Description

Problem/Abstract

With climate change increasing temperatures around the world, harmful algal blooms grow rapidly in waterways such as lakes and rivers. These blooms include blue-green algae, such as M. aeruginosa, which release harmful cyanotoxins such as microcystins. When ingested, microcystins can cause dangerous symptoms such as nausea, vomiting, liver failure, and even death. Smaller animals, such as dogs, are increasingly at risk due to their natural tendency to lick themselves after swimming, thus ingesting any harmful water toxins they might have encountered.

As the water becomes hotter due to climate change, algal blooms occur more often. This will expose more people and animals to harmful microcystins. As a result, developing safe and environmentally friendly ways to address 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 microcystins' first detection in Austin in 2019, toxic algal blooms have killed several dogs and even hospitalized a person in 2021. Current measures involve treating Lady Bird Lake with clay to reduce the amount of phosphorus that the algae “feed” on to bloom. The project is soon ending its run in 2026 with mixed results, with microcystin release being slightly reduced but not eliminated from the lake.

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). While these methods are effective, they can be expensive and require samples to be taken from the environment and tested inside a laboratory environment. Although bacteria such as Pseudomonas Aeruginosa have also been discovered to have the ability to degrade microcystins from its cyclic hepatotoxin form into linear non toxic substituents, little research has been done to implement these bacteria as a countermeasure to harmful algal blooms.

Our solution is to create an easy-to-use biosensor that can detect and potentially degrade microcystins in situ, therefore removing the requirement of training and expensive laboratory equipment. Utilizing previous research on biosensors, ADP1, and microcystins, our goal is to genetically modify Acinetobacter baylyi (ADP1), a naturally competent bacterium that has the inherent ability to transform extracellular DNA into its genome, to detect and degrade naturally occurring microcystins found in aquatic environments.

Our Goals

1. Producing in-house microcystins for testing

In order to reliably test ADP1's ability to sense microcystins, we decided to produce our own microcystins in-house by expressing the mcy genome within A. baylyi to produce MC-LR, the most toxic and most common microcystin strain found in algal blooms. These samples will provide a realistic positive control to test the effectiveness of our biosensors and degradation enzymes.

2. Creating microcystin-specific biosensors

To ensure the specificity of A. baylyi to the microcystin genome, we will incorporate a frameshifted coding sequence with homologous flanks matching ACIAD-2049, a common gene sequence found in MC-LR, into A. baylyi. The ADP1 cell will then exercise its natural competence to replace the “broken” sequence with a full in-frame ACIAD-2049, triggering a positive signal such as fluorescence or antibiotic resistance.

3. Engineer ADP1 to express microcystin-degrading enzymes mlrA and mlrD

Following detection of microcystins, we want ADP1 to utilize MC-LR specific enzymes that neutralize the toxic cyclic peptide into its less harmful linear substituents. We plan to do this via incorporation of both the mlrA and mlrA+mlrD genomes into ADP1, therefore expressing it. mlrD is a transporter protein that will engulf the microcystin into the cell, exposing it to the degrading enzyme, and mlrA is the enzyme that actively breaks the cyclic bond between the 4th position residue and ADDA residues.

4. Integrate and verify the effects of the Recombinase enzyme into A. baylyi

After successful verification of our engineered ADP1 biosensor, we want to examine the effects of Recombinase on ADP1's transformation ability. Recombinase is an enzyme that utilizes an ET pair exonuclease system to increase transformation frequency. This can be useful because in situ, mcy 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. Combine both DNA sensor and degradation

After both the biosensor and degradation parts are completed in separate areas of the A. baylyi genome, we plan to assemble both parts together. This final product would have the ability to both fluoresce in order to signal presence of microcystins inside water and subsequently degrade those detected microcystins into less harmful substituents.

Future Prospects

Having succeeded in creating a naturally competent biosensor inside A. baylyi, we hope that our research done in this project may serve as a blueprint and inspiration to future scientists pursuing environment and user-friendly methods of detection. Our modular design allows for detection of many different types of DNA unrelated to microcystins along with many different biosignaling cues other than antibiotic resistance or fluorescence.

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 harmful DNA or more specifically, cyanotoxins and microcystins, using Acinetobacter baylyi.

Summary

The UTexas 2025 team project focuses on engineering bacteria to express cyanobacteria detection and degradation abilities. The specific bacteria we are utilizing is the gram-negative Acinetobacter baylyi, or ADP1, due to the bacteria's inherent natural ability to transform extracellular DNA into its genome. As the project stands,we have created a microcystin (MC-LR) biosensor that expresses antibiotic resistance upon incorporation of the mcy gene cluster into ADP1, along with a safe and inexpensive way to grow microcystins in vitro, also through ADP1. Future plans involve incorporating the recombinase enzyme into our biosensor and investigating mlrA and mlrD expression inside A. baylyi.