Description

The Problem

With rising global temperatures, harmful algal blooms are becoming increasingly frequent in lakes and rivers. These blooms often include Microcystis aeruginosa, a cyanobacterium that produces microcystins, cyclic hepatotoxins capable of causing nausea, liver failure, or even death upon ingestion.¹ Animals such as dogs and livestock are especially vulnerable, as they have been observed consuming cyanobacteria-infested water despite access to clean sources.¹

As warming intensifies, so will the frequency of harmful algal blooms, exposing more people and animals to harmful microcystins.⁷ As a result, developing safe and environmentally friendly methods to detect and mitigate microcystins has become a growing priority.

Our Inspiration

Located just fifteen minutes from our campus, Lady Bird Lake is a 400-acre reservoir serving as a major recreation center for Austinites who enjoy paddleboarding, watersports, or taking their pets outside. Since 2019, cyanobacterial blooms there have caused multiple dog fatalities and at least one human hospitalization.² Ongoing remediation efforts (such as phosphorus-reducing clay treatments) have yielded only partial success, reducing bloom density but failing to fully eliminate cyanotoxins.³ Witnessing this persistent problem in our own community inspired our team to develop a biological platform that could both detect and degrade these toxins at the source.

Our Solution

Current methods for detecting microcystins utilize complex techniques that often require training such as Enzyme-Linked Immunosorbent Assays (ELISA) and Polymerase Chain Reaction (PCR).⁵ While these methods are effective, they can be expensive and require samples to be taken from the environment and tested inside a laboratory environment. Microcystin-producing DNA is also degraded over time, reducing the amount able to be easily detected. Although bacteria like Pseudomonas aeruginosa have also been discovered to have the ability to degrade microcystins from its cyclic hepatotoxin form into linear non toxic substituents,⁶ little research has been done to implement these bacteria as a countermeasure to harmful algal blooms.

To address this, we are engineering Acinetobacter baylyi ADP1, a naturally competent bacterium capable of integrating extracellular DNA directly into its genome,⁸ to serve as a modular biosensor and degrader. Our goal is a living system that can detect environmental DNA (eDNA) from microcystin-producing cyanobacteria and subsequently degrade the toxin itself, providing an inexpensive, deployable monitoring tool.

Figure 1. Detection of mcy eDNA using ADP1 biosensor. The homologous recombination schematic is simplified, refer to Figure 2 for a more detailed graphic.

Our Goals

1. Engineer microcystin-specific biosensors

To ensure the specificity of A. baylyi ADP1 to the microcystin genome, we will incorporate a “broken” mcy coding sequence containing a 10 base-pair deletion inside ADP1 in place of ACIAD-2049, a nonessential gene inside the A. baylyi genome. The ADP1 cell will then exercise its natural competence to identify the matching homology flanks and replace the frameshifted coding sequence inside ADP1 with a full, in frame mcy coding sequence. The removal of the 10 base-pair deletion will correct all downstream elements, then expressing a positive signal (eg. fluorescence or antibiotic resistance.)

Figure 2. Detailed Schematic of Homologous Recombination into ADP1.

2. Incorporate microcystin-degrading enzymes (MlrA and MlrD)

Following detection of environmental DNA (eDNA) from the mcy gene cluster in M. aeruginosa, we want ADP1 to utilize two enzymes from Sphingopyxis sp. that cleave the toxic cyclic microcystin into its less toxic linear form by a factor of around 1000.⁶ We plan to do this via incorporation of both the mlrA and mlrD gene sequences into ADP1, which will then be expressed. MlrD is hypothesized to transport microcystins into the periplasm, where MlrA cleaves the cyclic bond between the ADDA residue and the fourth amino acid, reducing toxicity by approximately three orders of magnitude.⁶

3. Produce microcystins in-house for validation

To reliably test ADP1's ability to detect microcystin-producing DNA and degrade microcystins, we aim to produce our own microcystins in-house by expressing the mcy gene cluster within A. baylyi ADP1 to produce MC-LR, the most commonly found microcystin in blue-green algal blooms.⁶ These samples will provide a reproducible, positive control for both biosensor calibration and degradation assays.

4. Enhance transformation efficiency with recombinase expression

Separately, we want to examine the effects of the RecAB recombinase system on ADP1's transformation frequency using smaller homology flanks. RecAB is a modified version of the RecET protein pair designed to be inducible and compatible with Acinetobacter baumannii.⁹ RecE is an exonuclease that exposes 3' single-stranded DNA to RecT, a single stranded binding protein that catalyzes recombination of the extracellular strand.¹⁰ The sped up reaction allows for increased transformation frequency using shorter homology flanks. This can be useful because in the environment, extracellular DNA is naturally degraded and the genome is often not available in its full length. Increased transformation frequency would improve detection of mcy in its shorter, degraded form.

5. Integrate biosensing and degradation modules

Once validated individually, we aim to combine both the sensing and degradation systems into a single A. baylyi ADP1 strain. This final construct will both signal the presence of microcystin-producing DNA upon detection and enzymatically degrade the associated toxins.

Future Prospects

Having succeeded in creating a naturally competent biosensor using A. baylyi, we hope that our research may serve as a blueprint for future scientists pursuing environment and user-friendly methods of detection pathogen or toxin detection. Our modular design can be easily adapted to detect many different types of DNA unrelated to microcystins. The modular design can be easily re-purposed to detect other environmental DNA sequences, such as antibiotic resistance genes, pathogens, or pollutant indicators, by simply swapping the homology flanks of the sensing region.

Due to time constraints, we were unable to insert the microcystin degrading enzymes mlrA and mlrD into the genome alongside the DNA sensing portion. We hope future teams continue our goal of synthesizing a composite part that is able to both detect and degrade cyanotoxins or more specifically, microcystins, using A. baylyi.

Summary

The 2025 Austin-UTexas iGEM team is engineering Acinetobacter baylyi (ADP1), a naturally competent Gram-negative bacterium, as a chassis for microcystin detection and degradation. Leveraging ADP1's ability to integrate extracellular DNA (eDNA) via homologous recombination, we constructed a biosensor to be embedded within its genome, targeting the mcy gene cluster from Microcystis aeruginosa. Successful recombination with the target sequence restores a disrupted reading frame, activating a selectable marker and confirming detection of microcystin-producing DNA.

In parallel, we developed an ADP1-based platform for safe, cost-effective in vitro microcystin production, enabling standardized positive controls for biosensor testing. Future work will focus on integrating a recombinase system to enhance recombination efficiency with short or degraded DNA fragments, and on expressing the microcystin-degrading enzymes MlrA and MlrD from Sphingopyxis sp. Together, these efforts aim to establish A. baylyi ADP1 as a modular chassis for environmental DNA sensing and detoxification.