Measurements
Introduction
In our iGEM 2025 project, precise measurements were essential for characterizing the arsenic biosensor's performance, ensuring reproducibility, and aligning with iGEM standards as outlined in the 2025 Judge Handbook. The system integrates the ArsR-regulated Pars promoter variants with the Broccoli RNA aptamer for fluorescence detection in a cell-free format, targeting arsenic in rice extracts. We prioritized quantitative assessments over qualitative ones, employing standardized protocols for plasmid quantification, cell-free expression, and fluorescence assays. These measurements, informed by literature such as Filonov et al. (2014) on Broccoli aptamer characterization and Senda et al. (2022) on T7 promoter tuning, evaluated sensitivity, specificity, and practical utility. Key innovations included designing non-coding filler sequences with balanced base composition (approximately 50% GC content) to minimize secondary structures, precise volume handling for small-scale reactions, and specialized microplates for 12.5 µL fluorescence readings.
Methods
Plasmid Design and Construction
Constructs were engineered in Benchling, featuring ArsR repressor, optimized Pars promoters (e.g., ParsOC2), and Broccoli reporter (WIST_REPORT_OC2). Filler sequences were inserted post-promoter to maintain structural integrity, with GC content adjusted to ~50% to avoid folding issues. Sense variants like WIST_SENSE_MedArsR_StrArsC_001_A were synthesized by Twist Bioscience and cloned into pTwist Amp Medium Copy vectors. Integrity was verified via restriction digestion (e.g., EcoRI, XbaI) and sequencing, per Stage 2 protocols.
Plasmid Amplification and Quantification
E. coli DH5α hosted plasmid amplification. Concentrations were determined using NanoDrop spectrophotometry, yielding 49.47–1205 ng/µL across variants. Calculations ensured optimal input for cell-free reactions: plasmid mass (ng) = concentration (ng/µL) × volume (µL), targeting 5 ng/µL final. Purity was confirmed by A260/A280 ratios (1.8–2.0). Precise pipetting (e.g., 1–10 µL tips) minimized errors in small volumes.
Cell-Free Expression System
The NEBExpress system facilitated expression in 12.5 µL reactions, scaled via percentage-based calculations (e.g., lysate: 24%, buffer: 50%). Components included T7 RNAP (2%), RNase inhibitor (2%), and DFHBI-1T fluorophore (40 µM). Reactions incubated at 37°C with durations adjusted iteratively (initially 2 hours, later refined to 45 minutes for plasmid 1 and 30 minutes for plasmid 2, and finally pivoted to 90 minutes).
Arsenic Stock Preparation and Dilution Series
Stocks of As(III) were diluted to 0–1000 ppb, with calculations for serial dilutions (e.g., C1V1 = C2V2). Safety protocols involved fume hoods and disposal guidelines.
Fluorescence Measurements
Kinetic readings used BioTek Synergy H1 (excitation: 469 nm, emission: 501 nm) in black-bottom 96-well plates suited for 12.5 µL volumes. Data spanned 0–90 minutes (n=3), with background subtraction. Methods drew from Filonov et al. (2014) for molar brightness and folding efficiency.
Interferent and Rice Extract Testing
Specificity was tested with interferents Copper, Lead and Iron. Rice extracts included a wild type and jasmine rice, both popular Thai varieties, homogenized and tested without spiking to assess natural arsenic levels, followed fluorescence protocols.
Data Analysis
Excel processed data for baseline correction, normalization, and statistical evaluations (e.g., t-tests, p<0.05). Iterative analysis of fluorometer outputs guided protocol refinements, embodying the Design-Build-Test-Learn (DBTL) cycle. For instance, early data from September 20 experiments indicated inconsistent signal development at lower arsenic concentrations,leading us to lower incubation times to 45 minutes for plasmid 1 and 30 minutes for plasmid 2 in subsequent runs to optimize signal capture. Similarly, observations of imbalanced reporter activation prompted adjustments in plasmid ratios, shifting from a 1:1 sense-to-reporter concentration to a 1:10 ratio for improved regulatory dynamics. Later datasets from September 25 revealed persistent baseline noise, which informed optimizations in DFHBI-1T concentrations (standardized to 40 µM) and enhanced background controls. By October 1-2, these iterative changes, including a pivot to full 90-minute incubations in the fluorometer, resulted in more consistent kinetic profiles, enabling progression to interferent and rice extract validations. This data-driven approach ensured progressive enhancements in assay reliability.
Sensitivity, Interferent and Rice Extract Fluorescent Raw Data October 2nd, 2025
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