Experimental Results

Introduction Module 1: Construction and Preliminary Optimization of Basic Sensing Circuit Module 2: Promoter Engineering and Component Innovation Module 3: Self-Assembly System and System-Level Optimization

DBTL Experimental Results from Cycle 1 to Cycle 7

Introduction

This page presents the experimental results from our DBTL (Design-Build-Test-Learn) cycles for arsenic biosensor development. The results are organized into three modules covering basic circuit construction, promoter engineering, and system-level optimization.

Module 1: Construction and Preliminary Optimization of Basic Sensing Circuit

This module focuses on the construction and initial optimization of basic arsenic-responsive circuits through three DBTL cycles, identifying core issues and eliminating interference factors.

Cycle 1: First-Round DBTL Development of Basic Plasmids V0-V1-V2

Plasmid Construction Results

Three recombinant plasmids were successfully assembled (pET46a-ArsR-sfGFP-V0, pET46a-PlacV-ArsR-ParsOC2-sfGFP-V1, pET46a-ParsOC2-ArsR-sfGFP-V2). Verification through restriction enzyme digestion, antibiotic resistance screening, colony PCR, and sequencing confirmed correct insertion of the elements (ArsR, sfGFP, and corresponding promoters) without sequence mutations or assembly errors.

Performance Testing Results

Using a multi-gradient NaAsO₂ treatment (0-300 ppb, 0-300 μM), the "fluorescence value (Fluo)/cell density (OD600)" correction ratio was measured by a microplate reader, yielding key data:

Key Findings:

V0 (No specific promoter, only ArsR-sfGFP): Primarily used to validate the "baseline level without regulation," it did not exhibit an effective arsenic response and served only as baseline control data.
V1 (PlacV-driven ArsR+ParsOC2 regulating sfGFP): At 300 ppb arsenic concentration, fluorescence intensity increased only 1.7-fold compared to the 0 ppb control. Background expression was high under arsenic-free conditions, and fluorescence response remained minimal after 6 hours of gradient induction, with insufficient signal amplification.
V2 (ParsOC2 drives ArsR+ParsOC2 regulating sfGFP): GFP background expression was significantly elevated in the absence of arsenic. Following arsenic treatment, fluorescence induction exhibited substantial error, failing to achieve the expected induction multiple. The overall "signal-to-noise ratio" was poor, rendering it unsuitable for sensing applications.

Figure 1: The simplified construction of V0, V1, V2 plasmid and result

Problem Identification

Core Conclusion: Based on literature reports and experimental data, it was determined that the "constitutive promoter-driven ArsR expression level" is the core variable. Both excessively high and low ArsR expression levels weaken the repression effect on the ParsOC2 promoter, leading to "background leakage" in downstream sfGFP expression. This finding provides direction for subsequent promoter optimization.

Cycle 2: Second-Round DBTL Iteration of Optimized Plasmids V3-V4

Plasmid Construction Results

Two optimized plasmids were successfully constructed (pET46a-PlacV-ArsR-ParsOC2-sfGFP-V3, pET-46a-PJ100-ArsR-ParsOC2-sfGFP-V4). Following C1's standardized molecular cloning workflow (restriction digestion, ligation, transformation, sequencing), it was confirmed that the ArsR coding sequence in V3 was optimally positioned, and the strong constitutive promoters (PJ100/PJ101/PJ102/PJ104/PJ111) was correctly inserted.

Performance Testing Results

Using the same multi-gradient NaAsO₂ treatment and "Fluo/OD600" detection method as C1, the performance differences between V3, V4, and C1's V1, V2 were compared. Key data are as follows:

Key Findings:

V3 (PlacV promoter + optimized ArsR coding sequence): No breakthrough performance improvement observed; residual background leakage persists, ruling out "ArsR protein sequence stability" as a core factor affecting background.
V4 (strong constitutive promoter driving ArsR): Significant performance variations among promoters, with PJ100 promoter yielding optimal results—enabling high-sensitivity detection (fluorescence induction up to 2.5-fold) while substantially reducing background expression under arsenic-free conditions, significantly improving signal-to-noise ratio and meeting fundamental arsenic sensing requirements; Other promoters (PJ101/PJ102/PJ104/PJ111) demonstrated inferior sensitivity and background control compared to PJ100.

Figure 2: Five different test promoter sequences

Figure 3: The result of 5 different promoters

Figure 4: Reaction mechanism construction and results

In-depth Analysis Findings

Core Conclusion: Integrating "promoter-repressor binding efficiency predictions" with consistent C1 and C2 data refutes the preliminary C1 conclusion — The core cause of high background leakage is not insufficient or excessive ArsR expression, but rather "inefficient binding of the ArsR repressor protein to the ParsOC2 promoter." Concurrently, further investigation is needed to rule out interference from induction conditions (arsenic concentration, induction duration) on response stability.

Cycle 3: DBTL Verification of Multi-Gradient Induction Experiments

Core Findings

Regardless of adjustments to induction concentration, duration, or initial OD values, the arsenic-free background levels in V3 and V4 showed no significant reduction, completely ruling out "unreasonable induction conditions" as a confounding factor.

Problem Identification

Core Conclusion: Based on AlphaFold3 protein-DNA binding predictions, it was confirmed that "amino acid conflicts exist at the ArsR-ParsOC2 binding interface," leading to weakened repression and downstream gene leakage. This necessitates shifting focus to "promoter sequence screening" rather than merely replacing promoter types.

Figure 5: Basic framework and AlphaFold predictions

Module 2: Promoter Engineering and Component Innovation

This module introduces the innovative "niche matching" strategy and develops novel promoter components with stronger compatibility through DBTL cycles.

Cycle 4: DBTL Verification of CML2 Natural Promoter

Key Findings

ParsCML2 exhibits stronger co-evolutionary adaptation and lower binding free energy compared to the CML2-derived ArsR, significantly reducing GFP background leakage to 8% under arsenic-free conditions. At arsenic concentrations ranging from 50 to 300 μM, its fluorescence intensity reached 4785–6000 a.u., representing 2.3–3.1 times that of the ParsOC2 system, while maintaining stable performance in the rice paddy microenvironment (pH 6.2–6.8, microaeric conditions).

Issue Identification

Core Conclusion: A single ParsCML2 variant cannot fully eliminate background fluorescence. A promoter library must be constructed via "random mutation + high-throughput screening" to identify variants with higher binding efficiency.

Figure 6: Application principles, results evaluation, and experimental findings

Cycle 5: DBTL Construction and Planning of Promoter Library (In Progress)

Core Findings

Currently, only library construction design and preliminary operations have been completed; screening and testing have not yet commenced.

Expected Objectives

Future Goals: Screen for ParsCML2 mutants exhibiting "higher ArsR binding efficiency and lower background leakage" to provide high-quality promoter elements for subsequent sensor optimization.

Figure 7: Sequences of ParsCML2 mutants

Module 3: Self-Assembly System and System-Level Optimization

This module integrates the innovative strategy of "AND-gate-like circuit" and attempts to solve the basal leakage problem from the "system level" through DBTL iteration.

Cycle 6: First-Round DBTL of AND-Gate-Like Self-Assembly System

Key Findings

The improved split-GFP variant system exhibits leakage efficiency below 1%, with negligible spontaneous background fluorescence in the absence of arsenic. It accurately distinguishes between "background signals" and "arsenic-induced signals" when detecting low arsenic concentrations, significantly enhancing detection specificity. However, a drawback is that fluorescence intensity increases slowly with rising arsenic concentrations (e.g., 200–300 μM), resulting in a relatively weak overall response signal.

Problem Identification

Hypothesis: We hypothesize two reasons for the weak fluorescence: First, although GFP fragments are expressed normally, impaired complementary interfaces lead to poor self-assembly. Second, GFP fragments degrade readily after expression, preventing sufficient accumulation for self-assembly.

Figure 8: Schematic diagram of the system

Cycle 7: DBTL for In Vitro Verification of GFP Fragments

Core Findings

Currently, plasmid structure design and preliminary construction have been completed. We purified the GFP1-10 (52.49 kDa) and the GFP11 (3.96kDa). Stability testing and fluorescence detection have not yet been conducted.

Figure 9: Expression and purification of green fluorescent protein (GFP1-10+GFP11): (A) PAGE gels of pre - and post-induction samples and PAGE gels of supernatant and pellet samples for each of GFP 1-10 +GFP 11.: lane 1,ladder ; lane 2, Pre-induction of GFP1-10; lane3, post-induction of GFP1-10; lane4,Pre-induction of GFP11; lane5, post-induction of GFP11; lane6 , precipitation of GFP1-10; lane7, precipitation GFP11; lane8, supernatant GFP1-10; lane9 , supernatant GFP11; lane10, mixed supernatant; (B) PAGE gels of purifying the target protein after mixing the two supernatants.: lane 1,ladder ; lane2, mixed supernatant; lane 3, flow through; lane 4, 20 mM imidazole eluent ; lane 5, 100 mM imidazole eluent ; lane 6, 200 mM imidazole eluent ; lane 7, 300 mM imidazole eluent ; lane 8, 500 mM imidazole eluent .

Expected Objectives

Future Goals: Significantly enhance fragment stability while preserving the self-assembly function of the GFP fragment, thereby addressing the issue of weak fluorescence intensity under high arsenic concentrations.

Summary of Experimental Progress

Through seven DBTL cycles organized into three modules, we have systematically addressed key challenges in arsenic biosensor development:

Key Achievements

Module 1: Identified the core issue of "inefficient ArsR-ParsOC2 binding" as the primary cause of high background leakage
Module 2: Demonstrated the superior performance of natural ParsCML2 promoter and initiated library construction for further optimization
Module 3: Developed an AND-gate-like self-assembly system with extremely low leakage (<1%) and initiated fragment stability optimization

Future Directions

Complete screening of ParsCML2 promoter library for higher binding efficiency variants
Optimize GFP fragment stability to enhance fluorescence response at high arsenic concentrations
Integrate optimized components into a final biosensor system for environmental testing