Week 3 HW: Lab Automation

Python Script for Opentrons Artwork

from opentrons import types

import string

metadata = {
    'protocolName': '{Ashraful} - Opentrons Art - HTGAA',
    'author': 'Ashraful',
    'source': 'HTGAA 2026',
    'apiLevel': '2.20'
}

Z_VALUE_AGAR = 2.0
POINT_SIZE = 0.75

sfgfp_points = [(-17.6,13.2), (-15.4,13.2), (-13.2,13.2), (-11,13.2), (-8.8,13.2), (11,13.2), (13.2,13.2), (15.4,13.2), (17.6,13.2), (-17.6,11), (-15.4,11), (-11,11), (-8.8,11), (8.8,11), (11,11), (13.2,11), (15.4,11), (17.6,11), (-15.4,8.8), (-13.2,8.8), (-8.8,8.8), (-6.6,8.8), (6.6,8.8), (8.8,8.8), (13.2,8.8), (15.4,8.8), (-13.2,6.6), (-11,6.6), (-6.6,6.6), (-4.4,6.6), (4.4,6.6), (6.6,6.6), (11,6.6), (13.2,6.6), (-11,4.4), (-8.8,4.4), (-4.4,4.4), (-2.2,4.4), (2.2,4.4), (4.4,4.4), (8.8,4.4), (11,4.4), (-8.8,2.2), (-6.6,2.2), (-2.2,2.2), (0,2.2), (2.2,2.2), (6.6,2.2), (8.8,2.2), (-6.6,0), (-4.4,0), (0,0), (4.4,0), (6.6,0), (-4.4,-2.2), (-2.2,-2.2), (0,-2.2), (2.2,-2.2), (4.4,-2.2), (-2.2,-4.4), (0,-4.4), (2.2,-4.4), (-2.2,-6.6), (0,-6.6), (2.2,-6.6), (-2.2,-8.8), (0,-8.8), (2.2,-8.8), (-2.2,-11), (0,-11), (2.2,-11), (-2.2,-13.2), (0,-13.2), (2.2,-13.2), (-11,-15.4), (-8.8,-15.4), (-6.6,-15.4), (-4.4,-15.4), (-2.2,-15.4), (0,-15.4), (2.2,-15.4), (4.4,-15.4), (6.6,-15.4), (8.8,-15.4), (11,-15.4), (13.2,-15.4), (-13.2,-17.6), (-11,-17.6), (-8.8,-17.6), (-6.6,-17.6), (-4.4,-17.6), (-2.2,-17.6), (0,-17.6), (2.2,-17.6), (4.4,-17.6), (6.6,-17.6), (8.8,-17.6), (11,-17.6), (13.2,-17.6), (15.4,-17.6), (-15.4,-19.8), (-13.2,-19.8), (-11,-19.8), (-8.8,-19.8), (-6.6,-19.8), (-4.4,-19.8), (-2.2,-19.8), (0,-19.8), (2.2,-19.8), (4.4,-19.8), (6.6,-19.8), (8.8,-19.8), (11,-19.8), (13.2,-19.8), (15.4,-19.8), (17.6,-19.8)]

point_name_pairing = [("sfgfp", sfgfp_points)]

# Robot deck setup constants
TIP_RACK_DECK_SLOT = 9
COLORS_DECK_SLOT = 6
AGAR_DECK_SLOT = 5
PIPETTE_STARTING_TIP_WELL = 'A1'

# Place the PCR tubes in this order
well_colors = {
    'A1': 'sfGFP',
    'A2': 'mRFP1',
    'A3': 'mKO2',
    'A4': 'Venus',
    'A5': 'mKate2_TF',
    'A6': 'Azurite',
    'A7': 'mCerulean3',
    'A8': 'mClover3',
    'A9': 'mJuniper',
    'A10': 'mTurquoise2',
    'A11': 'mBanana',
    'A12': 'mPlum',
    'B1': 'Electra2',
    'B2': 'mWasabi',
    'B3': 'mScarlet_I',
    'B4': 'mPapaya',
    'B5': 'eqFP578',
    'B6': 'tdTomato',
    'B7': 'DsRed',
    'B8': 'mKate2',
    'B9': 'EGFP',
    'B10': 'mRuby2',
    'B11': 'TagBFP',
    'B12': 'mChartreuse_TF',
    'C1': 'mLychee_TF',
    'C2': 'mTagBFP2',
    'C3': 'mEGFP',
    'C4': 'mNeonGreen',
    'C5': 'mAzamiGreen',
    'C6': 'mWatermelon',
    'C7': 'avGFP',
    'C8': 'mCitrine',
    'C9': 'mVenus',
    'C10': 'mCherry',
    'C11': 'mHoneydew',
    'C12': 'TagRFP',
    'D1': 'mTFP1',
    'D2': 'Ultramarine',
    'D3': 'ZsGreen1',
    'D4': 'mMiCy',
    'D5': 'mStayGold2',
    'D6': 'PA_GFP'
}

# Mapping for visualization colors
VISUALIZATION_COLOR_MAP = {
    'sfGFP': 'green',
    'mRFP1': 'red',
    'mKO2': 'orange',
    'Venus': 'yellow',
    'mKate2_TF': 'purple',
    'Azurite': 'blue',
    'mCerulean3': 'cyan',
    'mClover3': 'lightgreen',
    'mJuniper': 'darkgreen',
    'mTurquoise2': 'teal',
    'mBanana': 'gold',
    'mPlum': 'plum',
    'Electra2': 'navy',
    'mWasabi': 'lime',
    'mScarlet_I': 'darkred',
    'mPapaya': 'peachpuff',
    'eqFP578': 'brown',
    'tdTomato': 'tomato',
    'DsRed': 'indianred',
    'mKate2': 'darkmagenta',
    'EGFP': 'chartreuse',
    'mRuby2': 'firebrick',
    'TagBFP': 'slateblue',
    'mChartreuse_TF': 'darkseagreen',
    'mLychee_TF': 'palevioletred',
    'mTagBFP2': 'darkblue',
    'mEGFP': 'limegreen',
    'mNeonGreen': 'lawngreen',
    'mAzamiGreen': 'mediumseagreen',
    'mWatermelon': 'pink',
    'avGFP': 'forestgreen',
    'mCitrine': 'khaki',
    'mVenus': 'olivedrab',
    'mCherry': 'crimson',
    'mHoneydew': 'honeydew', # This will likely be too light
    'TagRFP': 'rosybrown',
    'mTFP1': 'dodgerblue',
    'Ultramarine': 'mediumblue',
    'ZsGreen1': 'springgreen',
    'mMiCy': 'peru',
    'mStayGold2': 'goldenrod',
    'PA_GFP': 'darkgreen'
}

volume_used = {
    'sfgfp': 0
}

def update_volume_remaining(current_color, quantity_to_aspirate):
    rows = string.ascii_uppercase
    for well, color in list(well_colors.items()):
        if color == current_color:
            if (volume_used[current_color] + quantity_to_aspirate) > 250:
                # Move to next well horizontally by advancing row letter, keeping column number
                row = well[0]
                col = well[1:]

                # Find next row letter
                next_row = rows[rows.index(row) + 1]
                next_well = f"{next_row}{col}"

                del well_colors[well]
                well_colors[next_well] = current_color
                volume_used[current_color] = quantity_to_aspirate
            else:
                volume_used[current_color] += quantity_to_aspirate
            break

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

    # Tips
    tips_20ul = protocol.load_labware('opentrons_96_tiprack_20ul', TIP_RACK_DECK_SLOT, 'Opentrons 20uL Tips')

    # Pipettes
    pipette_20ul = protocol.load_instrument("p20_single_gen2", "right", [tips_20ul])

    # Deep Well Plate
    temperature_plate = protocol.load_labware('nest_96_wellplate_2ml_deep', 6, 'Deep Well Plate')

    # Agar Plate
    agar_plate = protocol.load_labware('htgaa_agar_plate', AGAR_DECK_SLOT, 'Agar Plate')
    agar_plate.set_offset(x=0.00, y=0.00, z=Z_VALUE_AGAR)

    # Get the top-center of the plate, make sure the plate was calibrated before running this
    center_location = agar_plate['A1'].top()

    pipette_20ul.starting_tip = tips_20ul.well(PIPETTE_STARTING_TIP_WELL)

    # Helper function (dispensing)
    def dispense_and_jog(pipette, volume, location):
        assert(isinstance(volume, (int, float)))
        # Go above the location
        above_location = location.move(types.Point(z=location.point.z + 2))
        pipette.move_to(above_location)
        # Go downwards and dispense
        pipette.dispense(volume, location)
        # Go upwards to avoid smearing
        pipette.move_to(above_location)

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

    # Print pattern by iterating over lists
    for i, (current_color, point_list) in enumerate(point_name_pairing):
        # Skip the rest of the loop if the list is empty
        if not point_list:
            continue

        # Get the tip for this run, set the bacteria color, and the aspirate bacteria of choice
        pipette_20ul.pick_up_tip()
        max_aspirate = int(18 // POINT_SIZE) * POINT_SIZE
        quantity_to_aspirate = min(len(point_list)*POINT_SIZE, max_aspirate)
        update_volume_remaining(current_color, quantity_to_aspirate)
        pipette_20ul.aspirate(quantity_to_aspirate, location_of_color(current_color))

        # Iterate over the current points list and dispense them, refilling along the way
        for i in range(len(point_list)):
            x, y = point_list[i]
            adjusted_location = center_location.move(types.Point(x, y))

            dispense_and_jog(pipette_20ul, POINT_SIZE, adjusted_location)

            if pipette_20ul.current_volume == 0 and len(point_list[i+1:]) > 0:
                quantity_to_aspirate = min(len(point_list[i:])*POINT_SIZE, max_aspirate)
                update_volume_remaining(current_color, quantity_to_aspirate)
                pipette_20ul.aspirate(quantity_to_aspirate, location_of_color(current_color))

        # Drop tip between each color
        pipette_20ul.drop_tip()

Simulation

# Execute Simulation / Visualization
protocol = OpentronsMock(well_colors, VISUALIZATION_COLOR_MAP)
run(protocol)
protocol.visualize()

Post-Lab Questions

Find and describe a published paper that utilizes the Opentrons or an automation tool to achieve novel biological applications.

In the paper “An open-source, automated, and cost-effective platform for COVID-19 diagnosis and rapid portable genomic surveillance using nanopore sequencing” published in Scientific Reports, the researchers integrated a robotic liquid-handling system (Tecan Freedom EVO) to automate the MAVRICS RNA extraction workflow in a 96-well format. The robot performed magnetic bead–based RNA extraction, washing, and transfer steps with optimized pipetting and contamination-control measures, allowing high-throughput and reproducible processing of clinical samples. The automated extraction was then combined with in-house qRT-PCR diagnostics and the portable NIRVANA nanopore sequencing system for variant tracking. This automation significantly reduced human error and cross-contamination, increased testing capacity (up to thousands of samples per day), and enabled scalable, low-cost pandemic response—highlighting the importance of robotic tools in biosecurity, diagnostics, and rapid outbreak surveillance.

Write a description about what you intend to do with automation tools for your final project. You may include example pseudocode, Python scripts, 3D printed holders, a plan for how to use Ginkgo Nebula, and more.

Automated Workflow for Screening EcoRI Constructs in Cell-Free System

1. Echo – DNA Transfer  
Transfer codon-optimized EcoRI DNA constructs into designated wells with high precision.

2. Bravo – Master Mix Addition  
Add CFPS master mix containing buffers and cofactors uniformly to all wells.

3. Multiflo – Start Reaction  
Dispense cell-free lysate into each well to initiate protein synthesis.

4. PlateLoc – Seal Plate  
Seal the plate to prevent evaporation and contamination during incubation.

5. Inheco – Incubation  
Maintain optimal temperature (e.g., 37°C) for EcoRI protein expression.

6. XPeel – Remove Seal  
Open the plate safely to prepare for downstream reactions or measurements.

7. PHERAstar – Measure Output  
Quantify EcoRI activity on fluorescent DNA substrates to compare construct performance.