Week 3 Lab: Opentrons Art

Post-Lab Questions — Laboratory Automation & Final Project

Relevant Figures

Figure 1 — Automated Workflow Schematic

┌─────────────────────────────────────────────────────────────────────┐
│                  OT-2 Automated CFPS Screening Pipeline             │
│                                                                     │
│  DNA Library        CFPS Master Mix       Inducer Gradients         │
│  (384 constructs)   (cell extract +       (0 → 1000 µM)            │
│       │             energy system)               │                  │
│       │                  │                       │                  │
│       └──────────────────┴───────────────────────┘                  │
│                          │                                          │
│                    ┌─────▼──────┐                                   │
│                    │  OT-2 OT-2 │  ← Robotic dispensing            │
│                    │  Dispenser │     1–5 µL per well               │
│                    └─────┬──────┘                                   │
│                          │                                          │
│                    ┌─────▼──────┐                                   │
│                    │ 384-well   │  ← Reaction: 30°C, 6 h           │
│                    │   plate    │                                   │
│                    └─────┬──────┘                                   │
│                          │                                          │
│                    ┌─────▼──────┐                                   │
│                    │   Plate    │  ← GFP fluorescence               │
│                    │   Reader   │     (ex: 485nm / em: 520nm)       │
│                    └─────┬──────┘                                   │
│                          │                                          │
│                    ┌─────▼──────┐                                   │
│                    │  Python    │  ← Data analysis, dose-response   │
│                    │  Analysis  │     curve fitting, hit ranking    │
│                    └────────────┘                                   │
└─────────────────────────────────────────────────────────────────────┘

This figure represents the end-to-end automated pipeline: from DNA library preparation through robotic CFPS assembly, incubation, fluorescence readout, and computational analysis.

Figure 2 — Representative Dose-Response Heat Map (Conceptual)

        Inducer Concentration →
        0.01  0.1   1    10   100  1000  µM
       ┌─────┬─────┬─────┬─────┬─────┬─────┐
  C1   │ ░░░ │ ░░░ │ ▒▒▒ │ ▓▓▓ │ ███ │ ███ │  ← Strong biosensor
  C2   │ ░░░ │ ░░░ │ ░░░ │ ▒▒▒ │ ▓▓▓ │ ███ │  ← Medium response
  C3   │ ▒▒▒ │ ▒▒▒ │ ▒▒▒ │ ▒▒▒ │ ▒▒▒ │ ▒▒▒ │  ← Leaky (reject)
  C4   │ ░░░ │ ░░░ │ ░░░ │ ░░░ │ ░░░ │ ░░░ │  ← Non-functional
  C5   │ ░░░ │ ░░░ │ ▒▒▒ │ ███ │ ███ │ ███ │  ← Best hit: steep ON
       └─────┴─────┴─────┴─────┴─────┴─────┘
  
  Legend: ░ = Low fluorescence  ▒ = Medium  ▓ = High  █ = Maximum
  Rows = biosensor construct variants (C1–C5)
  Columns = inducer concentration

Each row represents a unique biosensor construct variant; each column a different inducer concentration. Construct C5 shows the steepest dose-response (ideal switch-like behavior) with minimal background — identified as the top hit for downstream validation.

References: Hossain et al. (2020) ACS Synth. Biol.; Pardee et al. (2014) Cell; Opentrons Protocol API v2 Documentation.