Week 3 HW: Lab Automation

Post-Lab Qeustions

1. Paper Review: Automation for Novel Biology

Usai et al. (2023) — “Design and biofabrication of bacterial living materials with robust and multiplexed biosensing capabilities” — Materials Today Bio (open access)

This paper uses a CELLINK INKREDIBLE+ 3D bioprinter, a pneumatic-based, dual-head, computer-controlled extrusion system, to fabricate engineered living materials (ELMs) with spatially arranged bacterial biosensors. The automation principle is similar to the Opentrons where CAD-designed structures are translated into G-code instructions that control precise deposition of biological materials at defined spatial coordinates.

The authors embedded engineered E. coli strains into an alginate/gelatin hydrogel bioink, then used the bioprinter’s dual printheads to deposit different strain-contained bioinks into adjacent separate compartments, creating material structure and biological pattern. Each strain contains a synthetic gene circuit that produces a visible color change (red fluorescent protein) in response to a specific chemical input including quorum sensing molecules (VAI/PAI), IPTG, and tetracycline.

The novel application is the multi-strain spatial logic that automation enables. They built several configurations that would be impossible to fabricate by hand at this precision:

  • A level-bar readout — four compartments containing the same VAI sensor but with different promoter strengths (from the Anderson promoter library), so each bar activates at a different chemical concentration threshold which can be used to estimate the analyte concentration range.
  • A shuriken-shaped AND gate — 4 compartments each have its own sensing system; one compartment is a boolean logic gate only turns red when both IPTG and VAI are present.
  • A cell-cell communication device — two strain types printed in adjacent compartments: a sender strain produces VAI when triggered, and the signal molecule diffuses through the hydrogel to activate a receiver strain (turns red) in an adjacent compartment, creating emergent spatial patterns based on geometry.

The materials were validated in real-world conditions including non-sterile soil, tap water, and clinical bronchial aspirate samples from P. aeruginosa-infected patients, demonstrating that automation-fabricated biosensing materials can function outside the lab. Cell viability persisted over 15 days with periodic subculturing, and the materials remained functional after one month of refrigerated storage.

This paper is directly related to my interest in wearable biosensor arrays using cell-free systems rather than living cells, but the core concept of spatially arranged biosensors that produce colorimetric outputs with built-in signal processing is shared. The automation-enabled precision of multi-compartment spatial arrangement from this paper is inspiring to me.

2. Automation Plan for Final Project

My idea for the final project is to use freeze-dried cell-free (FDCF) reaction spots deposited in a designed spatial array with layered computation capability. When rehydrated, sender spots produce a diffusible signal molecule that travels and activates receiver spots at neighboring positions, producing color. Inspired by the level-bar readout in the paper, receivers contain different concentrations of toehold switch sensor, giving it a different activation threshold. The emergent pattern is non-uniform gradient arising from spatial diffusion distance and engineered sensitivity thresholds.

The emergent pattern depends on three variables: sender/receiver distance, sender concentration, and receiver sensitivity. I intend to use automation in 2 possible ways:

1. Sender/receiver pair screening via Ginkgo Nebula (cloud lab)

Before spatial exploration, I need to identify which cell-free formulations work (individual validation) and which sender/receiver pairs successfully communicate (pair activation). This is suited for cloud lab automation:

  1. Echo transfer receiver toehold switch variants + trigger molecules (single validation), and sender reaction outputs + receiver variants (pair screening), into wells
  2. Bravo stamp in cell-free TX-TL master mix
  3. Multiflo dispense cell-free lysate to start expression
  4. PlateLoc seal the plate
  5. Inheco incubate at 30–37°C for 1–4 hours
  6. XPeel remove seal
  7. PHERAstar read colorimetric/fluorescent output

This screens: (a) which receivers produce color when triggered directly, and (b) whether the sender reaction produces enough signal to activate those validated receivers at realistic concentrations.

2. Diffusion-gradient screening via Opentrons OT-2

With validated sender/receiver pairs from Stage 1, I would use the Opentrons to deposit sender and receiver spots to test spatial configurations on my final material (possibly textiles) to find which produce the most compelling emergent patterns. I would need to design a 3D-printed holder for my material to mount on the OT-2 deck.

I used Claude to help understand, develop, and refine this automation plan.