Overview

As engineers, it is important that we follow a cycle based on learning and improvement to creat the best, most effective product we can. Therefore the following page describes our iteration through the design, build, test, learn cycle and how it applies to all aspects of our project.

Putrescine Biosensor

Reason

Objective:

• Engineer a genetic circuit that allows E. coli biosensors to detect putrescine concentration and convert it into an optical fluorescence output.

Need:

• A sensing mechanism that specifically responds to putrescine, produces a measurable fluorescence signal, and includes internal controls to ensure circuit reliability.

Research

Promoter Selection:

• Adopted a custom promoter design from published work that is regulated by the putrescine-sensitive protein PuuR.

Regulatory Mechanism:

• Studied how PuuR acts as a transcriptional repressor, binding to the promoter in the absence of putrescine and releasing upon ligand binding.

Design

Primary Circuit:

• Placed GFP downstream of the putrescine-sensitive promoter. In the absence of putrescine, GFP is repressed; in its presence, PuuR binds putrescine instead of the promoter, enabling GFP expression.

Secondary Circuit:

• Added a constitutive promoter upstream of the puuR gene to ensure continuous production of the PuuR protein. Coupled this with an mCherry reporter downstream of puuR to verify repressor production and provide a baseline fluorescence reference.

Dual-Color Output:

• Established a two-signal system: GFP as the dynamic putrescine-responsive signal, mCherry as a constitutive control for normalization.

Build

Plasmid Construction:

• Cloned the custom promoter, GFP, constitutive promoter, puuR gene, and mCherry reporter into a single plasmid, ensuring compatibility of expression elements.

Host Transformation:

• Introduced the plasmid into E. coli strains to generate stable putrescine biosensors.

Test

Functional Validation:

• Exposed biosensors to increasing concentrations of putrescine and confirmed that GFP fluorescence increased in proportion to putrescine levels.

Growth Assessment:

• Monitored growth kinetics of engineered plasmids to evaluate metabolic burden, revealing an extended lag phase in V004 likely due to uninduced GFP expression.

Induction Testing:

• Performed IPTG dose–response experiments to identify optimal induction levels without negatively impacting cell growth.

Biosensor Characterization:

• Exposed biosensors to varying putrescine concentrations, refined conditions to reduce clumping, and validated consistent fluorescence trends across replicates and culture formats.

Learn

Improvements:

• Identified unintended regulatory elements: the LacUV5 and reverse T7 promoters. These elements potentially affect system behavior and could contribute to unexpected expression patterns.

Optimization:

• Planned plasmid modifications to remove these extraneous promoters, refining construct design to ensure signal specificity and accurate characterization of the Putrescine-driven response.

Optical System

Reason

Objective:

• Develop a compact fluorescence optical system to accurately quantify GFP expression from putrescine-responsive bacteria.

Need:

• A system capable of exciting GFP at its optimal wavelength and capturing its fluorescence emission while minimizing ambient light interference and maximizing signal-to-noise ratio.

Research

Component Selection:

• Selected a high-power 465 nm monochromatic LED (OSRAM OSCONIQ P 3030) for GFP excitation, a 525 nm-specific photodiode (Marktech MTD5052D3) for emission detection, an aspheric lens for light focusing, and a HOYA Y52 longpass filter to reduce background interference.

Circuit Testing:

• Investigated photodiode sensitivity by building a simple op amp circuit (LMP7721, non-inverting configuration, gain = 2) with two 1 kΩ resistors to characterize response under controlled dark-box conditions.

Design

System Layout:

• Designed a compact layout positioning the excitation LED, aspheric lens, longpass filter, and photodiode to optimize GFP fluorescence detection while reducing direct LED leakage.

Thermal Management:

• Integrated a copper heatsink into the PCB-mounted LED design to manage thermal resistance during prolonged operation.

Build

Assembly:

• Assembled the optical components within a shielded enclosure to block stray light and preserve signal integrity.

Positioning:

• Mounted the 520 nm green LED directly above the sample chamber and aligned the 525 nm photodiode with focusing optics and filter to maximize emission capture efficiency.

Test

Calibration:

• Tested the system in a dark box, using a 520 nm green LED as a controlled excitation source to verify photodiode response and sensitivity.

Sensitivity Testing:

• Recorded photodiode voltage output while decreasing LED drive voltage in 0.1 V steps, producing a calibration curve of sensor response versus input light intensity.

Learn

Improvements:

• Identified the need for signal amplification between the photodiode and data acquisition system, addressed with an op amp gain stage.

PCB:

• Designed custom PCBs for stable LED mounting and wiring, improving reliability and ease of integration into the optical housing.

Hydrogel Tray and Mechanics

Reason

Objective:

• Develop a reliable housing method for putrescine-responsive bacteria that enables consistent daily sensing while preserving bacterial viability.

Need:

• A system that maintains living biosensors over multiple days, allows for simple handling by the user, and ensures correct alignment with the optical detection system.

Research

Hydrogel Formulation:

• Investigated alginate-based hydrogels for their ability to support bacterial viability while permitting diffusion of analytes needed for biosensing.

Cassette Design:

• Explored modular cassette approaches to allow multiple tests from a single preparation while minimizing contamination risk.

Design

Cassette Layout:

• Designed circular cassettes partitioned into seven compartments—one per day of the week—to provide daily testing without requiring hydrogel replacement.

Tray Mechanism:

• Created a sliding tray with an alignment notch to ensure cassettes are consistently and correctly positioned within the device.

Build

Fabrication:

• Manufactured hydrogel cassettes and trays using biocompatible materials that support bacterial function and withstand repeated handling.

Integration:

• Incorporated the cassette and tray system into the device housing, ensuring compatibility with the optical detection system and user workflow.

Test

Viability Testing:

• Confirmed that bacteria embedded in the alginate hydrogel remain viable and responsive across seven days of storage within the cassette.

Usability Testing:

• Verified that the sliding tray and notch system reliably aligns cassettes and simplifies hydrogel replacement by non-expert users.

Learn

Improvements:

• Identified the potential for expanding cassette designs to accommodate additional tests per week given long biosensor viability lifetime.

User Experience:

• Found that the sliding tray design significantly reduces handling errors, improving test consistency across users.

Graphical User Interface

Reason

Objective:

• Create an intuitive graphical interface that allows users to easily interpret biosensor outputs and track BV progression from FloraDX.

Need:

• A user-friendly display that can present complex fluorescence and pH data in a simple, actionable format, both on-device and via a companion app.

Research

User Needs:

• Studied how non-expert users interact with diagnostic devices, emphasizing clarity, minimal steps, and immediate interpretation of results.

Display Options:

• Compared different display technologies and identified the LILYGO T-RGB 2.1-inch LCD touchscreen as optimal for compact size, high visibility, and multi-color output.

Design

Interface Layout:

• Designed a touchscreen interface that displays numeric readouts for fluorescence intensity and pH alongside color-coded indicators (e.g., green = safe, red = alert).

Dual Interaction Modes:

• Developed both on-device GUI and a mobile app interface, ensuring consistency across platforms and giving users flexibility in how they access results.

Feedback Features:

• Incorporated simple prompts and confirmation messages on the LCD to guide users through testing steps and confirm cassette alignment or test completion.

Build

Integration:

• Embedded the LILYGO LCD touchscreen into the front panel of the device for direct access, wired to the microcontroller for real-time data transfer.

App Development:

• Built a companion app that syncs with the device via Bluetooth/Wi-Fi, allowing users to log and review historical test results.

Test

Usability Testing:

• Conducted trials with users to ensure the interface was clear, intuitive, and required minimal training to operate.

Performance Validation:

• Verified that the GUI correctly translated raw fluorescence and pH sensor data into accurate readouts and alerts.

Learn

Improvements:

• Determined that multi-color alerts alone were insufficient for some users, so numeric readouts and trend graphs were added for clarity.

User Experience:

• Found that combining simple color indicators with numeric precision boosted user confidence and improved repeatability in interpreting results.