Project Description
Project Description
In the summer of 2022, a devastating ecological disaster occurred on the Oder River. This major waterway in Central Europe flows through the Czech Republic, Germany, and Poland, where our team is based. It is Poland's second longest river, an important reservoir for biodiverse wildlife, and a critical waterway for supporting fishermen and their livelihoods. For many, the river represents not only a natural resource, but also a cherished part of their cultural heritage.
The disaster marked one of the most severe environmental crises ever witnessed in the history of European rivers. The scale of the catastrophe was staggering: fish mortality along the entire 560 km stretch of the affected river was estimated at 1650 tons, which constituted a 60% decline in the fish population from pre-disaster levels [1].
For a long time, there was significant misinformation about the causes of the disaster, which created unease within the local community. Many potential causes were proposed, including sewage dumps or mercury compounds, but due to slow detection systems, nothing could be said for sure while the Oder ecosystem suffered irreversible damage.
Eventually, the culprit was identified as Prymnesium parvum, commonly known as "golden algae," which is an algal species capable of causing harmful blooms [1]. Prymnesium parvum produces toxins that affect organisms with gills, often leading to large-scale fish kills.
Despite the existence of a number of rapid, molecular methods for detecting organisms in environmental samples, golden algae are still most widely identified on the basis of morphological features observed under a microscope[2]. This approach requires time, access to advanced equipment and specialized knowledge, all of which contribute to slow detection times and delays in preventive action. It is possible to detect the algae using PCR, but this method necessitates a thermocycler, making it impractical for field use.
The need to address the problem of inefficient detection has never been more urgent, as the threat of Prymnesium parvum blooms continues to loom over not only the Oder river, but also neighboring water bodies. Year after year, blooms keep reoccurring, posing a threat to previously unaffected ecosystems. After conversations with specialists and local community members, we realized that a faster, and more user-friendly detection method is a must.
Our project aims to create an easy-to-use lateral flow test that can quickly detect the presence of golden algae in water samples directly in-field. This test would be accessible to anyone interested in monitoring local water conditions and serve as a rapid screening tool for government agencies.
We want to further develop our test by designing a 3D-printed device and corresponding software that will allow quantitative readouts of fluorescence using a cell phone camera.
We will develop a platform that simplifies the creation of similar tests, thereby contributing to the synthetic biology community. By making detection with Cas13a proteins more accessible, we aim to support scientists worldwide and future iGEM teams, regardless of the target agent they want to detect.
Finally, we want to raise awareness about the environmental threat posed by golden algae blooms and involve the local community in discussions on how to prevent such disasters in the future. Our goal is to include all voices to protect and preserve the beautiful aquatic ecosystems in Poland and beyond.
Our project aims to apply the SHERLOCK (Specific High-Sensitivity Enzymatic Reporter Unlocking) method for the detection of Prymnesium parvum DNA in water samples [3]. In particular, we want to focus on the detection of the Oder strain of this organism.
The SHERLOCK platform is a modern synthetic biology tool that takes advantage of the properties of the Cas13a protein, an enzyme in the family of proteins used in the Nobel Prize-winning CRISPR-Cas system. The Cas13a protein is guided with high specificity to the sequence we want to detect, using crRNA.
The crRNA molecule is key to the assay's specificity. It consists of a DR (direct repeat) sequence and the spacer sequence, which is complementary to the target. When generating crRNA from DNA templates through in vitro transcription, the T7 polymerase promoter is placed upstream of the crRNA sequence, and the entire sequence is ordered as the DNA reverse complement. The crRNA molecule can be designed to uniquely identify the organism in question by targeting the ITS (Internal Transcribed Spacer) sequence in its genome [4].
Once bound to an organism's genetic material, the Cas13a protein becomes activated and exhibits a so-called “collateral” RNase activity, meaning that it non-specifically cleaves nearby single-stranded RNA molecules [3].
We take advantage of this activity in our assays by including synthetic RNA probes tagged with a fluorescent reporter and a quencher in the reaction mixture. If we see a fluorescent signal, it means that a portion of the reporters have been cleaved by Cas13a, indicating the DNA target was in the sample. This readout method relies on access to a fluorometer, so it is best for laboratory applications.
First, after collecting the water sample, we isolate the organism's DNA using either a genomic DNA isolation kit or an in-field method.
Then, to enhance the signal, we amplify the target DNA using recombinase polymerase amplification (RPA). This technique allows us to increase the DNA concentration under isothermal conditions, eliminating the need for a PCR thermocycler. In RPA, recombinase molecules help stabilize the bond between the primers and the amplified DNA, enabling the reaction to proceed even at temperatures below 37 °C.
Once the amplification is complete, we introduce all the SHERLOCK reaction components, including T7 RNA polymerase, which transcribes the target DNA into RNA. This step activates the crRNA:Cas13a complex, and the activated Cas13a protein starts cleaving RNA probes in the reaction mix.
For in-field testing, it's important to have a test readout format that doesn't rely on complex fluorimetric equipment. To meet this need, we developed the PrymFlow Lateral Flow Assay (LFA). Our goal was to create a portable detection system that's easy to use in the field, allowing for straightforward, qualitative testing with simple strips.
In PrymFlow, we use RNA reporters labeled with 6-Carboxyfluorescein (FAM) on one end and biotin on the other. This test is similar to those used for pregnancy or COVID-19 but differs in result interpretation.
Here, if the target is present, the T-line becomes visible, indicating a positive result. Meanwhile, the C-line serves as a control that is less visible when greater amounts of the target sequence are present. Gold nanoparticles (GNPs) with anti-FAM antibodies provide the visual indication of the test result.
Streptavidin, immobilized on the C-line, captures the biotin-labeled ends of the intact reporters. The reporters are captured on the C-line, and the binding of gold nanoparticles (GNPs) with antibodies makes only the C (control) line visible.
The reporters are cleaved by the activated Cas protein. As a result, GNPs with anti-FAM antibodies capture the FAM-labeled fragments, resulting in a strong T-line signal. This strong T-line indicates a positive result for Prymnesium parvum presence.
In summary, we aimed to create a test that can be used in the field to quickly and effectively screen water bodies for the presence of Prymnesium parvum.
Due to climate change and human activities, Harmful Algal Blooms (HABs) have become an increasingly urgent problem in today's world. These blooms not only threaten aquatic ecosystems globally but also negatively impact economies. Many different species of algae are capable of forming such blooms, with strains differing genetically, necessitating the rapid development of tools to detect them [5]. We believe that standardization, which lies at the heart of synthetic biology, can help solve this problem.
That's why we're developing SynLOCK—crRNA Synthesis System for SHERLOCK. Our goal is to make it easier for scientists and future iGEM teams to use Cas13 proteins for detection, no matter what they’re trying to find.
Our solution simplifies the process for scientists who need to order crRNA, whether as full-length RNA or a DNA template, whenever a new target sequence needs to be detected. With the current method, reordering is necessary when supplies run low, leading to time and cost inefficiencies. Most importantly, our approach streamlines the generation of new crRNA molecules and ensures the process is straightforward and easy to understand.
Our system introduces a DNA fragment, called a cassette, into a typical RFC10 and RFC1000 compatible plasmid backbone to allow for maximal flexibility of user cases. The cassette already contains the T7 promoter for in vitro transcription and the DR sequence compatible with the Cas13a protein, enabling scientists to only design the custom spacer sequence for their detection target.
The cassette also includes a purple chromoprotein reporter, assembled from basic parts, between the SapI cutting sites, allowing for easy insertion of custom spacer sequences using the SapI enzyme and straightforward screening of the colonies that have accepted the spacer.
Using the Type IIS standard avoids introducing any scars during assembly, ensuring we obtain the exact crRNA template sequence of our choice. The cassette also contains a BbsI cutting site to linearize the plasmid before in vitro transcription, ensuring the crRNA is of the correct length.
In summary, our platform allows scientists to rapidly amplify a plasmid containing all necessary system components in E. coli and then use Type IIS assembly to clone in a short sequence they want to detect. It allows for easy visual screening of the correct colonies and ordering of shorter sequences compared to the full crRNA template. This makes the process of generating crRNA cheaper and more streamlined.
We believe that our highly standardized and customizable system has strong potential to be a building block in creating fast and reliable detection tools by future iGEM teams and scientists worldwide.
In the future, we envision the result of our project as a set of easily accessible tools that bring together local community members, including fishermen, scientists, and policymakers, to swiftly detect Prymnesium parvum’s threats and take proactive steps towards their prevention.
We believe in the power of biology as an engineering discipline to enable us as scientists to tackle new issues as they arise. Together, we can restore a healthy environment by bringing science and community together to protect our waters. We're committed not only to conserving our planet, but also to shaping a future where innovation and environmental stewardship go hand in hand.
[1] Szlauer-Łukaszewska, Agnieszka, Łukasz Ławicki, Jacek Engel, Ewa Drewniak, Karol Ciężak, and Dominik Marchowski. “Quantifying a Mass Mortality Event in Freshwater Wildlife within the Lower Odra River: Insights from a Large European River.” Science of The Total Environment 907 (2024): 167898. https://doi.org/10.1016/j.scitotenv.2023.167898.
[2] Luo, Ningjian, Hailong Huang, and Haibo Jiang. “Establishment of Methods for Rapid Detection of Prymnesium Parvum by Recombinase Polymerase Amplification Combined with a Lateral Flow Dipstick.” Frontiers in Marine Science 9 (October 24, 2022): 1032847. https://doi.org/10.3389/fmars.2022.1032847.
[3] Kellner, Max J., Jeremy G. Koob, Jonathan S. Gootenberg, Omar O. Abudayyeh, and Feng Zhang. “SHERLOCK: Nucleic Acid Detection with CRISPR Nucleases.” Nature Protocols 14, no. 10 (October 2019): 2986–3012. https://doi.org/10.1038/s41596-019-0210-2.
[4] Binzer, Sofie Bjørnholt, Daniel Killerup Svenssen, Niels Daugbjerg, Catharina Alves-de-Souza, Ernani Pinto, Per Juel Hansen, Thomas Ostenfeld Larsen, and Elisabeth Varga. “A-, B- and C-Type Prymnesins Are Clade Specific Compounds and Chemotaxonomic Markers in Prymnesium Parvum.” Harmful Algae 81 (January 2019): 10–17. https://doi.org/10.1016/j.hal.2018.11.010.
[5] Durán-Vinet, B, K Araya-Castro, Tc Chao, Sa Wood, V Gallardo, K Godoy, and M Abanto. “Potential Applications of CRISPR/Cas for next-Generation Biomonitoring of Harmful Algae Blooms: A Review.” Harmful Algae 103 (March 2021): 102027. https://doi.org/10.1016/j.hal.2021.102027.
