Team:HKIS/Description
Project Description
Background
This year's iGEM project proved to be challenging. With the rise of Covid, we identified early on that it would be difficult to connect effectively with local residents and design a project centered around aiding a local problem. However, despite this setback, our team was firmly rooted in exploring Hong Kong's unique culture while possibly extending this implication to the global community.
When thinking about the origin and definition of traditional Hong Kong culture, the first thought that occurred to us was seafood and fisheries. Hong Kong was initially based as a small fishing village; only recently did it become "Asia's world city." With this genuinely spectacular evolution, we wanted to delve into the long-standing tradition that was molded to the city we see today.
Historical Context
The seafood industry in Hong Kong has thrived for over 700 years. The presence of seafood fueled the livelihood of thousands of fishers, growing into the industry that is so ingrained in Hong Kong’s vibrant aquaculture as we see today. Within this giant sector, the oyster industry has always accompanied the steady growth of Hong Kong. Oyster farming within Hong Kong is a tradition that has been passed down through generations. Traditionally, oysters are cultured by the bottom culture method with spat collected by laying rock, concrete tile, or post as a clutch on the mudflat in May or June. Oysters are also suspended from floating structures such as rafts, held in trays or strung. These rafts used to be a common sight all over Hong Kong, from Deep Water Bay to Lau Fau Shan, with styrofoam, oil drums, and polyethylene floats appearing in the misty haze.
However, despite many years of history, we found that oyster contamination is an issue yet to be solved. Even though oysters products appear everywhere, such as restaurants, grocery stores, and even the sprawling wet markets across the island, there is a clear market gap when it comes to detecting and purifying oyster contamination.
Solution Pt1: Detection
Oysters are often contaminated with bacteria vibrio spp., a common gram-negative bacteria found in marine environments. Furthermore, oysters can also be subject to contamination from sources such as heavy metals and other norovirus strains. Contamination takes place as oysters feed. When filtering water, Vibrio can become concentrated in oyster tissue, making it dangerous for consumption, especially when eaten raw. The issue is that farmers and aquaculturists have no way of determining whether or not their oysters are contaminated due to the fact that current commercially available systems require expensive machinery and extensive knowledge in biology to operate. Due to the fact that most oyster farmers do not possess a background in biology and cannot afford to purchase a PCR machine and other related equipment, they often sell products that aren’t guaranteed to be safe.
After interviewing with FDA Oyster Agriculture and Vibrio detection expert Dr. Jones, we have decided to only target Vibrios with pathogenic properties, which requires biomarkers for its pathogenicity. With an extensive review, we found out the most suitable genes and organisms to detect would be the
- Vibrio Vulnificus with vcgC gene as biomarker, which causes 50% fatality when infected and is responsible for 95% of all death
- Vibrio parahaemolyticus with TDH as biomarker, which commonly causes outbreaks
- Vibrio Cholerae with ctxA gene as biomarker.
Our solution is a Vibrio detection system based on three core technologies: CRISPR (Cas12a enzyme), Recombinase Polymerase Amplification (RPA), and Lateral Flow Detection (LFD). Among the series of CRISPR Associated (Cas) proteins, Cas9 is undoubtedly the most well-known, famous for its ability to accurately detect and cleave specific locations based on a protospacer adjacent motif (PAM) and guide RNA (gRNA). The Cas12a enzyme is very similar to Cas9 in that it too possesses the ability to recognize PAM and form complexes with gRNA. The difference between these two enzymes lies in their cleaving abilities; Where Cas9 cleaves a single target location, Cas12a is capable of trans-cleavage [1]. Trans-cleavage is the ability to cleave non-specific sites, which is what makes our detection work. The next core technology implemented is Recombinase Polymerase Amplification (RPA) [2]. RPA is an isothermal amplification method that allows for mass amplification of genetic material, similar to Polymerase Chain Reaction (PCR). PCR is the most commonly used amplification method, but due to the nature of PCR, the amplification process cannot occur without a machine. PCR machines are not only costly but also require an experienced technician to handle and maintain. RPA, on the other hand, can proceed with three core proteins at room temperature. Lastly, we utilized a lateral flow test for its cheap cost, rapid detection, and ease of use.
For distinct results, our lateral flow readout utilized Cas12a trans-cleavage in conjunction with fluorescein (FAM) and biotin-labeled ssDNA reporters [3]. (Fig. 1) Lateral Flow Assays utilize nanoparticles for visible result bands. By using gold nanoparticles coupled with anti-FAM antibodies, the lateral flow strip will present distinct positive and negative results on specific bands on the assay. (Fig. 3) The assay would have a sample zone, conjugated zone, control band, then a sample band. The sample zone would be where the sample is dropped; the conjugated zone is saturated with gold nanoparticles coupled with anti-FAM antibodies, ensuring that if a FAM-labelled reporter were to pass through, it would be bonded to by a particle. The control band would be saturated with streptavidin, which can anchor biotin-labeled complexes. (Fig. 2) Finally, the sample band would consist of anti-mouse antibodies that bind to the anti-FAM antibodies (mouse produced) coupled to the gold nanoparticles.
The protospacer adjacent motif (PAM) (Fig. 1) has proven to be a significant limitation in the sites we could use for detection [4]. The purpose of a PAM site is primarily to assist in unwinding dsDNA and forming an R-Loop, which is necessary for checking gRNA compatibility. However, during RPA, the strand displacing polymerase unwinds and separates the target DNA. Research has indicated that in these highly specific situations, unlike Cas9, Cas12a is still able to perform DNA cleavage regardless of the presence of a PAM site. (Fig. 4). Thus removing the PAM site limitation in our circumstance. This enables more options for gRNA segments and increases the potential detection accuracy of our LFA.
Solution Pt2: Purification
The second part of our project is purification. After designing a functioning detection kit, we wanted to create a product that could be used in the event of a positive readout. This product would have to do what current mainstream methods cannot: eliminate Vibrio without killing the oyster. Current mainstream methods include depuration (explicitly written by the FDA to be insufficient for bivalve contamination treatment), irradiation, and high-pressure processing. These methods lack a crucial feature in that they are incapable of thoroughly purifying bacterial contamination down to raw consumption standards without killing the oyster being purified. It is crucial for the oyster to be alive in most cases because once an oyster loses freshness, its price value diminishes rapidly.
Our solution for the purification of Vibrio contamination in oysters is for oyster containment water to be filled with the variant of an antimicrobial peptide called Tachyplesin I. We chose antimicrobial peptides over other antibiotics, or other chemicals is due to the fact that they are less susceptible to resistance mutation among bacteria compared to antibiotics [6]and also degrade rapidly, ensuring that no remaining active peptides will be ingested by consumers. While Vibrio’s innate resistance to AMPs may be an issue, due to the fact that Tachyplesin I is documented [7]to have antimicrobial capabilities against Vibrio bacteria, this issue is not important.
A more advanced version of our solution for this issue is an algae-based antimicrobial peptide expression system that can simultaneously serve as food for oysters as well as express antimicrobial peptides required for the elimination of Vibrio bacteria concentrated in oyster guts. This method is much more affordable for farmers, as the proposed transgenic algae can be cultivated in vast colonies and are therefore renewable. Not only this, by incorporating a surface display anchorage mechanism with short inflexible linkers [8], we can ensure that the expressed peptides do not inhibit further algae growth. Then, with linkers designed to be susceptible to enzyme cleavage by native enzymes found in an oyster’s gut, we can ensure that peptides will be activated in their gut and still eliminate present Vibrio bacteria. This is an effective workaround to the issue of a predicted negative feedback loop of algae growth where increased algae colony size expresses more AMPs which inhibits further algae growth. However, this solution has yet to be implemented and is our team's next step.
Sources
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