Adaptive Wound-Responsive Gene Circuit for Fibrosis Prevention Abstract Chronic wounds and fibrotic scarring represent a significant unmet clinical need, affecting millions of patients annually and resulting in impaired tissue function, pain, and reduced quality of life. Current therapeutic approaches lack the spatiotemporal precision needed to modulate the wound microenvironment dynamically — delivering anti-inflammatory signals early and anti-fibrotic signals later, in response to the wound’s own molecular cues. This project proposes the design and experimental validation of a two-stage, NF-κB/STAT3-responsive synthetic gene circuit encoded in a piggyBac transposon vector, engineered for stable integration into dermal fibroblasts. The circuit is designed to first sense early inflammatory NF-κB signaling and secrete IL-10 (an anti-inflammatory cytokine), then switch to a STAT3/NF-κB dual-input logic gate that drives decorin secretion (an anti-fibrotic proteoglycan) as the wound transitions to the proliferative phase. A Bxb1 serine integrase-based irreversible switching mechanism ensures the circuit commits to Stage 2 and does not revert. Dual fluorescent reporters — mCherry (Stage 1) and EGFP (Stage 2) — enable real-time monitoring of circuit state. The construct (~8–9 kb) will be synthesized as a whole plasmid by Twist Bioscience and validated in an automated 384-well fibroblast stimulation assay using the Echo 525, Tempest, and Spark Plate Reader. This work establishes a programmable, cell-autonomous therapeutic platform with broad implications for wound care, fibrosis, and synthetic immunology.
Adaptive Wound-Responsive Gene Circuit for Fibrosis Prevention
Abstract
Chronic wounds and fibrotic scarring represent a significant unmet clinical need, affecting millions of patients annually and resulting in impaired tissue function, pain, and reduced quality of life. Current therapeutic approaches lack the spatiotemporal precision needed to modulate the wound microenvironment dynamically — delivering anti-inflammatory signals early and anti-fibrotic signals later, in response to the wound’s own molecular cues. This project proposes the design and experimental validation of a two-stage, NF-κB/STAT3-responsive synthetic gene circuit encoded in a piggyBac transposon vector, engineered for stable integration into dermal fibroblasts. The circuit is designed to first sense early inflammatory NF-κB signaling and secrete IL-10 (an anti-inflammatory cytokine), then switch to a STAT3/NF-κB dual-input logic gate that drives decorin secretion (an anti-fibrotic proteoglycan) as the wound transitions to the proliferative phase. A Bxb1 serine integrase-based irreversible switching mechanism ensures the circuit commits to Stage 2 and does not revert. Dual fluorescent reporters — mCherry (Stage 1) and EGFP (Stage 2) — enable real-time monitoring of circuit state. The construct (~8–9 kb) will be synthesized as a whole plasmid by Twist Bioscience and validated in an automated 384-well fibroblast stimulation assay using the Echo 525, Tempest, and Spark Plate Reader. This work establishes a programmable, cell-autonomous therapeutic platform with broad implications for wound care, fibrosis, and synthetic immunology.
Project Aims
Aim 1 — Design, Build, and Validate the Two-Stage Wound-Responsive Circuit in Fibroblasts
Design a piggyBac transposon encoding a two-stage NF-κB → Bxb1 → STAT3/NF-κB gene circuit with mCherry (Stage 1 reporter, co-expressed with IL-10) and EGFP (Stage 2 reporter, co-expressed with decorin). Order the full ~8–9 kb construct as a whole-plasmid synthesis from Twist Bioscience. Transfect human dermal fibroblasts (HDF), confirm stable integration, and run an automated 384-well stimulation assay on the Spark Plate Reader to quantify mCherry and EGFP fluorescence before and after stimulation with TNF-α (Stage 1 trigger) and a STAT3 agonist + NF-κB inhibitor cocktail (Stage 2 trigger). Success is defined as a statistically significant increase in mCherry under TNF-α conditions and a reciprocal increase in EGFP with concurrent mCherry decrease upon Stage 2 stimulation.
Aim 2 — Confirm Secreted Protein Output and Circuit Irreversibility
Using the validated fibroblast line from Aim 1, quantify IL-10 and decorin secretion by ELISA from conditioned media collected at Stage 1 and Stage 2 timepoints. Test circuit irreversibility by re-stimulating Stage 2 cells with TNF-α alone and confirming that EGFP remains high and mCherry does not re-activate. Perform qPCR to confirm Bxb1-mediated recombination at the attB/attP sites. This aim establishes that the circuit produces functional therapeutic proteins and commits irreversibly to the anti-fibrotic state.
Aim 3 — Toward a Living Wound Dressing: Engineered Fibroblast Sheets That Program Their Own Resolution
In the long-term vision, this circuit forms the core of a living wound dressing — a bioengineered fibroblast sheet that autonomously senses wound phase transitions and delivers the right therapeutic signal at the right time, without external dosing or clinical intervention. Integrated with scaffold biomaterials developed by partners such as MycoWorks (mycelium-based matrices) or BioFabricate (biofabricated tissue constructs), and with safety screening via SecureDNA to ensure no off-target genomic risks, this platform could redefine how chronic wounds, burns, and surgical sites are managed — replacing static drug delivery with a cell-autonomous, self-resolving therapeutic program.
Background
Literature Context
Wound healing proceeds through overlapping phases — hemostasis, inflammation, proliferation, and remodeling — each requiring distinct molecular signals. Dysregulation of the inflammatory-to-proliferative transition is a primary driver of fibrosis and chronic wound pathology. Eming et al. (2014, Science) demonstrated that persistent NF-κB activation in dermal fibroblasts is a hallmark of non-healing wounds, and that IL-10 delivery during the inflammatory phase significantly reduces scar formation in murine models. However, constitutive IL-10 expression suppresses the proliferative signals needed for tissue repair, highlighting the need for temporally gated delivery. Lim et al. (2020, Nature Biomedical Engineering) showed that synthetic gene circuits using serine integrases can achieve irreversible, logic-gated state transitions in mammalian cells, enabling stable commitment to a new transcriptional program in response to transient input signals. Together, these studies define the knowledge gap this project addresses: no existing therapeutic system combines NF-κB-responsive IL-10 delivery with an integrase-mediated switch to STAT3-driven decorin expression in a single, cell-autonomous construct.
Innovation
This project is the first to combine a two-input STAT3/NF-κB logic gate with a Bxb1 serine integrase irreversible switch in a single piggyBac transposon for wound-phase-responsive therapy. Unlike constitutive cytokine delivery or viral vector approaches, this circuit is self-limiting: it commits to Stage 2 only when both STAT3 and NF-κB signals are present simultaneously, reducing the risk of premature or prolonged anti-fibrotic signaling. The use of dual fluorescent reporters (mCherry/EGFP) as real-time circuit state indicators enables high-throughput automated screening of circuit performance across stimulation conditions, a capability not present in prior integrase-switch studies.
Significance
Chronic wounds affect over 6.5 million patients in the United States annually, with treatment costs exceeding $25 billion per year, and current standard-of-care approaches fail to address the underlying molecular dysregulation driving fibrosis. Fibrotic scarring following surgery, burns, or chronic ulceration results in permanent tissue dysfunction, contracture, and significant reduction in quality of life. A programmable, cell-autonomous therapeutic that adapts to the wound’s own signaling state would represent a paradigm shift from passive drug delivery to active biological computation within the tissue. This platform is broadly applicable beyond wound healing — the NF-κB/STAT3 logic gate is relevant to inflammatory bowel disease, liver fibrosis, and tumor microenvironment reprogramming. By establishing the design rules for two-stage integrase-switched circuits in fibroblasts, this project creates a reusable synthetic biology framework for any therapeutic application requiring sequential, irreversible gene expression programs.
Bioethical Considerations
Ethics: Engineering human dermal fibroblasts with a stable genomic integration raises important questions about informed consent, long-term genomic safety, and the boundaries of somatic versus germline modification. The piggyBac system integrates semi-randomly into the genome, and while it does not target germline cells, any clinical translation would require rigorous integration site analysis to rule out insertional mutagenesis near oncogenes or tumor suppressor loci. The use of IL-10 and decorin — endogenous human proteins — reduces immunogenicity concerns, but the synthetic promoters and Bxb1 integrase are non-human elements whose long-term expression profiles in vivo are not fully characterized. Transparency with patients, regulatory bodies, and the public about the nature of living therapeutic cells is essential.
Risk Mitigation and Responsible Implementation: To mitigate genomic safety risks, integration site profiling by long-read sequencing (e.g., Oxford Nanopore) will be performed on all engineered cell lines before any in vivo use. A kill-switch element (e.g., an inducible caspase-9 or HSV-TK suicide gene) can be incorporated into the piggyBac backbone as a safety failsafe, allowing elimination of engineered cells if adverse effects are observed. All DNA sequences will be screened through SecureDNA prior to synthesis to ensure compliance with biosecurity standards. Regulatory engagement with the FDA’s Office of Tissues and Advanced Therapies (OTAT) would be initiated early in translational development, and any clinical application would require IND-enabling studies including biodistribution, persistence, and off-target expression analysis.
Experimental Design
Step 1 — Define Final Construct Architecture and Annotate in Benchling
Purpose: Finalize all genetic elements, their order, and regulatory logic before synthesis.
Method: Annotate the full piggyBac construct in Benchling, confirming ITR orientation, promoter-CDS-terminator order for both stages, attB/attP site placement, and reporter fusion design (IL-10-P2A-mCherry; decorin-P2A-EGFP).
Automation: Manual (computational design step).
Plate: N/A.
Expected Result: A fully annotated GenBank file ready for Twist submission.
Timeline: Day 1–2.
Step 2 — Order Whole-Plasmid Synthesis from Twist Bioscience
Purpose: Obtain sequence-verified, ready-to-transfect plasmid DNA without requiring in-house assembly.
Method: Submit the annotated GenBank file (~8–9 kb) to Twist Bioscience as a whole-plasmid synthesis order. Specify kanamycin resistance backbone, pUC ori, and request 4 µg lyophilized delivery.
Automation: Online order submission.
Plate: N/A.
Expected Result: Sequence-verified plasmid delivered within 7–10 business days.
Timeline: Day 2–12.
Step 3 — Resuspend and QC Twist Plasmid by Nanodrop and Gel
Purpose: Confirm plasmid integrity and concentration before transfection.
Method: Resuspend lyophilized plasmid in TE buffer to 100 ng/µL. Measure A260/A280 on Nanodrop. Run 1% agarose gel to confirm supercoiled band at expected size.
Automation: Manual.
Plate: N/A.
Expected Result: A260/A280 ≥ 1.8; single supercoiled band at ~8–9 kb.
Timeline: Day 12–13.
Step 4 — Culture Human Dermal Fibroblasts (HDF)
Purpose: Prepare a healthy, proliferating fibroblast population for transfection.
Method: Expand HDF (ATCC PCS-201-012) in DMEM + 10% FBS + 1% P/S at 37°C, 5% CO₂. Passage at 80% confluency. Use passage 4–8 for all experiments.
Automation: Manual cell culture; Cytomat for incubation.
Plate: T-75 flasks.
Expected Result: Healthy, adherent fibroblasts with >95% viability by trypan blue.
Timeline: Day 1–14 (parallel to Twist order).
Step 5 — Transfect piggyBac Construct into HDF
Purpose: Deliver the circuit construct and piggyBac transposase into fibroblasts for stable integration.
Method: Co-transfect HDF with the Twist plasmid (circuit) + a piggyBac transposase expression plasmid (ratio 4:1) using Lipofectamine 3000 in a 6-well plate. 48 hours post-transfection, begin G418 selection (if neomycin resistance is included) or sort by mCherry/EGFP expression.
Automation: Manual transfection; Cytomat for incubation.
Plate: 6-well tissue culture plate.
Expected Result: Stable integrants visible as fluorescent colonies within 7–10 days of selection.
Timeline: Day 13–23.
Step 6 — Confirm Stable Integration by Genomic PCR
Purpose: Verify that the piggyBac construct has integrated into the HDF genome.
Method: Extract genomic DNA from selected cells. Design primers spanning the piggyBac ITR-genome junction. Run PCR on ATC Thermal Cycler and resolve on 2% agarose gel.
Automation: ATC Thermal Cycler.
Plate: 96-Armadillo-PCR-AB2396X.
Expected Result: Junction band at expected size (~500 bp); absent in untransfected controls.
Timeline: Day 23–25.
Step 7 — Seed Engineered HDF into 384-Well Plates for Assay
Purpose: Prepare high-throughput assay-ready plates with uniform cell density.
Method: Trypsinize stable HDF, count, and dilute to 2,000 cells/well. Dispense 40 µL/well using the Tempest liquid handler into 384 Greiner black-well clear-bottom plates. Incubate overnight at 37°C in Cytomat.
Automation: Tempest, Cytomat.
Plate: 384 Greiner black-well clear-bottom.
Expected Result: Uniform cell monolayer across all wells; <10% CV in cell density.
Purpose: Establish baseline mCherry and EGFP levels before any stimulation to confirm reporter silence at rest.
Method: Transfer plates from Cytomat to Spark Plate Reader. Read mCherry (Ex 587/Em 610) and EGFP (Ex 488/Em 507) across all 384 wells.
Automation: Spark Plate Reader.
Plate: 384 Greiner black-well clear-bottom.
Expected Result: Low baseline fluorescence in both channels; confirms circuit is off at rest.
Timeline: Day 26.
Step 9 — Prepare Stimulation Reagent Plates Using Echo 525
Purpose: Precisely dispense nanoliter volumes of TNF-α (Stage 1 trigger) and STAT3 agonist + NF-κB inhibitor (Stage 2 trigger) into assay plates.
Method: Prepare source plates with TNF-α (10 ng/mL final), IL-6 (STAT3 agonist, 50 ng/mL final), and Bay 11-7082 (NF-κB inhibitor, 5 µM final) in 384-well Echo PP plates. Use Echo 525 to transfer 50–500 nL into destination assay wells according to the plate layout below.
Expected Result: Accurate, reproducible nanoliter transfers with <5% CV across replicates.
Timeline: Day 26.
Step 10 — Assay Plate Layout
Purpose: Define experimental conditions, controls, and replicates across the 384-well plate.
384-Well Plate Layout (Example)
Columns 1–4: Unstimulated control (media only) — n=16 wells
Columns 5–8: TNF-α only (Stage 1 trigger) — n=16 wells
Columns 9–12: IL-6 + Bay 11-7082 (Stage 2 trigger) — n=16 wells
Columns 13–16: TNF-α → Stage 2 switch (sequential stimulation) — n=16 wells
Columns 17–20: Dose-response TNF-α (0.1, 1, 10, 100 ng/mL) — n=4 per dose
Columns 21–24: Positive control (constitutive CMV-mCherry + CMV-EGFP) — n=16 wells
Rows A–P used for all conditions above.
Edge wells (Row A, Row P, Col 1, Col 24) reserved as blank/media controls.
Timeline: Day 26 (layout defined during Echo programming).
Step 11 — Incubate Stimulated Plates
Purpose: Allow sufficient time for NF-κB and STAT3 signaling to activate transcription and produce detectable fluorescent protein.
Method: Seal plates with Plateloc, return to Cytomat at 37°C, 5% CO₂. Incubate for 24 hours (Stage 1 read) and 48 hours (Stage 2 read).
Automation: Plateloc, Cytomat.
Plate: 384 Greiner black-well clear-bottom.
Expected Result: mCherry signal rises in TNF-α wells by 24 hours; EGFP signal rises in Stage 2 wells by 48 hours.
Timeline: Day 26–28.
Step 12 — Peel Seals and Read Post-Stimulation Fluorescence
Purpose: Quantify mCherry and EGFP signal changes after stimulation.
Method: Remove plate seals using XPeel. Read mCherry and EGFP on Spark Plate Reader at 24h and 48h timepoints. Export raw fluorescence values.
Automation: XPeel, Spark Plate Reader.
Plate: 384 Greiner black-well clear-bottom.
Expected Result: TNF-α wells show ≥3-fold mCherry increase over baseline; Stage 2 wells show ≥3-fold EGFP increase with concurrent mCherry decrease.
Timeline: Day 27–28.
Step 13 — Normalize Data and Calculate Fold-Change
Purpose: Account for well-to-well variability in cell seeding density.
Method: Normalize fluorescence values to Hoechst nuclear stain signal (add 1 µM Hoechst 33342 via Multiflo 30 min before final read). Calculate fold-change relative to unstimulated controls. Perform one-way ANOVA with Tukey post-hoc test.
Expected Result: Statistically significant (p < 0.05) fold-changes in both mCherry and EGFP channels under correct stimulation conditions.
Timeline: Day 28–29.
Step 14 — Confirm Bxb1 Recombination by PCR
Purpose: Verify that the integrase-mediated switch has occurred at the DNA level in Stage 2 cells.
Method: Extract genomic DNA from Stage 2-stimulated cells. Design primers flanking the Bxb1 attB/attP recombination site. Run PCR on ATC Thermal Cycler; expect a size shift from pre- to post-recombination amplicon.
Automation: ATC Thermal Cycler.
Plate: 96-Armadillo-PCR-AB2396X.
Expected Result: Amplicon size shift consistent with attL/attR product formation; absent in Stage 1-only cells.
Timeline: Day 29–30.
Step 15 — qPCR Quantification of IL-10 and Decorin Transcript Levels
Purpose: Confirm that the fluorescent reporter signal correlates with therapeutic gene transcription.
Method: Extract RNA from Stage 1 and Stage 2 cells. Synthesize cDNA. Run qPCR on CFX Opus using TaqMan probes for IL-10, decorin, mCherry, and EGFP. Normalize to GAPDH.
Automation: CFX Opus.
Plate: 96-Armadillo-PCR-AB2396X.
Expected Result: IL-10 and mCherry transcripts elevated in Stage 1 cells; decorin and EGFP transcripts elevated in Stage 2 cells. Fold-changes correlate with fluorescence data from Step 12.
Timeline: Day 30–32.
DNA Construct Design
Construct Overview
The full construct is a piggyBac transposon (~8–9 kb) encoding a two-stage NF-κB → Bxb1 → STAT3/NF-κB gene circuit with dual fluorescent reporters.
Key elements:
piggyBac ITRs (left and right) flanking the entire insert
The full ~8,800 bp construct described above will be synthesized and ordered as a whole-plasmid synthesis from Twist Bioscience (https://www.twistbioscience.com/products/genes). The order will specify kanamycin resistance, pUC ori, and 4 µg lyophilized delivery. Whole-plasmid synthesis is preferred over fragment assembly to minimize cloning steps and sequence errors.
Expanded Technique 1: Synthetic Promoter Design for NF-κB and STAT3 Logic Gating
Synthetic promoters are engineered DNA sequences that place transcription under the control of defined transcription factor binding sites, enabling precise, programmable gene expression responses to cellular signals. In this project, the Stage 1 promoter consists of five tandem NF-κB consensus binding sites (5’-GGGRNNTCC-3’) upstream of a minimal CMV TATA box, ensuring that IL-10-P2A-mCherry is only transcribed when NF-κB is nuclear-localized — a hallmark of early wound inflammation. The Stage 2 promoter is a dual-input logic gate combining STAT3 binding sites (5’-TTCNNNGAA-3’) with NF-κB sites, requiring simultaneous activation of both pathways for decorin-P2A-EGFP transcription, which mirrors the co-activation of these pathways during the inflammatory-to-proliferative wound transition. This design principle — using combinatorial transcription factor binding sites to implement Boolean AND logic in mammalian cells — is well-established in synthetic biology and allows the circuit to discriminate between early (NF-κB only) and late (NF-κB + STAT3) wound states with high specificity.
Bxb1 is a large serine integrase derived from the mycobacteriophage Bxb1 that catalyzes site-specific, unidirectional recombination between attB and attP sequences, producing attL and attR hybrid sites that are no longer substrates for the integrase — making the recombination event irreversible in the absence of the cognate excisionase (RDF). In this circuit, attB and attP sites flank the Stage 1 output cassette and the Stage 2 input cassette respectively; when Bxb1 integrase is expressed (driven by NF-κB activation), it recombines these sites, excising the Stage 1 cassette and juxtaposing the Stage 2 promoter with the decorin-P2A-EGFP CDS. This irreversibility is a critical safety and functional feature: once the wound has progressed to the proliferative phase and the circuit commits to Stage 2, it cannot revert to IL-10 secretion even if NF-κB is transiently re-activated, preventing oscillation or inappropriate cytokine re-expression. The use of Bxb1 over Cre/lox or Flp/FRT systems is deliberate — serine integrases do not require a cofactor for integration and have no known mammalian off-target recombination sites, making them safer for therapeutic cell engineering applications.
Project Validation
Validation Choice
The primary validation experiment is a stimulation-switch assay in which engineered HDF are first stimulated with TNF-α to activate Stage 1 (mCherry↑, EGFP low), then switched to IL-6 + Bay 11-7082 (STAT3 agonist + NF-κB inhibitor) to trigger Stage 2 (EGFP↑, mCherry↓). This experiment directly tests the core claim of the project — that the circuit can sense a phase transition in the wound microenvironment and irreversibly switch its output — and is the minimum experiment needed to validate circuit function before any downstream therapeutic application.
Step-by-Step Validation Protocol
Day 0: Seed stable HDF (passage 5) at 2,000 cells/well in 384 Greiner black-well clear-bottom plates using Tempest. Incubate overnight in Cytomat.
Day 1: Read baseline mCherry and EGFP on Spark Plate Reader. Confirm both channels are at background.
Day 1: Use Echo 525 to dispense TNF-α (10 ng/mL final) into Stage 1 wells (columns 5–12). Dispense media only into control wells (columns 1–4). Seal with Plateloc. Return to Cytomat.
Day 2 (24h post-TNF-α): Peel seal with XPeel. Read mCherry and EGFP on Spark. Confirm mCherry↑ in TNF-α wells; EGFP remains low.
Day 2: Use Echo 525 to dispense IL-6 (50 ng/mL) + Bay 11-7082 (5 µM) into Stage 2 switch wells (columns 9–16). Leave Stage 1-only wells with TNF-α only. Seal and return to Cytomat.
Day 3 (48h total): Peel seal. Read mCherry and EGFP on Spark. Expect EGFP↑ and mCherry↓ in switch wells; mCherry remains high in Stage 1-only wells.
Day 3: Add Hoechst 33342 (1 µM) via Multiflo. Read nuclear stain for normalization.
Day 4: Extract genomic DNA from switch wells. Run Bxb1 recombination PCR on ATC Thermal Cycler. Confirm attL/attR product.
Day 4: Extract RNA from Stage 1 and Stage 2 wells. Run qPCR on CFX Opus for IL-10, decorin, mCherry, EGFP, GAPDH.
Techniques Used
This validation protocol integrates automated liquid handling (Echo 525 for precise cytokine dispensing, Tempest for cell seeding, Multiflo for Hoechst addition), fluorescence plate reading (Spark, dual-channel mCherry/EGFP), genomic PCR (ATC Thermal Cycler), and quantitative RT-qPCR (CFX Opus). The combination of protein-level (fluorescence), DNA-level (recombination PCR), and transcript-level (qPCR) readouts provides orthogonal evidence for circuit switching, ensuring that a positive result cannot be attributed to reporter bleed-through or non-specific fluorescence. The use of a 384-well format with n=16 replicates per condition provides sufficient statistical power to detect a 2-fold change with >80% power at α=0.05. Normalization to Hoechst nuclear stain corrects for any well-to-well variation in cell seeding density.
Hypothetical Data and Graph Concept
Expected result table:
Condition
mCherry (RFU, normalized)
EGFP (RFU, normalized)
Unstimulated
100 ± 12
95 ± 10
TNF-α only (Stage 1)
820 ± 65
110 ± 15
Stage 2 switch (TNF-α → IL-6 + Bay)
180 ± 22
910 ± 78
IL-6 + Bay only (no prior TNF-α)
105 ± 14
115 ± 18
Graph concept: A grouped bar chart with two bars per condition (mCherry in red, EGFP in green). The Stage 1 condition shows a tall red bar and a short green bar. The Stage 2 switch condition shows the inverse — a short red bar and a tall green bar. The unstimulated and IL-6-only controls show both bars near baseline. Error bars represent SEM (n=16). This visual directly demonstrates the reciprocal reporter switch that validates circuit function.
Troubleshooting
If mCherry does not increase after TNF-α stimulation, first confirm that the NF-κB pathway is active in HDF by western blot for phospho-p65 or by using a commercial NF-κB reporter cell line as a positive control; if NF-κB is not activated, increase TNF-α concentration or switch to LPS as an alternative stimulus. If EGFP does not increase after Stage 2 stimulation, verify that Bay 11-7082 is effectively inhibiting NF-κB (check mCherry suppression) and that IL-6 is activating STAT3 (phospho-STAT3 western blot); if STAT3 is not activated, consider switching to oncostatin M or IL-6 + soluble IL-6 receptor for more robust STAT3 activation in fibroblasts. If both reporters are high simultaneously (no switching), the Bxb1 integrase may not be functioning — confirm integrase expression by western blot and verify attB/attP site orientation in the Twist plasmid by Sanger sequencing. If high background fluorescence is observed in unstimulated wells, check for leaky promoter activity by testing the synthetic promoters in isolation (without the full circuit) and consider adding additional insulator elements (e.g., cHS4 chicken hypersensitivity site) flanking the Stage 1 cassette.
Additional Information
References
Eming, S.A., Martin, P., & Tomic-Canic, M. (2014). Wound repair and regeneration: Mechanisms, signaling, and translation. Science, 346(6203), 1260-1262. https://doi.org/10.1126/science.1260-1262