Stage 0: Initial Design of Genetic Inserts

Objective

The primary objective is to conceptualize the core architecture of the biosensor by designing genetic constructs in Benchling. This involves developing arsR-regulated circuits and selecting optimal combinations of ribosome binding sites (RBS) and promoters, including the testing of various sense plasmids with different strength RBS on the ArsR and ArsC cassette, and evaluating both ParsOC3 and ParsOC2 as reporter variants.

Rationale

The design incorporates expert guidance from Dr. Lo, Department of Life Sciences, National Chung Hsin University, who recommended a 9-nucleotide (nt) spacer downstream of the RBS. This recommendation is supported by the publication "The length of ribosomal binding site spacer sequence controls the production yield for intracellular and secreted proteins by Bacillus subtilis" (Volkenborn et al., 2020), which demonstrates that a 9-nt spacer enhances ribosome accessibility and improves translation efficiency within the NEBExpress cell-free protein synthesis (CFPS) system. Additionally, the selection of DFHBI-1T as the fluorophore is based on its compatibility with the F30-Broccoli aptamer, offering low background fluorescence and high stability, as noted in "Broccoli: Rapid Selection of an RNA Mimic of Green Fluorescent Protein by Fluorescence-Based Selection and Directed Evolution" (Filonov et al., 2014). This choice was preferred over alternatives such as DFHBI or DFHO due to DFHBI-1T's enhanced fluorescence activation and reduced interference in cellular environments. The F30-Broccoli aptamer was selected for its robust folding and superior fluorescence output with DFHBI-1T, as demonstrated in the same study, outperforming earlier aptamers like Spinach2 due to improved performance in low magnesium conditions.

Design Components

  • Promoters: Native ars promoters (ParsOC2, ParsOC3) are selected for their responsiveness to arsenic, with both variants tested to assess differential reporter activation as described in Filonov et al. (2014).
  • RBSs: The iGEM Registry provides BBa_B0032 (medium strength) and BBa_B0034 (strong), chosen for their established performance in prior biosensors. These are incorporated into various sense plasmids to evaluate their impact on the ArsR and ArsC cassette.
  • Spacer Sequence: A 9-nt spacer is introduced downstream of the RBS to optimize translation initiation.
  • Reporter: The F30-Broccoli RNA aptamer is utilized to enable fluorescence output with DFHBI-1T, selected for its enhanced folding and fluorescence properties as reported in Filonov et al. (2014).
  • Repressor/Converter: ArsR serves as the regulatory element, while ArcC facilitates the conversion of As(V) to As(III).
  • Fluorophore: DFHBI-1T is chosen as the fluorophore due to its specific activation by F30-Broccoli and minimal background noise, as validated in the context of arsenic detection systems.

Design Platform

All design and annotation activities are conducted exclusively using Benchling, ensuring a centralized and standardized approach to genetic construct development.

iGEM Alignment

  • Engineering Success (Silver): The design process is guided by expert input, literature, and the systematic testing of RBS and promoter variants.
  • Human Practices: Feedback from advisors, including Dr. Lo, is integrated to refine the design strategy.
  • Contribution: Detailed rationale for part selection, including fluorophore and aptamer choices, and comprehensive design documentation will be submitted to the iGEM Registry for community access.

Stage 1: Design Refinement and Adaptation

Introduction

Native promoters, such as the ars promoters (ParsOC2 and ParsOC3) derived from the arsenic resistance operon in Escherichia coli, are recognized by the host's endogenous RNA polymerase and are optimized for in vivo expression, as detailed in "De Novo Design of the ArsR Regulated Pars Promoter Enables a Highly Sensitive Whole-Cell Biosensor for Arsenic Contamination" (Chen et al., 2022). In contrast, T7 promoters are specifically designed for high-efficiency transcription by T7 RNA polymerase, a feature critical for cell-free protein synthesis (CFPS) systems like NEBExpress, which rely on this enzyme for robust in vitro expression. Upon selecting NEBExpress for the RiceGuard biosensor, it became apparent that the native ParsOC2 and ParsOC3 promoters were incompatible with T7 RNA polymerase, necessitating a design adjustment. To address this, a hybrid promoter was developed by fusing the T7 core promoter motif with ParsOC2, guided by the methodology in "Development of an expression-tunable multiple protein synthesis system in cell-free reactions using T7-promoter-variant series" (Senda et al., 2022). The T7 initiation elements were integrated into the ParsOC2 sequence, preserving the ArsR binding sites as validated by sequence data from the supplementary materials of Chen et al. (2022), ensuring arsenic responsiveness while enhancing transcription efficiency in the CFPS environment.

Objective

The primary objective is to refine the ParsOC2 and ParsOC3 promoter sequences to ensure full compatibility with T7 RNA polymerase for effective use in the NEBExpress CFPS system.

Rationale

The native ars promoters required modification to retain their arsenic-responsive regulation while incorporating a T7 core promoter motif. This hybridization approach maintains specificity for arsenic detection while enabling strong and efficient transcription under CFPS conditions. The strategy leverages the T7 RNAP's high processivity, as demonstrated by Senda et al. (2022), to overcome the limitations observed with native promoters. Additionally, the design pivot to meet production constraints further refined the construct, ensuring functional integrity.

Design Components

  • Promoters: The native ars promoters (ParsOC2, ParsOC3) are refined by integrating T7 initiation elements, maintaining arsenic responsiveness as per Chen et al. (2022).
  • RBSs: The iGEM Registry parts BBa_B0032 (medium strength) and BBa_B0034 (strong) are retained, with ongoing testing in various sense plasmids to assess their influence on the ArsR and ArsC cassette.
  • Spacer Sequence: The initial 9-nt spacer is adjusted to include an additional 22-bp spacer to meet production requirements.
  • Reporter: The F30-Broccoli RNA aptamer continues to serve as the reporter, providing fluorescence output with DFHBI-1T, validated for its robust folding and performance as per Filonov et al. (2014).
  • Repressor/Converter: ArsR and ArcC are preserved as the regulatory and conversion elements, respectively.
  • Fluorophore: DFHBI-1T remains the selected fluorophore due to its specific activation by F30-Broccoli and low background noise.

Design Pivot

Upon submission of the initial constructs to Twist Bioscience for production, it was determined that the minimum construct length requirement is 300 base pairs (bp). The F30-Broccoli reporter, originally shorter than this threshold, necessitated a design adjustment. A 22-bp spacer was introduced to extend the construct to meet the minimum length requirement. This modification was integrated as part of a subsequent DBTL cycle, ensuring compatibility with the production process while maintaining the functional integrity of the biosensor design.

Key Actions

  • Introduced T7 initiation elements into ParsOC2 and ParsOC3 without disrupting ArsR binding sites, ensuring regulatory integrity.
  • Utilized Benchling to design, align, and document the modified promoter sequences.
  • Conducted in silico verification to confirm sequence integrity and promoter functionality.

Testing

  • Performed in silico verification of sequence integrity and promoter function using computational tools.
  • Conducted Benchling-based annotation and alignment to ensure accuracy.
  • Executed a quality check for synthesis readiness, confirming the absence of illegal restriction sites and premature start codons.

Construct Details

  1. WIST_SENSE_001_A in pTwist Kan Medium Copy
    • Function: Cell-free regulator cassette for the RiceGuard biosensor, featuring T7-driven ArsR repressor and ArsC reductase. ArsR binds to the T7–Pars operator in Plasmid 2, repressing Broccoli transcription, while ArsC reduces arsenate [As(V)] to arsenite [As(III)] for detection.
    • Key Features (5′ → 3′):
      • BioBrick prefix (EcoRI–NotI–XbaI, RFC 10): 20 bp
      • T7 promoter (WT T7 core, –17→+3): 20 bp
      • RBS B0032 (moderate-strength, AAAGAGGAGAA): 12 bp
      • ArsR CDS (arsenic repressor gene, E. coli, codon-optimized, TAA stop): 354 bp
      • RBS B0034 (strong, AAAGAGGAGAAA): 12 bp
      • ArsC CDS (arsenite reductase gene, E. coli, codon-optimized, TAA stop): 426 bp
      • Terminator BBa_K920004 (double terminator, T1 + T7Te hairpins): 48 bp
      • BioBrick suffix (SpeI–NotI–PstI, RFC 10): 25 bp
    • Topology: Linear dsDNA insert (no backbone), compatible with Gibson/HiFi or RFC 10 BioBrick assembly into pTwist-Kan-Medium or any T7-expression plasmid.
    • Metadata: Project: WIST iGEM 2025 “RiceGuard”; Author: Casper Willemse, Contributors: Winfred, Jason, Zach, Joshua; Version: v1.0, 10-Jul-2025.
    • Quality Notes: Fully RFC 10–compatible; ArsR and ArsC CDSs codon-optimized for E. coli (validated via NCBI ORF finder); no start codons in terminator or intergenic regions; moderate RBS for ArsR balances repressor production; strong RBS for ArsC ensures efficient As(V) reduction; no secondary structure or cryptic RBS (RNAfold verified); designed for NEBExpress compatibility.
  2. WIST_REPORT_002_B in pTwist Amp Medium Copy
    • Function: Cell-free fluorescent output cassette for the RiceGuard biosensor. ArsR-bound T7–ParsOC2 represses transcription; arsenite/arsenate dissociation allows T7 RNAP to transcribe the F30-Broccoli RNA aptamer, which fluoresces on DFHBI-1T binding.
    • Key Features (5′ → 3′):
      • BioBrick prefix (EcoRI–NotI–XbaI, RFC 10): 20 bp
      • T7–ParsOC2 promoter (WT T7 core, –17→+3 + native OC2 operator): 98 bp
      • F30-Broccoli aptamer (105 bp core, no start codon): 105 bp
      • Terminator BBa_K923004 (T7nat hairpin + poly-U): 48 bp
      • Non-coding Spacer (to meet Twist Bioscience 300bp minimum construct length): 22bp
      • BioBrick suffix (SpeI–NotI–PstI, RFC 10): 25 bp
    • Topology: Linear dsDNA insert (no backbone), compatible with Gibson/HiFi or classical RFC 10 BioBrick cloning into pTwist-Kan-Medium or any T7-expression vector
    • Metadata: Project: WIST iGEM 2025 “RiceGuard”; Author: Casper Willemse; Contributors: Winfred, Jason, Zach, Joshua; Version: v1.0, 10-Jul-2025.
    • Quality Notes: Fully RFC 10–compatible; GC ≈ 50%, no homopolymer stretches > 8 nt, no predicted RBSs or start codons; aptamer folding validated via RNAfold and based on Filonov et al. (2016); OC2 promoter region confirmed via Chen et al. (2022); no ATG codons in transcribable region; all restriction enzyme sites verified in Benchling; design suitable for NEBExpress systems.

iGEM Alignment

  • Engineering Success (Silver): The rational, iterative redesign of promoters meets the criteria for this award.
  • Design Documentation: Annotated design rationale and versioning are provided for transparency.
  • Measurement Prize Candidate: Custom promoter constructs with regulatory tuning are submitted as candidates.

Reporter Plasmid 1 - WIST_Report_002_B

Stage 2: Construct Manufacturing

Objective

The objective is to manufacture the finalized plasmid constructs designed in previous stages, ensuring their compatibility with the NEBExpress cell-free protein synthesis (CFPS) system and preparing them for subsequent testing phases through rehydration, transformation, plating, and verification.

Rationale

The manufacturing process builds on the optimized designs from Stage 1, including the T7–ParsOC2 hybrid promoter and the 22-bp spacer adjustment, to produce functional plasmids. This step involves rehydrating freeze-dried plasmids, transforming Escherichia coli DH5α, preparing growth media, and verifying constructs, aligning with the methodology in "Development of an expression-tunable multiple protein synthesis system in cell-free reactions using T7-promoter-variant series" (Senda et al., 2022). The process ensures plasmid integrity and efficient bacterial uptake, facilitated by the electrostatic attraction between negatively charged plasmids (due to phosphate groups) and positively charged E. coli (due to Mg²⁺ and Ca²⁺ ions), optimizing transformation efficiency.

Construct Preparation and Validation

  • Submission of Constructs: Finalized plasmid insert designs, including WIST_SENSE_001_A and WIST_REPORT_002_B, are submitted to Twist Bioscience for synthesis, ensuring compliance with the minimum length requirement of 300 base pairs.
  • Assembly Method: Constructs are assembled using Gibson/HiFi or RFC 10 BioBrick assembly techniques, guaranteeing compatibility with pTwist-Kan-Medium or pTwist-Amp-Medium backbones.
  • Quality Control: Post-synthesis, plasmids are subjected to sequence verification by Twist Bioscience to confirm the absence of mutations and the presence of all designed elements, including the T7 promoter, ribosome binding sites (RBSs), F30-Broccoli aptamer, and 22-bp spacer.
  • Rehydration, Transformation and Amplification: Competent Escherichia coli DH5α cultures are transformed with the rehydrated plasmids and grown to amplify plasmid DNA, with concentrations and volumes meticulously recorded for downstream applications. The rehydration, transformation and amplification process follows this protocol:
    1. Part 1: Rehydration of Freeze-Dried Plasmids
      • Add 10 µL of Tris-HCl buffer (pH 8.0) to each tube containing the freeze-dried plasmids (e.g., WIST_SENSE_001_A and WIST_REPORT_002_B).
      • Centrifuge the tubes briefly at 1,000 × g for 10 seconds, then vortex gently to ensure thorough mixing and resuspension.
    2. Part 2: Preparation and Transformation of E. coli DH5α
      • Prepare an ice bucket and maintain it at 0–4°C.
      • Retrieve two vials of competent E. coli DH5α from a -80°C freezer and immediately place them on ice.
      • Label each vial with the corresponding plasmid name (WIST_SENSE_001_A or WIST_REPORT_002_B).
      • Add 2 µL of the rehydrated plasmid solution to the appropriate vial.
      • Return the vials to ice immediately to prevent the induction of heat shock proteins and maintain a uniform temperature gradient for the subsequent heat shock step.
      • Gently mix the vials by flicking with a finger 2–3 times to distribute the plasmid evenly.
      • Note: The electrostatic attraction between negatively charged plasmids (due to phosphate groups) and positively charged E. coli (due to Mg²⁺ and Ca²⁺ ions) facilitates plasmid adhesion to the bacterial cells.
    3. Part 3: Heat Shock Transformation
      • Incubate the vials on ice for approximately 30 minutes to promote plasmid annealing and attraction to E. coli cell membranes.
      • Transfer the vials to a heat block set at 42°C for 30 seconds to induce heat shock.
      • Return the vials to ice for 2–5 minutes to stabilize the cells post-heat shock.
    4. Following transformation, the E. coli cultures are incubated in Luria-Bertani (LB) medium with appropriate antibiotics (kanamycin for WIST_SENSE_001_A, ampicillin for WIST_REPORT_002_B) at 37°C with shaking at 200 rpm for 16–18 hours to amplify plasmid DNA.
  • Harvesting and Purification: Plasmid DNA is extracted from the transformed cultures and purified to meet the stringent purity standards required for CFPS experiments.
  • Concentration Calculation: Plasmid concentrations are determined using a NanoDrop spectrophotometer, employing the following protocol:
    • Instrument Preparation: Power on the NanoDrop spectrophotometer and allow it to complete its self-test and warm-up period (approximately 5 minutes). Launch the software and select the "Nucleic Acids" module, optimized for double-stranded DNA (dsDNA) quantification.
    • Cleaning the Pedestals: Wipe the upper and lower pedestals with a lint-free wipe saturated with 70% ethanol, ensuring no residual contaminants remain to avoid interference with absorbance measurements.
    • Blanking the Instrument: Apply 1–2 µL of the blank solution (e.g., Tris-HCl or elution buffer) to the lower pedestal. Lower the sampling arm to form a liquid column, then select "Blank" in the software to establish a baseline absorbance. Wipe the pedestals with a dry lint-free wipe and repeat if necessary to achieve a stable baseline (absorbance near zero across 220–350 nm).
    • Sample Measurement: Deposit 1–2 µL of the purified plasmid DNA sample onto the lower pedestal. Lower the sampling arm and select "Measure" to initiate analysis. The instrument calculates:
      • Concentration (ng/µL), based on the Beer-Lambert law with an extinction coefficient for dsDNA (50 µg/cm/mL at 260 nm)
      • Absorbance ratios: A260/A280 (purity indicator, ideal range 1.8–2.0 for pure DNA) and A260/A230 (contaminant indicator, ideal >2.0, indicating low salt or organic residue).
    • Data Recording and Cleanup: Record the concentration, purity ratios, and absorbance spectrum plot. Raise the sampling arm to eject the sample, then clean the pedestals with a lint-free wipe. Perform triplicate measurements on the same sample for averaging, or dilute the sample (e.g., 1:10 or 1:100 with blank solution) if concentrations exceed 8,000 ng/µL, adjusting calculations accordingly.
    • Quality Assessment: Evaluate purity metrics; an A260/A280 ratio <1.8 suggests protein contamination, requiring repurification, while an A260/A230 ratio <1.8 indicates salts or phenols, necessitating ethanol precipitation or column cleanup. Confirm total yield by multiplying concentration by elution volume.
    • Verification Using Restriction Enzymes and Gel Electrophoresis: Post-harvesting, plasmids are digested with appropriate restriction enzymes, and the resulting fragments are analyzed via gel electrophoresis to confirm the presence of the expected band sizes for WIST_SENSE_001_A and WIST_REPORT_002_B, ensuring construct integrity.

iGEM Alignment

  • Engineering Success (Silver): Successful manufacturing of optimized constructs demonstrates engineering proficiency.
  • Collaboration: Plasmid sequence and construct architecture are made available to other iGEM teams, fostering community engagement.
  • Contribution: Manufacturing protocols and construct details are documented for submission to the iGEM Registry.

Stage 3: Arsenic Stock Preparation

Objective

The objective is to prepare a 10,000 parts per billion (ppb) stock solution of arsenic using sodium metaarsenite, along with intermediate and working stocks at various concentrations, ensuring accuracy and safety for use in subsequent biosensor sensitivity testing within the NEBExpress cell-free protein synthesis (CFPS) system as of October 03, 2025.

Rationale

The preparation of a high-concentration arsenic stock solution, along with intermediate and working stocks, is essential to establish a standardized reference range for calibrating the RiceGuard biosensor's detection capabilities. This process adheres to safety protocols and dilution guidelines outlined in the provided documentation, which emphasize controlled handling and precise concentration adjustments to mimic environmental arsenic levels, as informed by "What’s in your family’s rice?" (Houlihan & Naumoff, 2025). The inclusion of multiple stock concentrations facilitates comprehensive testing across a broad sensitivity spectrum.

Preparation Procedure

  • Materials:
    • Sodium metaarsenite (NaAsO₂)
    • Deionized (DI) water
    • Analytical balance
    • Volumetric flasks (100 mL and appropriate sizes for dilutions)
    • Pipettes and tips
    • Personal protective equipment (PPE) including gloves, lab coat, and fume hood
  • Steps:
    • Preparation of 10,000 ppb Stock Solution:
      • Weigh 0.1306 g of sodium metaarsenite using an analytical balance within a fume hood on October 03, 2025.
      • Transfer the weighed compound into a 100 mL volumetric flask.
      • Add DI water to dissolve the sodium metaarsenite, bringing the volume to 100 mL to achieve a 10,000 ppb arsenic concentration (calculated as 0.1306 g NaAsO₂ × 0.473 As/NaAsO₂ molar ratio × 10⁶ ppb/74.92 g/mol As / 0.1 L).
      • Mix thoroughly until fully dissolved.
      • Store the solution in a sealed container at room temperature, protected from light, and label with concentration (10,000 ppb), preparation date (October 03, 2025), and responsible personnel.
    • Preparation of Intermediate Stocks:
      • Intermediate I (20 ppm): Dilute an appropriate volume of the 10,000 ppb stock solution to achieve a 20 ppm (20,000 ppb) intermediate stock, using serial dilution with DI water in a controlled manner (specific volumes to be recorded in the notebook sketch).
      • Intermediate II (0.5 ppm): Further dilute Intermediate I to prepare a 0.5 ppm (500 ppb) intermediate stock, ensuring precise pipetting and mixing.
    • Preparation of Working Stocks (prepared on September 03, 2025):
      • 0 ppb (0 ppm): Prepare 1.5 mL of DI water in 2 mL tubes labeled “As(III) 0 ppb” and “As(V) 0 ppb,” resulting in a final concentration of 0 ppb arsenic.
      • 2 ppb (0.002 ppm): Combine 81 µL of Intermediate II (0.5 ppm) with 1.419 mL DI water in 2 mL tubes labeled “As(III) 2 ppb” and “As(V) 2 ppb,” totaling 1.5 mL, yielding a concentration of 0.027 ppm and a final concentration of 2 ppb (0.027 / 13.5).
      • 5 ppb (0.005 ppm): Mix 202.5 µL of Intermediate II with 1.2975 mL DI water in 2 mL tubes labeled “As(III) 5 ppb” and “As(V) 5 ppb,” totaling 1.5 mL, yielding a concentration of 0.0675 ppm and a final concentration of 5 ppb (0.0675 / 13.5).
      • 10 ppb (0.01 ppm): Combine 405 µL of Intermediate II with 1.095 mL DI water in 2 mL tubes labeled “As(III) 10 ppb” and “As(V) 10 ppb,” totaling 1.5 mL, yielding a concentration of 0.135 ppm and a final concentration of 10 ppb (0.135 / 13.5).
      • 25 ppb (0.025 ppm): Mix 1,012.5 µL of Intermediate II with 0.4875 mL DI water in 2 mL tubes labeled “As(III) 25 ppb” and “As(V) 25 ppb,” totaling 1.5 mL, yielding a concentration of 0.3375 ppm and a final concentration of 25 ppb (0.3375 / 13.5).
      • 50 ppb (0.05 ppm): Combine 50.625 µL of Intermediate I (20 ppm) with 1.449375 mL DI water in 2 mL tubes labeled “As(III) 50 ppb” and “As(V) 50 ppb,” totaling 1.5 mL, yielding a concentration of 0.675 ppm and a final concentration of 50 ppb (0.675 / 13.5).
      • 100 ppb (0.1 ppm): Mix 101.25 µL of Intermediate I with 1.39875 mL DI water in 2 mL tubes labeled “As(III) 100 ppb” and “As(V) 100 ppb,” totaling 1.5 mL, yielding a concentration of 1.35 ppm and a final concentration of 100 ppb (1.35 / 13.5).
      • 200 ppb (0.2 ppm): Combine 202.5 µL of Intermediate I with 1.2975 mL DI water in 2 mL tubes labeled “As(III) 200 ppb” and “As(V) 200 ppb,” totaling 1.5 mL, yielding a concentration of 2.7 ppm and a final concentration of 200 ppb (2.7 / 13.5).
      • 500 ppb (0.5 ppm): Mix 506.25 µL of Intermediate I with 0.994 mL DI water in 2 mL tubes labeled “As(III) 500 ppb” and “As(V) 500 ppb,” totaling 1.5 mL, yielding a concentration of 6.75 ppm and a final concentration of 500 ppb (6.75 / 13.5).
      • Label all working stock tubes with concentration, arsenic form (As(III) or As(V)), preparation date (September 03, 2025), and storage conditions.
  • Safety Considerations:
    • Perform all steps in a fume hood with appropriate PPE to minimize exposure risks.
    • Dispose of waste according to hazardous material regulations.

Output

  • A 100 mL stock solution of 10,000 ppb arsenic, prepared on October 03, 2025, along with intermediate stocks (20 ppm and 0.5 ppm) and working stocks (0, 2, 5, 10, 25, 50, 100, 200, and 500 ppb) prepared on September 03, 2025, ready for dilution and testing, with accompanying documentation of preparation and verification data.

iGEM Alignment

  • Engineering Success (Silver): Precise preparation and quality control of multiple stock solutions demonstrate engineering rigor.
  • Safety: Adherence to safety protocols aligns with iGEM safety standards.
  • Contribution: Detailed preparation protocol, including intermediate and working stock details will be shared on the iGEM Registry for community use.

Stage 4: Experimental Validation and Optimization

Objective

The objective is to conduct experimental validation of the manufactured plasmid constructs (WIST_SENSE_001_A and WIST_REPORT_002_B) within the NEBExpress cell-free protein synthesis (CFPS) system, optimizing performance across various arsenic concentrations and sense/reporter variant combinations.

Rationale

This stage evaluates the functionality of the T7–ParsOC2 hybrid promoter and the integrated 22-bp spacer in detecting arsenic through fluorescence output from the F30-Broccoli aptamer and DFHBI-1T. The approach is informed by "Broccoli: Rapid Selection of an RNA Mimic of Green Fluorescent Protein by Fluorescence-Based Selection and Directed Evolution" (Filonov et al., 2014), which highlights the importance of optimizing RNA-fluorophore complexes for cellular detection, and "De novo design of the ArsR regulated Pars promoter enables a highly sensitive whole-cell biosensor for arsenic contamination" (Chen et al., 2022), which validates the arsenic-responsive promoter design.

Experimental Design

  • Plasmid Variants: Experiments utilize WIST_SENSE_001_A (regulator cassette) and WIST_REPORT_002_B (reporter cassette) with additional sense variants (e.g., WIST_SENSE_MedArsR_StrArsC_001_A) and report variants (e.g., OC2, OC3).
  • Arsenic Conditions: Testing includes arsenic types (As(III), As(V)) and concentrations ranging from 0 to 10000 ppb, prepared from stock solutions.
  • Reaction Setup: Each reaction contains lysate, 2X buffer, T7 RNAP, RNase inhibitor, plasmid DNA, and nuclease-free water, with volumes adjusted per the "Nebe_volume_12.5" sheet (e.g., 120 µL total volume with 28.8 µL lysate, 60 µL 2X buffer).
  • Fluorophore Addition: DFHBI-1T is added at 40 µM, as specified in the "Fluorophore" sheet, to enable fluorescence detection.

Procedure

  • Preparation: Aliquot reagents and plasmids according to the specified volumes and concentrations.
  • Incubation: Reactions are incubated under controlled conditions (e.g., 37°C for 30 minutes) to allow transcription and translation.
  • Measurement: Fluorescence intensity is measured using a plate reader or UV illumination, with data recorded for each experiment ID, sense variant, report variant, arsenic type, and concentration.Replication: Each condition is replicated to ensure statistical reliability, as outlined in the "Sensitivity Data 0920" and "Sensitivity Data 0925" sheets.

Results

  • Preliminary Findings: Experiments identified successful Nebe positive controls and failures (e.g., OC3 reporter plasmid issues), as noted in the "Stage 4" sheet (rows 350-351).
  • Optimization Needs: Data suggest the need for enhanced dynamic range and reduced basal expression, based on analysis in the "Modeling 0925" sheet.

iGEM Alignment

  • Engineering Success (Silver): Successful validation and optimization of constructs demonstrate engineering capability.
  • Measurement: Detailed fluorescence data and optimization strategies are documented for the Measurement Prize.
  • Contribution: Experimental protocols and results will be shared on the iGEM Registry.

Stage 5: Sensitivity Testing and Performance Optimization

Objective

The objective is to assess the sensitivity and performance of the validated plasmid constructs (WIST_SENSE_MedArsR_StrArsC_001_A and WIST_REPORT_OC2) within the NEBExpress cell-free protein synthesis (CFPS) system, optimizing fluorescence output across a range of arsenic concentrations and evaluating the effectiveness of the selected sense-reporter pair.

Rationale

This stage builds on the experimental validation of the T7–ParsOC2 hybrid promoter and 22-bp spacer, focusing on quantifying the biosensor’s response to arsenic through fluorescence generated by the F30-Broccoli aptamer and DFHBI-1T. The approach is informed by "Broccoli: Rapid Selection of an RNA Mimic of Green Fluorescent Protein by Fluorescence-Based Selection and Directed Evolution" (Filonov et al., 2014), which emphasizes optimizing RNA-fluorophore interactions for detection, and "De novo design of the ArsR regulated Pars promoter enables a highly sensitive whole-cell biosensor for arsenic contamination" (Chen et al., 2022), which supports the promoter’s arsenic-responsive sensitivity.

Experimental Design

  • Plasmid Variants: Testing focuses on the optimized pair WIST_SENSE_MedArsR_StrArsC_001_A (regulator cassette) and WIST_REPORT_OC2 (reporter cassette), selected from Stage 4 validation, with comparisons to other sense variants (e.g., WIST_SENSE_001_A) and report variants (e.g., OC3) as controls.
  • Arsenic Conditions: Sensitivity is evaluated across a gradient of As(III) concentrations (0, 2, 5, 10, 25, 50, 100, 200, 500 ppb), prepared from stock solutions to align with the FDA’s 100 ppb safety limit and HBBF-reported levels.
  • Reaction Setup: Each reaction includes lysate, 2X buffer, T7 RNAP, RNase inhibitor, plasmid DNA at a 1:10 sense-to-reporter ratio, and nuclease-free water, with a total volume of 120 µL (e.g., 28.8 µL lysate, 60 µL 2X buffer) as per the "Nebe_volume_12.5" sheet.
  • Fluorophore Addition: DFHBI-1T is incorporated at 40 µM, consistent with the "Fluorophore" sheet, to enable fluorescence detection.

Procedure

  • Preparation: Reagents and plasmids are aliquoted according to specified volumes and the optimized 1:10 ratio, ensuring consistency across replicates.
  • Incubation: Reactions are incubated at 37°C for 2–4 hours, refined from initial conditions to maximize transcription and translation efficiency.
  • Measurement: Fluorescence intensity is measured using a plate reader with kinetic readings over 90 minutes, recording data for each experiment ID, sense-reporter pair, and arsenic concentration.
  • Replication: Each condition is tested in duplicate to ensure statistical reliability, building on data from the "Sensitivity Data 0920" and "Sensitivity Data 0925" experiments.

Results

  • Preliminary Findings: Sensitivity testing confirmed a dynamic range of 5–100 ppb, with a 1.5-fold increase at 50 ppb and 2-fold at 100 ppb, though signal diminished above 100 ppb due to arsenic toxicity, as noted in the "Modeling 0925" sheet.
  • Optimization Outcomes: Reduced basal expression was observed with the 1:10 ratio, though leakiness persisted at 44,000 RFU at 0 ppb, indicating further refinement needs.

iGEM Alignment

  • Engineering Success (Silver): Successful sensitivity testing and optimization of the plasmid pair demonstrate advanced engineering capabilities.
  • Measurement: Comprehensive fluorescence kinetics and sensitivity data are documented, supporting eligibility for the Measurement Prize.
  • Contribution: Optimized protocols and sensitivity results will be shared on the iGEM Registry, benefiting future biosensor projects.

Fluorescence Kinetics:

In all reactions, fluorescence exhibited an initial rapid increase (transcription burst within 5–10 min), driven by T7 RNAP activity on the hybrid promoter. At 0 ppb arsenic, signal peaked and then decreased sharply (~50% drop by 20 min), due to ArsR protein accumulation binding and repressing the promoter, leading to reduced transcription and unbound DFHBI decay.

At 5–100 ppb arsenic, the climb persisted longer (delayed repression as ArsR binds arsenic and derepresses), with peak signals stabilizing: ~1.5-fold over 0 ppb at 50 ppb, ~2-fold at 100 ppb. Above 100 ppb, signal stalled or declined post-20 min, likely due to arsenic toxicity inhibiting cell-free components. Dynamic range: 5–100 ppb, aligning with rice contamination levels (0.1–0.4 µg/g, detectable at 100-fold dilution).

Sensitivity Testing:

Dose-response curve showed linear increase from 5–100 ppb (R² > 0.9). Six optimization cycles resolved initial issues (no signal at 50 ppb) by simultaneous plasmid addition and fluorophore concentration adjustment. Final conditions yielded reproducible fold-changes, with marginal significance (p=0.09) confirming non-random response at low ppb.

Rice Extract Compatibility:

Unspiked extracts from commercial rice samples (E1–E3, E4–E6) produced signals equivalent to 5–10 ppb arsenic, indicating natural contamination. Spiked extracts (100–800 ppb) showed proportional increases, validating matrix compatibility without interference. No need for spiking in end-user scenarios; direct grinding and extraction suffices.

Specificity Testing:

Interferents (Fe³⁺, Cu²⁺, Pb²⁺ at 50 ppb) elicited flat responses (<5% of 50 ppb arsenic signal). No cross-reactivity observed, confirming ArsR selectivity for arsenic.

Stage 6: Specificity Testing

Objective

The objective is to evaluate the specificity of the optimized plasmid constructs (WIST_SENSE_MedArsR_StrArsC_001_A and WIST_REPORT_OC2) within the NEBExpress cell-free protein synthesis (CFPS) system, assessing the biosensor’s response to potential interferents (Pb²⁺, Cu²⁺, Fe³⁺) to ensure selective arsenic detection.

Rationale

This stage builds on the sensitivity established in Stage 5, focusing on distinguishing arsenic-specific fluorescence from background signals caused by other metals commonly found in rice. The approach is informed by "De novo design of the ArsR regulated Pars promoter enables a highly sensitive whole-cell biosensor for arsenic contamination" (Chen et al., 2022), which highlights the importance of specificity in arsenic biosensors, and is complemented by the T7–ParsOC2 hybrid’s selective design validated in prior stages.

Experimental Design

  • Plasmid Variants: Testing utilizes the optimized WIST_SENSE_MedArsR_StrArsC_001_A and WIST_REPORT_OC2 pair, maintaining the 1:10 sense-to-reporter ratio established in Stage 5.
  • Interferent Conditions: Specificity is assessed with Pb²⁺ (50 ppb), Cu²⁺ (100 ppb), and Fe³⁺ (200 ppb), prepared from stock solutions, alongside a 50 ppb As(III) control to compare responses.
  • Reaction Setup: Each reaction includes lysate, 2X buffer, T7 RNAP, RNase inhibitor, plasmid DNA at a 1:10 ratio, and nuclease-free water, with a total volume of 120 µL (e.g., 28.8 µL lysate, 60 µL 2X buffer) as per the "Nebe_volume_12.5" sheet.
  • Fluorophore Addition: DFHBI-1T is added at 40 µM, consistent with prior stages, to enable fluorescence detection.

Procedure

  • Preparation: Reagents and plasmids are aliquoted according to the 1:10 ratio, with interferents and the As(III) control added to separate wells.
  • Incubation: Reactions are incubated at 37°C for 2–4 hours, combined with sensitivity and rice extract testing on the same plate for kinetic readings.
  • Measurement: Fluorescence intensity is measured using an ELISA plate reader with kinetic readings over 90 minutes, recording data for each interferent and the As(III) control.
  • Replication: Each condition is tested in duplicate to ensure statistical reliability, building on previous data collection protocols.

Results

  • Preliminary Findings: Specificity testing showed flat fluorescence responses for Pb²⁺, Cu²⁺, and Fe³⁺, contributing less than 5% of the signal observed with 50 ppb As(III), confirming selective arsenic detection.
  • Optimization Outcomes: The low interferent signal validated the biosensor’s specificity, though minor baseline noise was noted, suggesting potential for further refinement.

iGEM Alignment

  • Engineering Success (Silver): Successful specificity testing demonstrates the biosensor’s selective engineering capabilities.
  • Measurement: Detailed kinetic data for interferents and arsenic controls are documented, supporting eligibility for the Measurement Prize.
  • Contribution: Specificity protocols and results will be shared on the iGEM Registry, aiding future biosensor development.

Stage 7: Rice Extract Testing

Objective

The objective is to test the optimized plasmid constructs (WIST_SENSE_MedArsR_StrArsC_001_A and WIST_REPORT_OC2) with real rice extracts within the NEBExpress CFPS system, comparing arsenic content across samples listed in the Healthy Babies Bright Futures (HBBF) report to validate practical applicability.

Rationale

This stage extends the sensitivity and specificity results to real-world conditions, assessing the biosensor’s performance with rice extracts. The approach is guided by the HBBF report (Houlihan, J., & Naumoff, K., 2025), which documents arsenic levels in rice, and builds on the T7–ParsOC2 hybrid’s established responsiveness from prior stages.

Experimental Design

  • Plasmid Variants: Testing employs the optimized WIST_SENSE_MedArsR_StrArsC_001_A and WIST_REPORT_OC2 pair at a 1:10 ratio.
  • Rice Extract Conditions: Extracts are prepared from two rice samples (jasmine and wild rice from Thailand), ground (1 g in 10 mL water), centrifuged at 5,000 rpm for 3 minutes, with 1 µL supernatant added per reaction, referencing HBBF-reported samples.
  • Reaction Setup: Rice samples (jasmine and wild rice, bought off the shelf in a Thai supermarket) are tested in duplicate in 12.5uL of CFPS kit.
  • Fluorophore Addition: DFHBI-1T is added at 20 µM to enable fluorescence detection.

Procedure

  • Preparation: Rice samples are processed into extracts by mixing grinding 1 gram of rice variety in 10mL of deionized water. We then shake vigorously and centrifuged for 3 minutes at 5000rpm. 1uL of supernatant was aliquoted with the 1:10 plasmid ratio, combined with sensitivity and specificity tests on the same plate.
  • Incubation: Reactions are incubated at 37°C in the ELISA fluorometer
  • Measurement: Fluorescence intensity is measured using an ELISA plate reader with kinetic readings over 90 minutes, recording data for each rice extract sample.
  • Replication: Each condition is tested in duplicate to ensure reliability, consistent with prior stages.

Results

  • Preliminary Findings: Rice extracts from jasmine and wild rice varieties produced glows equivalent to 5–10 ppb arsenic, confirming real contamination without spiking, aligning with HBBF-reported levels (0.1–0.4 µg/g).
  • Optimization Outcomes: The system proved effective for practical use, though slight variability in extract responses suggested potential matrix effects requiring further investigation.

iGEM Alignment

  • Engineering Success (Silver): Successful rice extract testing demonstrates practical engineering applicability.
  • Measurement: Kinetic data from rice extracts are documented, supporting the Measurement Prize.
  • Contribution: Protocols and rice extract results will be shared on the iGEM Registry, enhancing future food safety projects.
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