Team:HKUST/Description

ShellBi

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Description

A Prokaryotic Biosensor for Shellfish Toxins

What It Means to Us...

Our home city, Hong Kong, is an archipelago of 236 islands. Surrounded by the South China Sea, it would not be difficult to see why seafood is an integral part of our diet! In terms of seafood consumption per-capita, Hong Kong ranks second in the whole of Asia.​ Each of us consumes 66.5 kg of seafood on average every year, which is more than three times the global average [1].​ And in 2020 alone, the estimated production was about 116,000 tonnes valued at $2.7 billion dollars [2]. Focusing on only one of these seafoods, shellfish is not only eaten here, but also harvested!

Shellfish is our territory’s hallmark and we wish to address the issues linked with what’s in the interest of so many Hongkongers! In Hong Kong, it is a popular hobby to harvest and eat fresh shellfish in public beaches - straight out of the ocean. Despite the attractive texture and flavour of fresh shellfish, we are aware of the different forms of poisoning that could be caused by them. From our survey it is concerning that some local farmers and most consumers are not even aware of the risks associated with eating raw shellfish. After interviewing government departments in Hong Kong responsible for fisheries (AFCD HK), we learn that there are barely any regulations in place to screen for shellfish toxins. So, this year, our interdisciplinary team of 14 dedicated students will utilize a synthetic biology approach to simplify the process of toxin detection in shellfish, ensuring health and safety for our local community!

Project Background

Shellfish, specifically bivalve mollusks such as clams, mussels, and oysters are filter feeders, which feed on small phytoplankton and zooplankton. With intensifying climate change, Harmful Algal Blooms (HABs) are occurring more frequently, making toxin-producing phytoplankton (algae and cyanobacteria) more abundant. Shellfish accumulate the toxins as they feed on the phytoplanktons. Some shellfish eliminate them as waste but some shellfish, for example butter clams, can accumulate these toxins as a defence mechanism for up to 2 years [3]!

Photo by Kai Schumann

Paralytic shellfish toxins, specifically saxitoxin, tetrodotoxin, and brevetoxin, are life threatening neurotoxins that cause Paralytic Shellfish Poisoning (PSP) by blocking the sodium channels in nerve cell membranes [4]. Symptoms of PSP range from lightheadedness, headache, and diarrhea, all the way to flaccid paralysis, respiratory failure, and even death [5]. The fatality rate of PSP varies on the dose consumed, could be as low as 6% or as high as 44% and mostly affects young children and people with poor access to healthcare the most [8].

CALL-TO-ACTION

“ Currently, there are no known methods available for detoxifying live contaminated shellfish in a way that is safe, fast and economically feasible, nor reliable quick test to determine the presence of PSP in shellfish flesh.”

- Centre for Food Safety, The Government of HKSAR

Most cases of shellfish poisoning may easily go unreported since mild reactions could be easily misconstrued as common food poisoning, and the treatment is almost always symptomatic. This issue is alarming since there is no antidote to paralytic shellfish toxins. Contaminated shellfish neither looks nor smells or tastes different. The toxin is also heat stable so regular cooking methods cannot eliminate the toxin [5]. So, the best option is to prevent humans from eating it.

One such case was reported very recently by the Centre for Health Protection, HK. The headline in the figure reports “One male and female purchased conches online, got blurry eyesight and felt dizzy after consuming. Suspected of catching shell toxin with numbing properties”.

Current Methods

The current methods used for detection of Paralytic Shellfish Toxins are the Mouse-Poison Bioassay (MBA) and High Performance Liquid Chromatography (HPLC). Both these methods suffer major drawbacks either in terms of ethics and sensitivity, or in cost and accessibility.

MBA

This method, accepted as the “gold-standard of toxin testing” by the FDA and still widely used in developing countries and low budget detection centres, is a qualitative toxicological screening technique. Mice are injected with the food extract and their resulting death or survival is an indication of if the food is safe to be consumed. The method has been conducted without optimisation or universal protocols leading to a “gross misuse” of laboratory animals and of scientific methodology [6]. Atypical results arise due to procedural artifacts and the method is unequivocally unethical [7]. Regardless, this method is still widely conducted, especially on harvest for export purposes given that most countries acknowledge this assay while the other non-animal alternatives might be debated still.

Advantages

✓ Easy to perform
✓ Direct readout
✓ Fair estimation of effects on human consumers

Disadvantages

✗ Unethical
✗ Slow and low throughput
✗ Non-specific
✗ Low sensitivity
✗ Low reproducibility

HPLC

This is a widely adopted equipment in analytical chemistry. HPLC can separate, identify, and perhaps even quantify compounds in a sample at high accuracy. The equipment could be used to analyse shellfish samples for the presence of toxins. However, this high-end analytical equipment faces challenges in regards to acceptance for frequent use or in low-budget testing centres.

Advantages

✓ Ethical
✓ High throughput
✓ High sensitivity and accuracy

Disadvantages

✗ Very expensive
✗ Requires intensive training and lengthy procedures
✗ Not a bioassay, cannot draw relations of impact on humans

Note: Despite known demerits of a mouse bioassay, it still remains as a gold standard due to the fact that some analogues of marine biotoxins of the DSP-type are not available as reference substances, and so the alternative methods can not be validated. In the EU at least, the mouse bioassay is unable to be replaced by other sophisticated methods [9].

Project Inspiration

We decided to create a prokaryotic biosensor for the detection of Paralytic Shellfish Toxins (PST) that would offer the best of both worlds. This biosensor would combine the advantages of both, mouse bioassay and HPLC being ethical, easy to use, and that could test samples at a large scale with a predicted high sensitivity.

Our biosensor, nicknamed ShellBi, is heavily inspired by a paper published by Pomati et al. (2004) titled “Effects of Saxitoxin (STX) and Veratridine Antagonism on Prokaryote Na+/K+ Influx- A prokaryotic based STX bioassay”. In this paper, the author describes the response of prokaryotic organisms (C. raciborskii and A. fischeri) towards the paralytic shellfish toxin — saxitoxin. Generally, prokaryotes are not used in studies relating to neurotoxins since they do not quite respond to the toxin drugs the same way as eukaryotes (humans) do. However, this research, led by Dr. Brett Neilan, indicated that the response of certain prokaryotes towards PSTs can be made observable if the effect of the toxin is antagonised [10]. In the study, the prokaryotic species responded to the PST saxitoxin upon being treated with an antagonist drug, Veratridine. This made it possible to use prokaryotes for our chassis (For detailed interaction, see Engineering”).

Engineering page

Our biosensor builds on the ideas highlighted in the study and engages the two component system from E. coli to a direct readout for the presence of paralytic shellfish toxins. The cells will appear green if there is no toxin in the shellfish sample. Conversely, if the toxin is present, the cells will glow red.

How ShellBi Works…

Paralytic Shellfish Toxins block ion channels of prokaryotes. Veratridine (VTD) is an antagonist drug that opens the channels, causing an ion influx. According to the aforementioned paper [10], when cells are exposed to veratridine, there is an immense ionic flux into the cell leading to metabolic and defects. On the contrary, if saxitoxin (a PST) is exposed to prokaryotic cells, even after the addition of Veratridine, the intracellular Na+ and K+ ions reduce in concentration, essentially rescuing the cells.

We have engineered the Two Component System in E. coli bacteria to respond to these intracellular ionic changes. We express reporter fluorescence proteins under relevant promoters- OmpF and OmpC promoters that are activated at different osmolarities. If PST is present, the ion concentration reduces and the promoter for low osmolarity gets activated. If PST is not present, VTD will cause cells to undergo an ion influx, increasing intracellular osmolarity, leading to the expression of reporter under promoter for high osmolarity. The cells eventually glow red or green. Red for toxin present, green for toxin absent.

ShellBi advantages

✓ Ethical
✓ Cheap
✓ High throughput
✓ Easy implementation
✓ Detection of wide range of toxins
✓ Direct indication of biological response

References:

1. “Sustainable Seafood.” World Wide Fund for Nature, WWF HK.

2. “Capture Fisheries.” Agriculture, Fisheries and Conservation Department, The Government of Hong Kong Special Administrative Region, 2017.

3. Clark, R F et al. “A review of selected seafood poisonings.” Undersea & hyperbaric medicine : journal of the Undersea and Hyperbaric Medical Society, Inc vol. 26,3 (1999): 175-84.

4. “Paralytic Shellfish Poison (PSP)“. Doh.Wa.Gov, 2021.

5. “Food Contaminants- Paralytic Shellfish Poisoning (PSP) Toxins”. Centre for Food Safety, The Government of Hong Kong Special Administrative Region, 2007.

6. Combes, Robert D. “The mouse bioassay for diarrhetic shellfish poisoning: a gross misuse of laboratory animals and of scientific methodology.” Alternatives to laboratory animals : ATLA vol. 31,6 (2003): 595-610.

7. Mouse Bioassay Not Suitable As A Reference Method For The Regular Analysis Of Algae Toxins In Mussels. Federal Institute Of Risk Assessment, 2005, pp. 1-7.

8. Ansdell, Vernon. "Seafood Poisoning". Travel Medicine, 2019, pp. 449-456. Elsevier.

9. Application of six detection methods for analysis of paralytic shellfish toxins in shellfish from four regions within latin america. (2020). Marine Drugs, 18(12), 616.

10. Pomati, Francesco et al. “Effects of saxitoxin (STX) and veratridine on bacterial Na+ -K+ fluxes: a prokaryote-based STX bioassay.” Applied and environmental microbiology vol. 69,12 (2003): 7371-6.

PROJECT

• Description
• Design
• Engineering
• Proof of Concept
• Implementation
• Safety
• Contribution
• Notebook

WETLAB

• Experiments
• Results

DRYLAB

• Model

REACH

• Human Practice
• Collaboration
• Communication
• Education
• Sustainable

PARTS

• Basic Parts
• Composite Parts

TEAM

• Team
• Attributions