Team:HKUST/Human Practices

ShellBi

Project
Description Design Engineering Proof of Concept Implementation Safety Contribution Notebook
Wet Lab
Experiments Results
Dry Lab
Model
Reach
Human Practice Collaboration Communication Education Sustainable
Parts
Basic Parts Composite Parts
Team
Team Attributions

Human Practices

Connecting Our Project to the World

Overview

From the start, we knew the importance of bringing our science research to the outside world. By integrating Human Practices to every aspect of our project, we were able to finetune our biosensor design for the people it affects the most: the stakeholders of the shellfish industry. Here is our timeline

Learn more about our journey from learning to educating below

IHP

Education

Collaboration

Integrated Human Practices

We wanted to create a Prokaryotic Biosensor for the detection of Paralytic Shellfish Toxins. Knowing that we could not separate our project from the outside world, we embraced the shellfish industry as a global giant, and reached out to the chain of stakeholders from farmers to consumers and everyone in between. In order to fully understand the scope of the problem we are combating, we needed to communicate with these stakeholders throughout the whole process of ideating and developing our biosensor. These individuals include:

1. Experts in the field of shellfish toxins

We reached out to many scholars who study the Paralytic Shellfish Toxins (PST) to gain guidance with regards to the biotechnological design and developing a proof-of-concept for our product. They were able to give us advice specific to our project, which is more effective than only relying on literature.

Throughout our project, we have communicated with professors with different areas of expertise.

Qinglu Zeng | Hong Kong

Qinglu Zeng is an associate professor at the Hong Kong University of Science and Technology in the Department of Ocean Science, as well as the director of the Marine Microbial Ecology Laboratory. His focal point of research is on Cyanobacterium, Cyanophage, and Marine Ecology, and has had 36 publications in this field.

We reached out to Prof. Zeng as we were trying to consider a viable chassis for our biosensor. In our meeting, he had advised us to not use Cyanobacteria, due to limitations offered by their slow growth rate as compared to E. coli and also care and handling of appropriate culture conditions. Engineering anything with a slow doubling time is tedious, and by nature of our project and the competition, most importantly, we wished to get as much data as possible in regards to the engineering rather than spending time optimising growth conditions of the chassis. Key takeaway

• ❒ Eliminate Cyanobacteria species as a chassis choice

Engineering page

Stanley Lau | Hong Kong

Stanley Lau is an associate professor in the Department of Ocean Science at the Hong Kong University of Science and Technology. His focal point of research is in ​​Marine Bacteria and Environmental Microbiology. He has had 76 publications on a variety of topics related to ecosystems.

We found out that Prof. Lau worked with Vibrio fischeri, a gram-negative bacteria commonly found in marine environments. Our iGEM team reached out to him to see if V. fischeri would be a viable chassis for our project as there was literature evidence for our drug antagonism concept working in this organism.

Prof. Lau advised us that, even if using a particular bacteria is possible, it may not be the best option for us. V. fischeri is a model organism for studies involving biofilms and quorum sensing, however, besides being able to maintain the species, we also need to first knockout its native bioluminescence and then further engineer it. All of this would take enormous time and effort and at our level, it would not be suggested to move a whole circuit native to another organism in another chassis. Key takeaway

• ❒ Eliminate V. fischeri as a choice of chassis

Aifeng Li | China

We had the opportunity to interview professor Aifeng Li from the Ocean University of China. He has authored and co-authored 58 scientific journals. His expertise is in Harmful Algal Blooms, Phycotoxins, and Seafood Safety, which we thought was extremely relevant to our project direction.

Our meeting with Prof. Li was on July 5th. We spoke about the significance of the toxins and how our biosensor would be able to help mitigate the risks associated with Shellfish Poisoning. He himself works on the HPLC method for identifying and researching PSTs and he helped us compare this technology to other existing methods. We discussed with him the repercussions of the Mouse-Poison Bioassay and he informed us that despite being unethical and not quite reproducible, a bioassay is a more desired method for food quality assurance agencies to employ since a bioassay could tell the exact response a complex biological entity would have towards a toxin. An isolated, in vitro method could never allow us to draw such an assumption. This gave us confidence on our project ShellBi, which also offers a direct receptor-based bioassay. He was pleased with the idea of having a ‘yes/no’ system for detection of PSTs if it was targeted to farmers and other public, but also mentioned that if the target was to be expanded to researchers, it would be more favourable to make the biosensor signal more quantitative.

He also was concerned about false positives and false negatives with a biosensor. In his experience working with the highly sensitive HPLC, in order to trust a biosensor, he would be skeptical about false results. This got us thinking about incorporating the antisense RNA mechanism to suppress noise in the circuit and prevent the overlap of signals, i.e. have discrete ‘red’ and ‘green’ signals to indicate the presence and absence of toxin, respectively. Key takeaway

• ❒ Made us aware of the advantage of our project: being a bioassay
• ❒ We incorporated an antisense RNA mechanism to prevent false results from the biosensor

Engineering page with Antisense RNA information

Brett Neilan | Australia

One of the papers we based our project on was titled “Effects of saxitoxin (STX) and veratridine on bacterial Na+ -K+ fluxes: a prokaryote-based STX bioassay”, co-written by Professor Brett Neilan. It made us aware of the Saxitoxin-Veratridine antagonism that our project exploits.

Brett Neilan is a molecular biologist and microbial chemist, affiliated with the University of Newcastle. He has authored and co-authored 432 scientific journals, and his main focus is on Toxin Biosynthesis and Synthetic Biology.

After reaching out to him, we have had monthly virtual meet ups with Brett Neilan since our first contact in July. He was keen to discuss our project with us and to see our progress.

At the time of our first meeting on July 8th, our project was still focussed heavily on only the detection of Saxitoxin, as that was the Shellfish Toxin in the paper that had the antagonism with Veratridine. However, Professor Neilan pointed out that - since all Paralytic Shellfish Toxins work on interfering with the sodium channels in neurons - our biosensor should be able to test for a wider variety of toxins if we shifted our focus. We also noticed that Tetrodotoxin (TTX), another type of Paralytic Shellfish Toxin, was more prevalent locally, and decided that widening our target toxin would benefit our project.

In our second meeting on August 10th, he recommended that in order to find a potential chassis, we create a phylogenetic tree with different bacterial species, matching them based on the similarity of their voltage-gated ion channels. Before the meeting, we conducted a BLASTp search (sequence alignment program) in many bacterial species against nav1.7 human sodium channels, as we wanted to know if any bacteria expresses a channel that is similar to nav1.7. We found the results to be inconclusive. When bringing this up, Prof. Neilan suggested using a phylogenetic tree. This is so that we can see the evolutionary relationship between the voltage-gated sodium channels in different organisms.

For our phylogenetic tree, we conducted our own secondary meta-analysis, and picked out sodium, potassium, and calcium channels from many different organisms that were both prokaryotic and eukaryotic. We conducted a phylogenetic analysis that took us about 3 weeks to generate significant results. Between our second and third meeting with Dr. Neilan, we had maintained continued communication. After showing him our first tree, he recommended that we run 1000 times more “bootstraps” on a few specific organisms, and see if we could pin-point a specific chassis to use.

Our third meeting was on September 10th, and after updating Prof. Neilan in regards to our progress in wet lab and dry lab, we wanted to know what our phylogenetic tree’s results meant for our project. Some were Archaea bacteria, Cyanobacteria, but there was one that stood out for us: Bacillus halodurans. From this meeting on we wanted to use B. halodurans as our potential chassis, as at that time it seemed to be the best choice.

In our final meeting on October 7th, we gave the last update in regards to our project. We were happy to know that Prof. Neilan was genuinely interested in testing our circuit as they have access to the Paralytic Shellfish Toxins that Hong Kong does not allow us to use. We could send them our circuit for testing with the toxins, signing an MTA and an NDA to safeguard our project. More information click below link.

Also, after months of trying to find a suitable chassis for our biosensor, we found out that we could use E.coli. Conducting our own research and analysis for this justification, we found that E. coli would be the most suitable chassis for our project. We have linked our reasoning here :  

Engineering: choice of chassis

Key takeaway

• ❒ Shifted initial focus from detection of only Saxitoxin to detection for most Paralytic Shellfish Toxins
• ❒ The ideal chassis for us to use → E. coli
• ❒ Created a phylogenetic tree

With the advice and comments given to us from these various sources, we became more confident with our project, our choice of chassis, and added changes into our circuit design.

Antisense RNA information

Chassis information

Although they are experts in their scientific field, we wanted to know what our home city, Hong Kong, does about the issue of Paralytic Shellfish Poisoning. Hence the next link in the industry:

2. Government bodies which focus on safety regulations for food and the environment

To further understand how the Government is managing PSTs as well as the current significance of PSTs in Hong Kong, we reached out to government bodies who may give us insight into the limitations of current regulations and how our product could help overcome them.

Aquaculture Fisheries Division | Hong Kong

We interviewed Dr. Chung Wai Ki, Terence from the Fisheries and Conservation Department Headquarters in Hong Kong. Dr. Chung is part of the Aquaculture Fisheries Division. Our main take-away was there are no current laws for testing of PSTs in Hong Kong. They do not test directly for toxins in shellfish, but only determine the concentration of algae/ organisms, then assuming a high concentration correlates to the presence of toxins.

On July 8th, we learnt that there were limited laws and regulations in place in Hong Kong in regards to PSTs, or any other shellfish toxin. We realised that, especially in Hong Kong, there was a “gap in the market” in regards to toxin testing for shellfish, as the government themselves do not necessitate conducting these types of tests regularly.

After our meeting, we found a statement from the Centre for Food Safety on the website of the Government of HKSAR, saying “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.” That reconfirmed our faith of being able to fill in a spot in the Hong Kong market, as our proposed solution is safe, fast, affordable, and direct testing of Paralytic Shellfish Toxins in shellfish. Key takeaway

• ❒ Learnt that there are very little regulations for Paralytic Shellfish Toxins
• ❒ Our project could help fill a spot for in regards to implementation in Hong Kong

3. Commercial shellfish farmers

We believe it is important to understand the needs of shellfish producers as they are a major stakeholder in the shellfish industry. Understanding their needs helped us see the current limitations of PST testing from their perspective, and helped us shape our product to meet their needs.

We wanted to see how the first link in the production chain, shellfish farmers, thought about our project plan, so we visited a local oyster farm in Hong Kong.

陳祥記 (Chen Cheung Kee) is the biggest oyster farm in Hong Kong, and has been in operation for more than 50 years. Their line of expertise is from oyster seedling cultivation to packaging, and they specialise in raw oysters, golden oysters, and oyster sauce. Even though they are a family business, they distribute oysters to major restaurants around Hong Kong.

We met with Ms. Chen, the daughter of Chen Cheung, on their oyster farm on June 29th, and we learnt so much about the oyster farming process in Hong Kong, from how long it takes for one cycle of oyster harvesting, to what is the ideal harvest season, and how they distribute their oysters to different countries. There are cases of harmful algal blooms near their farm but they are irregular due to changes in salinity of the water.

After sharing our project idea with Ms. Chen, even though they were not quite aware of Paralytic Shellfish Poisoning and its effects, they told us they expect our product to be fast and sensitive to the toxin. We decided to incorporate the SCAFFOLD and AUTO-INHIBITION into our circuit to make the biosensor more robust and fast.

Scaffold information

Our next meeting with Ms. Chen was on Sept 27th. As our target end-users were ultimately shellfish farmers, we decided to ask them more in regards to implementing our project into the shellfish industry. They told us that they think there is only a very small market for our biosensor in the farms, as shellfish aren't tested very frequently. We were also told that if we really wanted to expand in this market, our product had to be simple to use, quick, and affordable. Key takeaway

• ❒ We learnt that speed of detection was priority, hence changed our circuit to include auto-inhibition and a scaffold mechanism.
• ❒ Decided to not only focus on shellfish farmers as our target end-user

We learnt that in Hong Kong, the farmers themselves do not engage in water quality and shellfish toxins testing, but rather the buyers and distributors collect their own samples for testing. The farmers are not informed of the testing results, and only know if their shellfish can be sold or not. After this interview, we found that there is another stakeholder in the shellfish industry that we initially neglected: the testing labs.

4. Testing and diagnostic laboratory

As we wish to implement our project into the real world by making a biosensor for paralytic shellfish toxins, we decided to reach out to a testing and diagnostic laboratory that also works on sodium channel-blocking toxins.

Mark Van Asten | Australia

Mark Van Asten is the Managing Director of “Diagnostic Technology”, and has 30 years of experience in the medical diagnostics and life science industry. For the past 20 years, he has commercialised products that detect the presence of toxin-producing species in algae.

We have had 2 fulfilling meetings with him. In our first meeting on August 4th, he talked about his experience in the market of diagnostics. Mark reminded us that as scientists, we tend to make things complicated, trying to do everything and fix everything, however, we need to be aware of how the public would react to it, and how our final product, the biosensor, would be used. We discussed current competition we had in the market, and discussed whether or not we should change our potential target end user, i.e. from farmers to lab scientists or researchers. He stated that our product could be used for more things other than toxin testing, such as in anesthetics research, and that keeping an open mind would be very useful for us.

For our second meeting on September 23rd, we were again reminded of existing technologies so we can find the “gap in the market” that we could potentially fill. We focused on the selling points of our product, which is that we could bring the advantages of a direct bioassay and use them to detect a wider range of shellfish toxins. Worried about getting backlash from the end users as we would be using Veratridine, which could be toxic at higher doses, we told him about wanting to replace Veratride with salt, coming to this conclusion from our own wet lab data. Mark supported our decision, and suggested that instead of using salt, we could consider using an osmolyte such as Glycine or Trehalose instead.

Safety page

Key takeaway

• ❒ Helped us realise that our target market can be expanded
• ❒ Exposed us to existing solutions to find how we can compete
• ❒ Allowed us to think of more marketable solutions while allowing the circuit to work

5. The Consumer

The last link in the shellfish industry chain are of course the consumers. We wanted to see what the general public knew about shellfish poisons and methods to test for them, so we created a survey and sent it out to the locals.

More than 100 responses were surveyed, and the results were quite shocking. Most people didn't know about shellfish toxins, much less that there were different types, and the majority of participants didn’t know about harmful algal blooms either. Hence we decided there is an opportunity for education and science communication.

Through the survey we conducted, we learnt that 71.3% of participants eat shellfish regularly, and they all eat a variety of shellfish. More than 60% of the participants are not aware of shellfish poisoning or of any types of shellfish toxins, and the majority of participants were not aware of the existence of harmful algal blooms.

We really found this quite alarming, as there is an obvious gap in the general public’s understanding. We want people to understand our project, but before we can achieve that, they must know the main background issues that our project is based on. In order to generate a better understanding, we decided to try and educate different levels of the public.

Education page

Symposium

A platform for sharing synthetic biological ideas is important to us, and with 11 Hong Kong iGEM teams competing this year, we decided to fill in that gap. We hosted the Hong Kong iGEM symposium this year, inviting 8 of Hong Kong’s iGEM teams to share not only their project ideas, but also their resources. The in-person symposium was hosted successfully on HKUST’s campus on August 11th 2021, and we were honored to have Professor King Chow (HKUST) and Professor Becki Kuang (HKUST) as guest speakers to inspire and educate our student audience.

The iGEM teams that participated were HKU, CityU, GTC, UCCKE, HKUST, CPU-WYY-WFN, HKIS, and SSC. It was a full day event, from 9:00 to 18:00, in one of the lecture theatres at the academic concourse of the HKUST campus. There were more than 80 participants that attended live, and we conformed with the COVID regulations that were in place on our HKUST campus.

The format was similar to the Jamboree presentations, where each team would present their ideas in 20 minutes, and allow time for feedback, questions, and comments on each other’s projects. We also left plenty of time for a mingling session after all the presentations, taking that time to communicate and learn more about how other teams are functioning, and about their journey through iGEM so far. We also invited several ex-iGEM team members and advisors from the undergraduate iGEM teams, and they gave comments to each group in regards to their projects. During the symposium, there was also a talk in regards to the iGEM Entrepreneurship Program Innovation Community (EPIC) from one of our ex-iGEMers at HKUST. They talked about how we could expand and grow our project into a start up even after the Jamboree.

It was a very fulfilling experience for us, as you could see for each individual team, whether they were an undergraduate or high school team, the amount of effort and dedication put into their project ideas and execution. We took this opportunity to collaborate and network with local iGEM teams, sharing ideas, asking questions, and creating discourse in order to improve on our projects. Through the Symposium, we were able to communicate with a few iGEM teams in regards to collaborating further. Special thank you to our guest speakers

Professor
King CHOW

Acting Dean of Students of Hong Kong University of Science and Technology

Department of Chemical and Biological Engineering

Principal investigator for HKUST iGEM team of 2021

Professor
Becki Yi KUANG

Department of Chemical and Biological Engineering

Hong Kong University of Science and Technology

Event Photos

Attached below is our rundown and brochure of the event!

Sustainable Development Goals (SDG)

1. SDG 6: Clean Water and Sanitation

As our biosensor serves to detect unsafe levels of toxin in shellfish, this can double as an indicator as to the water quality of the marine environment the shellfish resided in. This contributes to minimizing the spread of the toxins and potentially the prevalence of harmful algal blooms (eutrophication) as the correct authorities can then be made aware of the poor water conditions. This as such contributes to the 6th SDG, specifically target 6.3 which involves improving water quality, wastewater treatment, and safe reuse.

Although different parts of the world may have their own methods of testing the water, the regular testing for shellfish batches may serve as an additional reassurance and may serve as assistance for further data collection. Shellfish farms may also be found in more rural areas wherein water may not be regularly tested. The government may as such work with them, and or the testing labs that work with them, to get frequent reports as to the safety of water levels.

2. SDG 8: Decent Work and Economic Growth

Our biosensor can serve as preliminary testing, as such allowing farmers to be able to send their batch of shellfish to bigger markets with confidence that the product is safe for consumption. This serves to increase efficiency in the supply chain as in the case where the shellfish are safe to eat, further testing will not be in vain, and in the case where the shellfish are not safe to eat, swift action can be made to seal off the area, and better preparation can be made.

The estimated testing time is also more rapid than current testing methods which as such minimizes the time consumed at the initial stages of the supply chain. This coincides with target 8.4, as it contributes to resource efficiency in consumption and production, as less resources will be used than when the quality of the shellfish is known further up the supply chain. In addition to this, our biosensor also contributes to increasing employment in the economy, as personnel will be required to produce and manage our biosensors. This may contribute to target 8.1 which advocates for sustainable economic growth as both the increased efficiency in production and employment would contribute to the economy positively.

3. SDG 12: Responsible Consumption and Production

The mouse poison bioassay has been utilized as standard of testing for many different experiments, including the detection of marine biotoxins in shellfish. Although it has been greatly beneficial to the world of science, contributing to the discovery of a magnitude of knowledge about biological life, this method may be seen as unsustainable as raising the animals will take a greater amount of resources as compared to producing bacteria for our biosensors.

There has additionally been a moral dilemma that revolves around the method as the mice used for testing are often subjected to immensely harmful and torturous chemicals. As such, we believe that by using our biosensor to replace this unethical method, particularly contributing to target 12.6 which involves encouraging companies, specifically those of whom are involved in the shellfish industry, to adopt sustainable practices and sustainability reporting

4. SDG 14: Life below Water

Our biosensor contributes to preserving biodiversity. As we will be able to test for the existence and prevalence of toxins in shellfish (whether or not it is safe for human consumption), we will also be able to identify whether there are safe levels of toxins, and the dinoflagellates which produce them, in the bodies of water which the shellfish came from. This is of concern considering that the toxins can negatively impact the marine life which feeds off of the shellfish and other filter feeders in the ecosystems as the toxins accumulated may also hurt their population.

This may create an imbalance in the ecosystem which may in turn affect the rest of the marine life. In cases where these toxins are found, measures can be taken in order to minimize the spread, and the correct conservation authorities can be informed of the situation. As such, our biosensor also contributes to the 14th SDG, specifically target 14.A, being to increase scientific knowledge, research, and technology for ocean health, since our biosensor helps with the collection of information regarding conditions of the marine ecosystems, as well as target 14.2 which is to protect and restore ecosystems, as our biosensor will be able to help protect the ecosystems.

References:

1. Goal 6: Clean water and sanitation. The Global Goals. (n.d.).

2. Goal 8: Decent work and economic growth. The Global Goals. (n.d.).

3. Goal 12: Responsible consumption and production. The Global Goals. (n.d.).

4. Goal 14: Life below water. The Global Goals. (n.d.).

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