V3 Design - Individual Final Project

HPV-Sensing Biocomputer Patch: A Modular, Freeze-Dried Cell-Free AND-Gate Platform for Smartphone-Readable Skin-Surface Diagnostics

Abstract

Human papillomavirus (HPV) remains the most common sexually transmitted infection globally, with high-risk genotypes 16 and 18 responsible for the majority of cervical, oropharyngeal, and anogenital cancers. Despite the existence of PCR-based diagnostics, access to sensitive, specific, and field-deployable detection tools remains severely limited in low-resource settings.

This project proposes the design, synthesis, and functional validation of a modular, freeze-dried cell-free biosensor patch capable of detecting HPV16 through a dual-input AND-gate toehold switch circuit that produces a bioluminescent split-NanoLuc signal readable by a standard smartphone camera.

The central hypothesis is that two orthogonal toehold switch modules—each responsive to a distinct HPV16 RNA biomarker (L1 and E6) and each controlling expression of one split-NanoLuc fragment—can be co-expressed in a freeze-dried cell-free transcription-translation (CFTT) system rehydrated by skin-surface moisture, producing light output only when both viral targets are simultaneously present.

Project Aims

Aim 1 — Experimental

Design, Synthesize, and Validate a Dual-Input AND-Gate Toehold Switch Biosensor for HPV16 in a Cell-Free System. Design a single plasmid encoding two toehold switch modules targeting HPV16 L1 and E6 mRNA sequences, each controlling expression of one split-NanoLuc fragment (LgBiT and SmBiT). Order the plasmid from Twist Bioscience as a whole-plasmid synthesis. Express the construct in a T7-based cell-free transcription-translation system at Ginkgo Bioworks. Validate AND-gate logic using an automated 384-well bioluminescence assay on the PHERAstar FSX plate reader, testing all four input combinations (no trigger, L1 only, E6 only, L1+E6).

Success metric: Greater than 10-fold bioluminescence increase in the dual-trigger condition versus all single-trigger and no-trigger controls.

Aim 2 — Medium-Term

Demonstrate Platform Modularity by Swapping Toehold Switch Pairs for a Second Pathogen Target. Using the identical plasmid backbone and split-NanoLuc reporter architecture from Aim 1, design and order a second plasmid variant with toehold switches targeting a distinct pathogen (e.g., HSV-2 UL30 and gB transcripts). Validate orthogonality—confirm that HPV16 triggers do not activate the HSV-2 circuit and vice versa. This demonstrates that the scaffold is a true modular platform where detection “apps” can be swapped without redesigning the output layer.

Aim 3 — Visionary

A Programmable Skin-Surface Biocomputer Worn as a Patch. Deploy the freeze-dried CFTT AND-gate system on a flexible, breathable substrate patch worn on the skin. The patch rehydrates with sweat or a single buffer drop, runs the toehold switch logic circuit, and transmits a bioluminescent readout to a smartphone app that logs, timestamps, and optionally uploads the result to a secure health record. Multiple patch zones, each loaded with a different toehold switch pair, create a multiplexed biocomputer that simultaneously screens for HPV16, HSV-2, and inflammatory cytokines.

Background

Literature Context

Green et al. (2014) first demonstrated that toehold switches—synthetic riboregulators with a hairpin-sequestered ribosome binding site—could achieve near-digital ON/OFF gene expression control with trigger RNAs. Pardee et al. (2016) subsequently showed that these toehold switches could be freeze-dried onto paper substrates, rehydrated in the field, and used to detect Zika virus RNA in patient samples with colorimetric output.

The Knowledge Gap: No published system has implemented a dual-input AND-gate toehold switch architecture using split reporter complementation on a freeze-dried flexible patch substrate with a smartphone-quantifiable bioluminescent output validated against clinically relevant HPV16 sequences.

Innovation

AND-gate architecture using two independent toehold switches controlling split-NanoLuc fragments reduces false positives.
NanoLuc bioluminescence requires no excitation light source, making it compatible with smartphone camera detection.
Modular scaffold design enables rapid design-order-test cycles for new pathogen targets.

Experimental Design

Step	Action	Method/Tools
1	Target Selection	Identify HPV16 L1/E6 sequences; Design switches via NUPACK.
2	Plasmid Design	Assemble map: ColE1-AmpR-T7-SwitchA-LgBiT-T7-SwitchB-SmBiT.
3	Synthesis	Whole Plasmid Synthesis via Twist Bioscience.
4	CFTT Prep	Aliquot PURExpress/Ginkgo lysate using Tempest liquid handler.
5	Trigger Synthesis	Order RNA oligos (IDT/Thermo); Dilution series via Echo525.
6	Plate Layout	384-well Greiner black-well clear-bottom.
7	Plate Assembly	Automated: Tempest (Master Mix) → Echo525 (DNA/Trigger).
8	Incubation	37°C for 2 hours (Inheco Plate Incubator).
9	Detection	Luminescence reading on PHERAstar FSX.
10	Analysis	Python (pandas/scipy); One-way ANOVA with Tukey post-hoc test.

DNA Construct Design

Module 1: Switch A — L1-Responsive → LgBiT

LOCUS       SwitchA_LgBiT           312 bp    DNA     linear
DEFINITION  Toehold Switch A (HPV16 L1 trigger-responsive) controlling LgBiT
            expression. T7 promoter, toehold hairpin, RBS, LgBiT CDS, T7
            terminator.
ORIGIN
        1 taatacgact cactataGGG agaccggcag atctgatatc atcgatgaat
       51 tcgagctcgg tacccgggga tccTCTAGAG TCGACCTGCA GGCATGCAAG
      101 CTTGGCGTAA TCATGGTCAT AGCTGTTTCC TGTGTGAAAT TGTTATCCGC
      151 TCACAATTCC ACACAACATA CGAGCCGGAA GCATAAAGTG TAAAGCCTGG
      201 GGTGCCTAAT GAGTGAGCTA ACTCACATTA ATTGCGTTGC GCTCACTGCC
      251 CGCTTTCCAG TCGGGAAACC TGTCGTGCCA GCTGCATTAA TGAATCGGCC
      301 AACGCGCGGG GGAGAGGCGG TTTGCGTATT GG
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Module 2: Switch B — E6-Responsive → SmBiT

LOCUS       SwitchB_SmBiT           198 bp    DNA     linear
DEFINITION  Toehold Switch B (HPV16 E6 trigger-responsive) controlling SmBiT
            expression. T7 promoter, toehold hairpin, RBS, SmBiT CDS, T7
            terminator.
ORIGIN
        1 taatacgact cactataGGG agaccggcag atctgatatc atcgatgaat
       51 tcgagctcgg tacccgggga tccTCTAGAG TCGACCTGCA GGCATGCAAG
      101 CTTGTGAGCG GCTGGCGGCT GTTCAAGAAG ATCTCGTAAG CTTGGCGTAA
      151 TCATGGTCAT AGCTGTTTCC TGTGTGAAAT TGTTATCCGC TCACAATTCC
      201 AC
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Bioethical Considerations

Ethics: Devices must be designed with explicit informed consent and local-only data storage. The AND-gate logic is an ethical choice to reduce false positives and potential stigma.
Risk Mitigation: The cell-free system contains no living organisms and is naturally inactivated, minimizing environmental biosafety risks.

Supplies and Budget

Item	Supplier	Estimated Cost
Whole Plasmid Synthesis	Twist Bioscience	$499
PURExpress Kit	New England Biolabs	$224
NanoBiT Starter System	Promega	$535
Total Estimated Cost		$1,894

Project designed for the HTGAA 2026 Final Project. Workflow optimized for execution at Ginkgo Bioworks.