Week-03-HW:Lab Automation

Post-Lab Question 1: Published Paper Description

Automated High-Throughput DNA Assembly using Opentrons For this assignment, I researched how the Opentrons OT-2 is utilized to automate Golden Gate Assembly for synthetic biology applications. A prime example of this is the widespread use of the OT-2 in laboratories to assemble combinatorial genetic libraries. Instead of researchers manually pipetting thousands of small-volume reactions—which is highly prone to human error and fatigue—the Opentrons robot is programmed to precisely distribute vector backbones, inserts, buffers, and enzymes into 96-well or 384-well plates.

The novel biological application here is the rapid prototyping of genetic circuits. By automating the liquid handling, researchers can test hundreds of promoter-gene-terminator combinations simultaneously. This volumetric precision (down to 1µL) is critical when working with expensive master mixes. This high-throughput approach drastically accelerates the design-build-test-learn (DBTL) cycle, allowing for the faster development and optimization of engineered therapeutic bacterial strains.

Post-Lab Question 2: Final Project Automation Plan

Project Idea: High-Throughput Screening of Therapeutic Genetically Modified Bacteria For my final project, I intend to use lab automation tools to screen and characterize therapeutic genetically modified bacteria. The goal is to test how different engineered strains produce localized therapeutic proteins (or neutralize specific disease targets) under various simulated physiological conditions.

Hardware Setup:

Opentrons Temperature Module: To simulate human body temperature (37°C) during the assay.

96-Well Culture Plates: To run multiple strain variations and condition gradients simultaneously.

Liquid Reservoirs: To hold biological inducers, simulated tissue buffers, and bacterial growth media.

Automation Workflow:

Media Distribution: The Opentrons precisely distributes specific media and buffer gradients across a 96-well plate to simulate different microenvironments (e.g., specific gut pH levels).

Inoculation: The robot automatically inoculates these wells with various strains of therapeutic genetically modified bacteria from a master library plate.

Induction: Specific chemical or biological inducers are deposited into the wells to trigger the genetic therapeutic circuits of the bacteria.

Incubation: The Temperature Module maintains the culture at 37°C for growth and therapeutic expression.

Measurement: The plate is moved to a plate reader (like a PHERAstar) to measure fluorescence or absorbance, comparing the therapeutic response of each strain.

Example Python Pseudocode for Opentrons OT-2:

Python def run(protocol): # Load tip racks and pipettes tips_20ul = protocol.load_labware(‘opentrons_96_tiprack_20ul’, ‘1’) pipette = protocol.load_instrument(‘p20_single_gen2’, ‘right’, tip_racks=[tips_20ul])

# Load modules and labware
temp_module = protocol.load_module('temperature module gen2', '4')
culture_plate = temp_module.load_labware('corning_96_wellplate_360ul_flat')

bacteria_library = protocol.load_labware('corning_24_wellplate_3.4ml_flat', '5')
inducer_reservoir = protocol.load_labware('nest_12_reservoir_15ml', '6')

# Set temperature to simulate human body conditions (37°C)
temp_module.set_temperature(37)

# Select the first 12 wells for the screening assay
wells_to_test = culture_plate.wells()[:12]

pipette.pick_up_tip()

for well in wells_to_test:
    # 1. Inoculate well with the therapeutic genetically modified bacteria
    pipette.aspirate(10, bacteria_library['A1'])
    pipette.dispense(10, well)
    
    # 2. Add specific biological inducer to trigger therapeutic response
    pipette.aspirate(2, inducer_reservoir['A1'])
    pipette.dispense(2, well)
    
    # 3. Mix the culture thoroughly
    pipette.mix(3, 10, well)
    
pipette.drop_tip()