Week 3 HW: Lab Automation

Assignment: Python Script for Opentrons Artwork

This section documents my process of creating bio-art of the Markhor, Pakistan’s national animal, using the Opentrons OT-2 liquid handling robot.

1. Image Generation

I started by using Google Gemini to generate a high-quality image of a Markhor with a clean white background to serve as the reference for my design.

Markhor Original Markhor Original

2. Pixel Art Conversion

I then requested the AI to convert the high-resolution image into pixel art, which is necessary for the robot to dispense discrete “dots” of bacteria.

Markhor Pixel Art Markhor Pixel Art

3. Coordinate Generation via GUI

I uploaded the pixel art to the Opentrons Art GUI. The initial generation included many colors, so I tweaked the design to limit it to three primary fluorescent colors: Blue, Green, and Red. During this stage, I also manually added the Markhor’s ears, which were not correctly captured in the original AI image.

Opentrons GUI Preview Opentrons GUI Preview

4. Python Scripting & Debugging

Using the Echo CSV coordinates from the GUI, I utilized Google Gemini (both the web interface and the integrated assistant in the HTGAA Colab) to write the final Python script.

The process involved significant debugging due to:

  • Color Swapping: Adjusting the mapping to ensure the robot pulled the correct color from the source wells (A1, B1, C1).
  • API Syntax: Fixing NameError and KeyError related to the labware setup and coordinate indexing in the simulator.

Finally, I successfully simulated the design, ensuring the Markhor pattern was centered and clearly defined.

Google Colab Simulation Google Colab Simulation

Post-Lab Questions: Research & Automation Strategy

1. Published Research Paper Analysis

I have reviewed the following paper regarding advanced laboratory automation:

Paper Name: Automating life science labs at the single-cell level through precise ultrasonic liquid sample ejection: PULSE

Link: https://doi.org/10.1038/s41378-024-00798-y

General Overview

  • The paper introduces PULSE (Precise Ultrasonic Liquid Sample Ejection), a novel laboratory automation platform designed for biological experiments at the single-cell level.
  • While current lab automation excels at bulk samples, it often lacks the precision and biocompatibility required for single-cell work, which can mask critical cellular heterogeneity.
  • PULSE overcomes these limitations by using focused acoustic waves to eject nanoliter-sized droplets containing reagents or individual cells from a compact, disposable chip.
  • This technology transforms standard titer plates into high-density microdroplet arrays, allowing researchers to programmably print “nanodrop pixels” with specific numbers of cells.
  • The platform integrates software that synchronizes over 100 parameters, enabling automated and scalable workflows.

Major Applications

The authors demonstrate the versatility of PULSE across three major areas:

  1. Bio-fabrication: Precise deposition of different cell types to create 3D spheroids and patterned constructs.
  2. Precision Gating: Isolating single cells in nanodrops to track behavior over time (e.g., response to drugs) and retrieving them for downstream analysis.
  3. Deterministic Array Barcoding: Directly linking a single cell’s observable phenotype (via imaging) to its genotype (via sequencing) using pre-allocated, addressable primers.

Key Findings

  • High Precision: The platform can print single cells at speeds of 5-20 cells per second with an accuracy of 90.5-97.7%.
  • Biocompatibility: RNA sequencing revealed that the acoustic ejection process causes no significant differential gene regulation up to 24 hours post-treatment, outperforming FACS.
  • Accuracy: In deterministic barcoding experiments, the platform achieved a 95.6% accuracy in matching cell fluorescent phenotypes to genotypes.

2. Final Project Ideas

I am exploring three core directions for my final project, focusing on recombinant production, metabolic engineering, and industrial biocatalysis.

Idea 1: Recombinant Production of BmK CT Peptide for Glioma Therapy

  • The Concept: To engineer E. coli K-12 for the high-yield production of the 35-residue mature BmK CT scorpion peptide.
  • The Approach: Utilizing Twist Bioscience for codon-optimized gene synthesis and pET-series vectors for efficient recombinant expression.
  • Impact: Establishing a scalable bio-manufacturing pipeline for a peptide with high affinity for brain tumor cells to facilitate further oncological research.

Idea 2: CryoDesign Yak: AI-Engineered Cold-Active Enzymes

  • The Concept: Harnessing the unique metagenomic diversity of the Himalayan Yak microbiome to engineer psychrophilic enzymes for energy-efficient industrial bioprocessing.
  • The Approach: Utilizing ESM-2 Transformers for structural motif identification and ProteinMPNN for inverse folding to optimize catalytic loop flexibility.
  • Impact: Enabling room-temperature industrial catalysis (15°C–25°C) to slash heating energy demand, contributing to the EU Net Zero 2050 goal.

Idea 3: Automated Bio-manufacturing of Bryostatin-1 Precursors

  • The Concept: Engineering a microbial chassis for the sustainable production of Bryostatin-1, a potent marine-derived anti-cancer precursor.
  • The Approach: Implementing the biosynthetic gene cluster into a laboratory-friendly host and using Opentrons automation for screening optimal metabolic flux.
  • Impact: Transitioning from destructive marine harvesting to a sustainable, automated bio-manufacturing paradigm.

Lab Automation Strategies

Automation Strategy for Idea 2: CryoDesign Yak (Cold-Active Enzymes)

My automation strategy focuses on a Closed-Loop ‘Design-Build-Test’ cycle for validating AI-designed cold-adapted enzymes. Precision in thermal management is the primary constraint for this project.

  • Build (Ginkgo Nebula):
    • I will utilize the Echo Acoustic Liquid Handler to nanodisperse synthetic DNA variants into 384-well plates.
    • This will be coupled with the Bravo Automated Liquid Handling Platform to stamp in chilled cell-free protein synthesis (CFPS) reagents.
  • Test (Local Opentrons):
    • Since psychrophilic enzymes are heat-labile, I will use a 3D-printed chilled-block holder (maintained at 10°C) on the Opentrons deck.
  • Process Flow:
    • Opentrons P20 will perform high-accuracy dispensing of chromogenic substrates into the expressed enzyme variants.
    • The Inheco Thermoshaker module will be used for controlled incubation at ambient ‘cold-start’ temperatures (15°C).
    • Data will be captured via an integrated plate reader to measure the kinetic rate ($\Delta$ Abs / $\Delta$ time).

Automation Strategy for Idea 3: Bio-manufacturing of Bryostatin-1 Precursors

This strategy aims to automate the metabolic engineering and screening of Bryostatin-1 precursors in a laboratory-friendly microbial host. Since the biosynthetic gene cluster (BGC) is large and complex, automation is essential to find the optimal expression balance.

  • Design & Build (Ginkgo Nebula):
    • I will utilize the Echo Acoustic Liquid Handler to assemble combinatorial libraries of BGC components, including promoters, RBS, and biosynthetic modules.
    • The Bravo Automated Liquid Handling Platform will be used for rapid transformation and plating of the engineered chassis.
  • Test & Optimize (Local Opentrons):
    • I will use the Opentrons OT-2 to automate the metabolic screening of small-molecule precursors.
  • Process Flow:
    • Opentrons P300 will handle the precise serial dilution of feeding precursors (e.g., acetate/propionate units) into the culture media.
    • The Heater-Shaker Module will be programmed for automated induction and micro-fermentation at optimal temperatures.
    • Post-fermentation, the Opentrons will perform an automated extraction protocol (solvent addition and supernatant collection) for downstream analysis via an integrated HPLC or plate reader.