Team:HKUST/Implementation

ShellBi

Project
Description Design Engineering Proof of Concept Implementation Safety Contribution Notebook
Wet Lab
Experiments Results
Dry Lab
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Implementation

From Bench to the Field

Overview

In Hong Kong, aquaculture includes marine fish culture, pond fish culture, and oyster culture. In 2020 production from the aquaculture sector was 3,322 tonnes valued at $127 million which was 3 per cent in weight and 4 per cent in value of the total fisheries production [1]. With such a significant demand for fisheries products, there must be better ways to test the harvest. Currently, in Hong Kong, there are no regulations for the testing of the harvest apart from oyster depuration carried out by only a few farms accredited under a new agricultural hygiene scheme by AFCD (Agriculture, Fisheries and Conservation Department) [1]. Although often, one may encounter news articles concerning poisoning associated with shellfish consumption, most cases are labeled as ‘food poisoning’ since the symptoms are not distinct for paralytic shellfish poisoning if the toxins are consumed in low doses.

Figure 1: Fisheries and Oyster Farms distribution in Hong Kong

Pressing Need

Many countries also do not have established labs to test for the PSP because it had not previously been detected in their vicinity. After our own interview with a Hong Kong Agriculture, Fisheries, and Conservation Department (AFCD) representative, we even found that government facilities in Hong Kong have not mandated any toxin screening of harvest. The Food and Environment Hygiene Department (FEHD) in Hong Kong is responsible for issuing hygiene certificates for batch imports of seafood. Still, they can only advise local farms to stop harvesting if anything unusual is found, “They cannot mandate,” said Dr. Chung, Fisheries officer, AFCD. However, Dr. Chung mentioned that the main reason for the lack of regulation is that they have not yet faced any dire situation regarding PSP.

Figure 2: Sai Kung Seafood, Hong Kong

With the constant increase in global warming, there are increasing cases of Harmful Algal Blooms, as seen in Figure 3. The augmentation of HABs complemented by an increase in seafood demand puts many people at risk of being affected with Paralytic Shellfish Poisoning. In fact, a study conducted by Finch, Boundy, and Harwood (2018) raised the alarm towards the acute toxicity of paralytic shellfish toxins given the additive effects of (here) Saxitoxin and Tetrodotoxin [2]. With so many new cases of contaminated shellfish coming up in several European countries, North America, and Latin America, it is quintessential that every risk-prone country is equipped with at least a basic form of testing for these potent neurotoxins [3].

Figure 3: Increase in Harmful Algal Blooms in the past decade

With ShellBi, we wish to provide farmers with access to a relatively direct, easy, and cheap method for testing Shellfish toxins. After having a word with local oyster farmers in Hong Kong, the general consensus was that they would be extremely willing to adopt such a testing mechanism on farms as this will not only allow them to get a safer harvest in the market but could also benefit their business since tested shellfish could be sold for a higher value.

Target Audience

Shellfish farmers

Initially, we aimed the biosensor to be used on-site by the farmers themselves. After interviewing our local oyster farms, we learnt that most large-scale farms use a -30°C storage for the shellfish. This storage temperature is also highly suitable for our biosensor components. If all components can be favourably stored, they could easily carry out the procedure for testing of toxins.

Food testing laboratories

Although our biosensor can be targeted towards farmers for on-site use, this will only be viable for large scale farms with on-site facility of well-equipped, hygienic and appropriate storage. Since the biosensor involves living cells, there are temperature and handling requirements that would be unrealistic for all harvesters to follow. Instead, we propose the target audience of our biosensor to also be low-budget food testing facilities. It would also be significant as a stepping stone towards shellfish testing in a place like Hong Kong, where no testing regulations exist due to lack of viable technology. Our product may offer a preliminary test for the harvest.

Drug and Anaesthetic Research

ShellBi, being living engineered cells, offers a stable and self-sustaining source of ‘receptor-based solution’ for researchers in the field of anesthetics and drug development. They could utilize this technology as a cheap and versatile mechanism for preliminary testing of the activity of potential new drugs. The fluorescent readout could be an indication of whether a prospective anaesthetic drug can block ion channels. Since the cells offer a ‘receptor based assay’, they are not limited by detection probes. Hence, multiple drugs could be tested using our biosensor.

Figure 4: Stakeholders and target audience

Why ShellBi

ShellBi provides a more ethical, cheaper, and direct biological method for the testing of toxins. One advantage ShellBi holds against most other non-biological methodologies is its capability to detect a wide range of toxins. For example, gene-based or antibody-based tests all rely on having specificity towards only one predetermined target compound, whereas ShellBi is based on the receptor-binding ability of a toxin; hence, it can detect not just one but several different toxins and their variants with the same equipment and protocol. However, to compete against the other solutions, we require extensive testing and improvement.

From The Eyes of a Consumers

Kit content

Figure 5: Shellbi Kit content

Procedure

Figure 6: Procedure for Shellbi Kit

Note: Veratridine could potentially be replaced with an osmolyte such as Glycine or Trehalose solution to offer a much safer and cheaper testing kit. However, we are yet to find which one offers the best alternative in terms of speed of reaction and enhancement of biosensor signal.

Market Penetration Consideration

Market Comparison

	SHELLBI	HPLC	MOUSE-POISON BIOASSAY	ELISA	RECEPTOR BINDING ASSAY
Cost	Cheap Bottleneck being drug Veratridine	Very Expensive Requires intensive machinery	Cheap Require mice management No standard number of mice	Intermediate - expensive Requires specific antibody	Intermediate - expensive Requires purified cell membrane
Operations	Easy Requires proper handling of Veratridine Maintain cells at favourable temperature Time: To be confirmed	Difficult Heavy machinery Requires trained personnel Lengthy procedures Time: 60 minutes	Easy - intermediate Injecting shellfish samples No standard protocol Maintaining mice in facility Time: 1-2 days	Easy Standard protocols Quick readout Time: within minutes	Easy Standard protocol Similar activity as ELISA Time: within minutes
Reagents	GM cells Cell culture medium Drug veratridine	Solvent for extraction (HCl/ Acetic acid, Acetonitrile/ Formic acid) HPLC column Carrier gas [4]	Lab mice Solvent, acetone	Purified antibody Testing kits available	Purified cell components Testing drugs
Ethicality	Ethical Prokaryotic cells	Ethical Dry Lab testing, chromatography methods	Unethical Huge number of mice sacrificed No standard protocol to monitor	Ethical Requires animals for generating antibody, but ELISA tests are ethica	Ethical
Specificity	To be determined	High specificity Quantitative detection of toxins	Low specificity Only observes the lethality in mice Artifacts could present false positives	Intermediate Antibody could bind analogues of toxins Background may be high	Intermediate- high Toxins direct reaction to binding of a receptor
Detection limit	To be determined	8 μg STX equiv. Kg-1 [5]	400 μg STX equiv. Kg-1 [8]	0.015 ng/mL [7]	100 μg STX equiv. kg-1 [8]
Directness	Intermediate Bioassay but not on mammalian components	Low Only tells which PST is present and quantifies	High Direct relation between effect on mice and humans	Low Only detect specific type of PSP	Intermediate Only tells if toxin can bind receptor, not effects

A comparison between existing solutions is presented in this table. Despite the existence of new methods, the standard testing method is still the widely used Mouse-Poison Bioassay. This is due to the significant time and money required to set up, validate, and deploy these alternatives. The choice of method is largely dependent on the performance of the approach as well as the targeted export market destination. For example, Latin American regions who want to export mussels to the US, will have to employ one of three LC-FLD methods: MBA, RBA, or PCOX [5]. Given that the mouse poison bioassay had been a gold standard for all this while, most export products would be tested by MBA which is extremely ethically questionable. If all of the current mouse poison tests are replaced with alternatives, we could save more than 15,000 mice annually [5].

We reached out to Mark VanAsten, founder of Diagnostic Technology, who has over 30 years of experience in the innovation of clinical diagnostic and life-science based solutions for Australia and New Zealand markets, to evaluate the viability of technology like ShellBi in the market. One of his products is centered on determining shellfish toxins as a measure of water quality near shellfish farms. His field liaison and expertise were extremely helpful for us to gain insights into how ShellBi could perform in the market.

One major issue that was eye-opening for us was that all these diagnostic technologies are ‘destructive technologies’ for a shellfish farmer. Naturally, there is a reluctance in accepting any new technology for a harvester whose livelihood depends on daily sales unless the testing via the method is mandated publicly. To reach that stage, one needs to establish proper controls and convince the authorities that our technology is at par with current detection methodologies and offers more benefits than those in place.

Another consideration is that we must apply for a license if we utilize any well-established genetic part, for example, the Tet-off system.

Our Goals

Short Term Goals

• ❒ Make a fool-proof system (See below)
• ❒ Make a qualitative system for detection of neurotoxins
•   • Boolean: Yes/No sensor
• ❒ Prepare a kit for ShellBi and package with all safety considerations
• ❒ Advertise and give prototype to low budget testing centres that uses mouse-poison bioassay in Asia
• ❒ Collect feedback and monitor the market response

Long Term Goals

• ❒ Patent ShellBi
• ❒ Compete in the market against other solutions
• ❒ Attempt to have quantitative measure for concentration of toxins
•   • Intensity relative to concentration

To make a Fool-proof System...

Establishing Control

Response Threshold

Establishing Control

Conceptually, since the fluorescence color readout from cells is based on their intracellular osmolarity, if we were to deliver the cells suspended in any medium, the cells would be fluorescing a particular color depending on the medium osmolarity. If that is the case, it would be difficult to establish a control, i.e., how do we know the cells are glowing a color due to the sample being tested or the medium they initially were growing in? Hence, we propose using a ‘Master-off switch’ component in our cells.

Mechanism of master-off switch

Figure 7: Tet off system in the absence of doxycycline

Figure 8: Tet off system in the presence of doxycycline

A tetracycline-off switch forms the heart of this mechanism. Tetracycline-off (Tet-off) system is a method for inducible gene expression where expression of genes could be turned off with the addition of tetracycline, or its derivative, doxycycline. If we encode an antisense RNA to both eGFP and mRFP1 under a Tet-off promoter, in a resting state, the antisense fragment would be constitutively expressed against the two possible colors, and the cells, theoretically, should not have a fluorescence or a weak signal if at all.

A Tet repressor at its native state would suppress the expression of tetracycline promoter. However, TetR, when fused with VP16 which is a transcriptional activation domain from herpes simplex will activate the Tetracycline promoter. When tetracycline or doxycycline is added, it will bind to the TetR-VP16 fusion protein thus precluding its binding to the tetracycline promoter resulting in an off state of the tetracycline promoter.

We would provide the users with a standard buffer that contains doxycycline along with other reagents. The user may grind the shellfish and mix the buffer with the sample. When the sample (which now contains doxycycline) is added to the cells, the expression of the two antisenses stops. The addition of veratridine follows this step. The color production in the cells could now start. Since the shellfish sample and veratridine are added to the cells, the fluorescence readout would only be a result of osmolarity change induced in the cells due to the shellfish sample.

The background signal can be suppressed due to the antisense RNA encoded under the promoters of the Two-Component system, and the robustness of the signal can be enhanced with a scaffold.

Further considerations

Interference: We need to consider what the ‘buffer’ would contain. If we are to add a whole shellfish sample to the cells, we need to make sure that the other contents from the shellfish do not interfere with the cellular response and gene interaction. Hence, the buffer must contain a chelating agent for other possible osmolytes and contaminants that can influence the cell response.

Response time: We are aware that if we utilize the Tet-off system, the response time for the biosensor may extend over 24 hours. Moreover, although according to literature, the ion influx can dramatically change starting from 15 mins up to 1 hour, the fluorescence proteins also require several hours to be expressed and matured. We have optimized this as far as possible, but it is only through steady effort and testing, can we be sure of the direction of improvement. Based on these identifiable factors, the biosensor might require at least 24 hours to give a significant, reliable readout. We could also use a Xeomycin induced promoter which could potentially offer a more rapid response.

Response Threshold

Currently, another challenge we face is the detection limit of the biosensor. Since we cannot access drugs, we cannot test for the response threshold of the biosensor ourselves. Professor Brett Neilan, The University of New Castle, Australia, has generously offered to test our engineered circuit with appropriate drugs required as they have the permit to access the drugs. For more information, please see “human practices”.

Nonetheless, we predict that we would need to finetune the promoter response for our circuit such that the Red fluorescence is observed up to a certain level of osmolarity in the cell to accommodate for samples containing different amounts of toxins. According to the dry lab modeling, the PompF promoter activity peaks at a low concentration over a narrow range (Figure 8).

Figure 9: Fluorescent readout against the range of intracellular osmolarity

However, it would be ideal if PompF is active over a broader range of osmolarity to prevent false-negative results. Another reason we need to finetune the response threshold is because there are over 40 variants of Saxitoxin, at least that have been identified. Each of those could have different channel binding efficiencies, leading to discrepancies in reading and false measurements.

To finetune the circuit, first, we would need to map out the effect varying concentrations of toxin (At low and high extremes) have on a cell's internal osmolarity. The activity of the OmpF promoter (PompF), which drives the RFP for a “positive signal” (Toxin present), would have to match that range.

The function of PompF can be modulated by changing the sequence of specific regions on the promoter- by enhancing the activity of positive regulators F1, F2, and F3; and reducing the activity of the negative regulatorF4 region. If the sequence of the negative regulator is altered such that its affinity towards OmpR is reduced, the promoter suppression would theoretically occur at a much higher concentration of OmpR, which renders the promoter a higher tolerance to increased osmolarity. This attempt will surely be promising befitting with the findings of the paper by Koga-Ban et al (1983) where a particular mutation in OmpF promoter region, resulted in overexpression of OmpF while concomitant suppression of OmpC [9].

Figure 10: OmpF binding model with mutation in the F4 site

Similarly, the OmpC promoter would require modifying its regulatory units to limit the overlap of signals.

References:

1. “Marine Fish Culture, Pond Fish Culture and Oyster Culture.” Agriculture, Fisheries and Conservation Department The Government of the Hong Kong Special Administrative Region,

2. Finch , Sarah, et al. “The Acute Toxicity of Tetrodotoxin and Tetrodotoxin⁻Saxitoxin Mixtures to Mice by Various Routes of Administration.” Toxins, U.S. National Library of Medicine, 23 Oct. 2018

3. F;Blanco L;Rodríguez LP;Vieites JM;Cabado AG; “Phycotoxins in Marine Shellfish: Origin, Occurrence and Effects on Humans.” Marine Drugs, U.S. National Library of Medicine, 29 May 2018.

4. Harju, Kirsi, et al. “Optimization of Sample Preparation for the Identification and Quantification of Saxitoxin in Proficiency Test Mussel Sample Using Liquid Chromatography-Tandem Mass Spectrometry.” MDPI, Multidisciplinary Digital Publishing Institute, 25 Nov. 2015.

5. Turner, Andrew D., et al. “Application of Six Detection Methods for Analysis of Paralytic Shellfish Toxins in Shellfish from Four Regions within Latin America.” MDPI, Multidisciplinary Digital Publishing Institute, 3 Dec. 2020.

6. Combes, Robert D. “The Mouse Bioassay for Diarrhetic Shellfish Poisoning: A Gross Misuse of Laboratory Animals and of Scientific Methodology - Robert D. Combes, 2003.” SAGE Journals, 1 Nov. 2003,

7. “Saxitoxins (PSP) (EC 2002/225 Compliant), ELISA, 96 Tests.” Publications.

8. Ruberu, Shiyamalie, et al. “Receptor Binding Assay for the Detection of Paralytic Shellfish Poisoning Toxins: Comparison to the Mouse Bioassay and Applicability under Regulatory Use.” Taylor and Francis Online, 8 Sept. 2017,

9. Koga-Ban, Y et al. "Mutation Causing Overproduction Of Outer Membrane Protein Ompf And Suppression Of Ompc Synthesis In Escherichia Coli". Journal Of Bacteriology, vol 155, no. 3, 1983, pp. 1110-1115. American Society For Microbiology.

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