 0, 0, 1, 1, 1, 1, 1, 0, 0, 0],
    [0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 1, 0, 0, 1, 1, 1, 1, 1, 0, 1, 0, 0, 1, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0],
    [0, 0, 0, 0, 0, 1, 0, 0, 1, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 1, 0, 0, 0, 0, 1, 0, 0, 0, 0, 0, 0, 0, 0],
    [0, 0, 0, 0, 0, 0, 0, 1, 0, 0, 1, 0, 0, 1, 1, 0, 1, 0, 1, 0, 0, 1, 0, 0, 1, 1, 0, 1, 0, 1, 0, 0, 0],
    [0, 0, 0, 1, 1, 0, 1, 0, 0, 0, 0, 0, 0, 0, 0, 1, 0, 1, 0, 1, 0, 0, 1, 0, 1, 1, 1, 1, 0, 1, 0, 0, 0],
    [0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0],
    [0, 0, 0, 0, 1, 1, 1, 1, 1, 0, 0, 0, 0, 0, 0, 0, 0, 1, 1, 0, 0, 1, 0, 0, 0, 1, 1, 1, 1, 1, 0, 0, 0],
    [0, 0, 0, 0, 1, 1, 1, 0, 1, 0, 1, 0, 0, 0, 0, 0, 0, 1, 1, 0, 0, 0, 0, 0, 1, 1, 0, 1, 1, 1, 0, 0, 0],
    [0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 1, 1, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0],
    [0, 0, 0, 0, 1, 1, 1, 1, 0, 0, 1, 1, 0, 0, 0, 1, 1, 1, 0, 0, 0, 1, 1, 0, 1, 1, 1, 0, 1, 1, 0, 0, 0],
    [0, 0, 0, 0, 1, 0, 1, 0, 0, 0, 0, 1, 0, 0, 1, 1, 1, 1, 0, 0, 0, 1, 0, 0, 1, 1, 1, 0, 0, 1, 0, 0, 0],
    [0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 1, 1, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0],
    [0, 0, 0, 1, 0, 0, 0, 1, 0, 0, 1, 1, 0, 0, 0, 0, 0, 0, 0, 0, 0, 1, 0, 0, 0, 0, 0, 1, 0, 0, 0, 0, 0],
    [0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 1, 1, 0, 0, 0, 0, 0, 0, 0, 0, 0, 1, 0, 0, 1, 0, 0, 1, 0, 0, 0, 0, 0],
    [0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0],
    [0, 0, 0, 1, 0, 1, 0, 1, 1, 0, 1, 0, 0, 1, 1, 0, 0, 0, 0, 0, 0, 1, 0, 0, 1, 1, 1, 1, 0, 1, 0, 0, 0],
    [0, 0, 0, 1, 0, 1, 0, 0, 0, 0, 0, 1, 0, 1, 0, 0, 0, 0, 0, 1, 0, 0, 0, 0, 1, 0, 0, 1, 0, 0, 0, 0, 0],
    [0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0],
    [0, 0, 0, 1, 1, 1, 1, 1, 0, 0, 0, 1, 0, 1, 1, 1, 1, 1, 0, 0, 0, 1, 1, 0, 0, 1, 1, 0, 0, 1, 0, 0, 0],
    [0, 0, 0, 1, 0, 0, 0, 1, 0, 0, 0, 1, 0, 1, 0, 0, 0, 0, 1, 0, 0, 1, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0],
    [0, 0, 0, 1, 1, 1, 1, 1, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0],
    [0, 0, 0, 1, 1, 1, 1, 1, 0, 0, 0, 1, 0, 0, 0, 1, 0, 0, 1, 0, 0, 1, 0, 0, 1, 0, 1, 0, 0, 1, 0, 0, 0],
    [0, 0, 0, 1, 0, 0, 0, 1, 0, 0, 1, 1, 0, 1, 1, 0, 1, 0, 0, 0, 0, 0, 0, 0, 1, 1, 0, 0, 0, 1, 0, 0, 0],
    [0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0],
    [0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0],
    [0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0],
]


def run(protocol):
  ##############################################################################
  ###   Load labware, modules and pipettes
  ##############################################################################

  tips_20ul = protocol.load_labware('opentrons_96_tiprack_20ul', TIP_RACK_DECK_SLOT, 'Opentrons 20uL Tips')
  pipette_20ul = protocol.load_instrument("p20_single_gen2", "right", [tips_20ul])
  temperature_module = protocol.load_module('temperature module gen2', COLORS_DECK_SLOT)
  temperature_plate = temperature_module.load_labware('opentrons_96_aluminumblock_generic_pcr_strip_200ul', 'Cold Plate')
  color_plate = temperature_plate
  agar_plate = protocol.load_labware('htgaa_agar_plate', AGAR_DECK_SLOT, 'Agar Plate')
  center_location = agar_plate['A1'].top()
  pipette_20ul.starting_tip = tips_20ul.well(PIPETTE_STARTING_TIP_WELL)

  ##############################################################################
  ###   Helper functions
  ##############################################################################

  def location_of_color(color_string):
    for well, color in well_colors.items():
      if color.lower() == color_string.lower():
        return color_plate[well]
    raise ValueError(f"No well found with color {color_string}")

  def dispense_and_detach(pipette, volume, location):
    above_location = location.move(types.Point(z=location.point.z + 5))
    pipette.move_to(above_location)
    pipette.dispense(volume, location)
    pipette.move_to(above_location)

  ##############################################################################
  ###   T-Rex QR Code patterning
  ##############################################################################

  PIXEL_SIZE_MM = 2.0   # spacing between pixels (mm)
  VOLUME_UL = 0.5       # 0.5 uL per drop (~117 uL total, fits in 200uL PCR strip well)
  COLOR = 'Green'

  # Collect (row, col) for all black pixels
  black_pixels = []
  for row in range(len(QR_PIXELS)):
    for col in range(len(QR_PIXELS[row])):
      if QR_PIXELS[row][col] == 1:
        black_pixels.append((row, col))

  # One tip, one color - aspirate in batches of 20uL (max pipette volume)
  pipette_20ul.pick_up_tip()
  source = location_of_color(COLOR)

  for i in range(0, len(black_pixels), 20):
    batch = black_pixels[i:i+20]
    vol = min(len(batch) * VOLUME_UL, 20)
    pipette_20ul.aspirate(vol, source)

    for row, col in batch:
      # Map pixel (row,col) to plate coords: center at (16,16), 2mm per pixel
      x_mm = (col - 16) * PIXEL_SIZE_MM
      y_mm = (row - 16) * PIXEL_SIZE_MM
      loc = center_location.move(types.Point(x=x_mm, y=y_mm))
      dispense_and_detach(pipette_20ul, VOLUME_UL, loc)

  pipette_20ul.drop_tip()

Protocol Code Block 2 — Simulation / Visualization

Copy this into the second code block (runs after the first):

# Execute Simulation / Visualization -- don't change this code block
protocol = OpentronsMock(well_colors)
run(protocol)
protocol.visualize()

Notes

233 black pixels — uses ~117 µL total (0.5 µL per drop); aspirated in batches of 20 µL; fits in 200 µL PCR strip well.
Single color (Green) — monochrome design; change COLOR to 'Red' or 'Orange' if desired
2 mm pixel spacing — fits within ~64 mm on a 90 mm plate
One tip per color — avoids cross-contamination; only one color used here

Standalone Protocol File

For Opentrons App validation or direct upload: trex_qr_protocol.py

Google Colab Notebook

HTGAA26 Opentrons Colab — make a copy and paste the protocol code blocks above into the notebook.