Introduction

BostonU iGEM 2025 set out to develop an at-home monitoring system for bacterial vaginosis (BV) using the tools of synthetic biology. After conversations with medical professionals and evaluating the current literature, the team identified putrescine as a relevant and measurable biomarker for vaginal microbiome health. A repressor circuit from the literature that is induced by putrescine was adapted for our use and built into a whole-cell biosensor in E. coli. That biosensor can then be suspended in a hydrogel for use with our combined hardware-biology platform. The device is able to monitor for bacterial vaginosis with better specificity and accuracy by encoding the logic of Amsel’s criteria, the current favored method for diagnosing BV, in the device using an electronic pH sensor.

Using this device, we can bring at-home vaginal microbiome testing to users at a relatively low cost. Our hardware and circuit design make this modular platform a jumping-off point for at-home vaginal health monitoring, enabling future iGEM teams to push forward in critical areas of women’s health research.

Putrescine Detection

Using a circuit design inspired by Selim et al. (2022) [1], the team successfully designed, constructed, and cloned a putrescine sensing genetic circuit (Figure 1) in NEB® 5-alpha Escherichia coli, confirmed by whole-plasmid sequencing. An additional construct with a second constitutive promoter instead of the repressed pHyb(1B) promoter was also constructed as a positive control.

The plasmids were then cloned into BL21(DE3) E. coli for testing and the sensing circuit was able to detect levels of putrescine down to 10 mM from stock samples in LB media. The circuit produces a quantifiable response in 10 hours.

Hydrogel Suspension

To deploy whole-cell biosensors in an at-home device, the team turned toward agar hydrogels as a medium for sustaining bacterial viability. Testing in the agar hydrogels was carried out in an internal E. coli strain that constitutively expresses eGFP under the strong pVeg1 promoter. We first tested the effect of resuspension in heated media, a requirement for the agar solids, on growth and fluorescence in regular LB liquid media. The results indicate that there was almost no effect on growth over 60 hours. However, there was a small effect on activity after around 20 hours, indicating a small loss in signal for our use case.

We tested different media conditions and found that standard LB nutrient concentration and 1% (w/v) agar were ideal for maintaining growth and signal strength. E. coli resuspended in PBS grew almost as well as cells in regular LB media, but their fluorescence expression remained lower than those in LB over the course of the experiment. The GFP signal strength was significantly lower in the hydrogel wells than in the liquid media, likely due to interfering effects of the solid agar matrix. Because of this, we stuck to 1% or 1.5% (w/v) agar for future experiments.

To test how well the agar hydrogel sustained E. coli viability over time, we took our optimal hydrogel conditions and suspended cultures for one week. After around 24 hours, the GFP signal strength plateaued and remained stable over the course of the week. This shows that agar hydrogels are a viable option for using whole-cell biosensors in deployable devices. There were some issues with agar hydrogels drying out over the course of the experiment. We found that covering the gels completely helped in most cases, but future testing is needed to optimize the size and conditions of the hydrogel.