Individual Final Project

BioLight — Final Project Update

April 14, 2026 | HTGAA 2026 Individual Final Project

Short Final Project Description

My final project develops a light-responsive genetic circuit in E. coli that expresses fluorescent protein, using LED light to map projected photographic images to a biological substrate on agar plates.

Custom-built LED exposure hardware controls light exposure, activating the engineered biosensor to achieve high-resolution, wide-gamut images appearing through protein expression in transformed bacteria.

The resulting workflow will serve as a framework for community makerspace activities and a platform for ongoing optogenetic imaging research.

Project Aims

Aim 1 — Experimental

• Engineer and validate a light-responsive fluorescent protein expression system in E. coli
• Success measured by fidelity and tonal resolution of the expressed fluorescent image relative to the projected visual image

Aim 2 — Development

• Translate the validated bio-circuit into an integrated imaging platform
• Custom LED exposure hardware, 3D printed components, and software protocols
• Connect analog light to digital tools, back to biological output
• Explore how a cell-free system and automated lab production could increase productivity
• Custom-design and build of light projection system including:
  • Raspberry Pi 5 as the primary controller
  • LED light array for controlled blue light exposure
  • Wavelength sensor for real-time spectral verification
  • OpenCV machine vision algorithms for luminosity measurement
  • Environmental sensors including temperature monitoring
  • Cycle timer to regulate and automate exposure sequences

Aim 3 — Visionary

• Establish a framework for experiential learning in synthetic biology within community makerspaces
• Long-term extension into machine vision interpretation of biosensor expression patterns
• LLM and neural network integration for image recognition and biosensor pattern analysis

Aim 1

Aim 1a — pBioLight x2 (primary)

pBioLight-1B-eLightOn-v1, designated pBioLight x2, is the primary construct for Aim 1a and the fastest path to first image. It is a 2,201 bp circular single-plasmid system designed in Benchling and ordered via Twist Bioscience clonal gene synthesis in a pUC19 backbone with AmpR selection. The eLightOn system uses a LexA408 DNA binding domain fused to RsLOV, a light-oxygen-voltage domain that undergoes a conformational change upon 450nm blue light activation, releasing repression of the pColE408 promoter and driving sfGFP expression.

Circuit architecture

J23106 constitutive promoter → LexA408 DBD (P40A/N41S/A42S, codon-optimized) → RsLOV 176aa (528bp, codon-optimized) → KV linker → pColE408 promoter → BBa_B0034 RBS → sfGFP → rrnB T1/T2 terminators

Key properties

• GC content: 48.98%
• 2 ORFs confirmed, no direct repeats
• Light activation: 450nm blue light
• Dynamic range: ~10,000×
• No external reagents required — the system uses FMN, a molecule E. coli naturally produces, as its light-sensing cofactor. This simplifies the workflow compared to systems like CcaS/CcaR that require externally supplied chromophores.
• Restriction cut sites flanking sfGFP enable future color swapping without redesigning the full circuit, supporting expansion toward wide-gamut multi-color biological imaging through Aim 2 and beyond

Appendix — Optogenetic Systems Evaluated

All systems below were evaluated for use in the BioLight platform. eLightOn was selected as the primary system for pBioLight x2. Systems marked with ★ remain viable parallel tracks.

Images

Light Responsive Plasmid Desgin (Asimov Schematic + Adobe Firefly + Gemini)

Light Projection Labware - Gemini

References

• Li et al. 2020, Nucleic Acids Research 48(6):e33, doi:10.1093/nar/gkaa044 — eLightOn system
• Levskaya et al. 2005, Nature 438, 441–442, doi:10.1038/nature04405
• Jayaraman 2016, PMC5001607
• Tabor Lab, Rice University — jtabor@rice.edu — pJT119b, pSR43.6r, pSR58.6 CcaS/CcaR optogenetic system
• Addgene pJT119b #50551, pSR43.6r #63197, pSR58.6 #63176