Implementation

Overview

    To make a living by using the local resources is common wise for the mass. So, seafood has brought up the costal people, among which shrimp is a kind of significant marine product particularly rich in protein, calcium, and astaxanthin. Since the outbreak in 2009 (1), acute hepatopancreatic necrosis disease (AHPND), infecting the digestive gland of shrimp, has spread rapidly around the world, causing massive shrimp mortality and severe economic losses to shrimp aquaculture. The pathogen causing this disease is Vibrio parahaemolyticus (Vp). Using drugs and antibiotics is the common approach to deal with AHPND currently, which neither specifically targets Vp nor prevents the Shrimp from death. What’s more, it would also perturb the ecological microenvironment and increase the risk of antibiotic resistance. In order to solve this problem, we design a set of Operable Magic, which is a comprehensive solution for AHPND, protecting shrimps by means of synthetic biology.

Description

    AHPND is mainly caused by the infection of Vp which involves the virulence genes, pirA and pirB (for more details please see the page prevention part in Integrated Human Practice). However, not all the Vp carry the virulence genes. Thus, it is not the Vp, but the genes, pirA and pirB, should be the target that our project focuses on. Our scheme of the project consists of the detection, prevention, and treatment parts.

Detection

    We designed a series of devices to achieve the goal of detection of virulence genes, and these devices would be integrated into a toolbox. There are two syringes in the toolbox used for lysis of bacteria from water samples and enrichment of DNA, respectively. Then, the DNA are transferred to a thermostatic device (37 °C) for amplification based on Recombinase Polymerase Amplification (RPA). And a cell-free sensing system is employed to transcribe DNA into RNA, capture targe RNA (pirA and pirB), trigger the ribozyme splicing, and finally emit fluorescence (please see Design for details) (Fig. 1) which could be recorded by the built-in camera. Next, the recorded data would be uploaded to the cloud account and analyzed by our model to generate a report. Users could download the report by logging in to the account on the supporting mobile app and evaluate the possibility of AHPND.
Fig. 1 Mechanism of ribozyme-splicing based signal transform of RENDR.

Treatment

    Treatment is conducted by engineering E. coli which would be colonized in the shrimp’s gut. Tail tubular protein A (TTPA) and tail tubular protein B (TTPB) of Vp phage are displayed on the surface of OMVs, which could specifically bind to Vp via the Vp0980 (Fig. 2). The rhamnose artificially released into the water bodies induces the high-level secretion of OMVs. The OMVs contain plasmids expressing endolysin edl060, which is also induced by rhamnose. Endolysin edl060 could be leaded by LMT to the peptidoglycan layer of Vp and destroy the layer, thus achieving the purpose of treatment.
Fig. 2 Schematic diagram for treatment strategy.

Prevention

    Prevention is performed by the engineered E. coli colonized in the shrimp’s gut, too. The colonized E. coli could secrete outer membrane vesicles (OMVs), the surface of which is anchored by FET or LvAPN1 by the linking of protein INP or ClyA. Domains of FET and LvAPN1 could competitively bind to PirA and PirB, achieving the prevention of AHPND (Fig. 3).
Fig. 3 Binding between rFET or rLvAPN1 and PirA/PirB.

Who Are the End Users?

    Our project can be of great benefit to shrimp farmers, technicists, and researchers.

Shrimp farmers

    They are the most immediate users who would benefit from our project to cope with AHPND based on the three parts (detection, prevention, and treatment strategies). Compared with the methods used presently, our detection strategy not only provides unprecedented speed (2.5 h per test), cost (30.1￥per test), and precision but also frees from the large and valuable instrument and complex operation process. In the prevention and treatment, the functional E. coli can be colonized in the shrimp gut to provide long-term and effective protection at a low-cost. Thus, this comprehensive method can not only provide an effective strategy to reduce economic losses but also reduce the risk of antibiotic abuse.

Technicists

    They are indirect users who would benefit from our detection strategy. Technicists often provide support for the aquaculture farmers whose ponds are located in the village far from the city. So, they can only bring the sample of water and tissue back to the company for further analysis and give the advice to the farmer on another day, which would miss the best time to take action. Our portable detection toolbox and rapid detection speed will make the field test possible so that technicists can make a decision and provide guidance for shrimp farmers.

Researchers

    Our comprehensive strategy provides a paradigm in the detection, prevention, and treatment of AHPND. This can be an invaluable platform for research groups (and iGEM teams) around the world to develop more and more kits, devices, and engineered strains to cope with other pathogenic microorganisms and viruses.
    As we learned from the interview with shrimp farmers, technicists in Mata company and Fujian Aquatic Technology Extension Station (see Human Practice for details), there’s no rapid detection method and effective treatment strategy for AHPND available for fisherfolks. Also, current prevention methods of AHPND are ineffective and pose a potential threat to public health. Therefore, our strategy can provide an effective method for shrimp farmers to get over AHPND.

How to use?

Detection

    As shown in Fig. 4, samples of the pond were collected and put into the syringe, and mixed with the lysis solution for 1 min. By injecting the sample solution into the second syringe, cell lysis was removed from the solution. The nucleic acid in the water sample was then enriched with fiber paper while other components were discarded. The following steps were conducted in the thermostatic black box. The paper was put into the RPA reaction mix for amplification of the target gene. After 10 min of RPA reaction, the sample was then added into the cell-free system. The data was collected in the built-in camera of the box and subsequently uploaded to the cloud account for analysis. Finally, the analysis report can be checked on phones with the app to monitor and evaluate the situation of AHPND.
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Fig. 4 Schematic diagram of detection strategy .

Treatment

    If the shrimps have suffered from AHPND, rhamnoses will be put into the fodder to induce the high level secretion of OMVs and the expression of endolysin, which would be secreted simultaneously and targeted to the Vibrio parahaemolyticus (see Design for details ). The expression of endolysin would kill the pathogen and achieve the treatment of AHPND.

Prevention

    The engineered bacteria were firstly added into the fodder and colonized on the gut of shrimps, which could realize the expression of OMVs. The proteins displayed on the surface of OMVs can competitively bind with the toxin to prevent AHPND.

Safety

    As the genetically engineered organisms (GMOs) are colonized directly in the shrimps’ intestines, the kill switch (Fig. 5) is essential for biosafety concerns. Therefore, before harvesting the shrimps, the kill switch is activated under the induction of arabinose and kills the GMOs. By cleaving the mRNA of GMOs inside, MazF can inhibit protein synthesis and kill the bacteria, guaranteeing biosafety.
Fig. 5 Arabinose-induced MazF expression

Risk assessment

    Since engineered bacteria are used when the project is implemented, there are potential risks to public health and the environment. Therefore, before we put our devices into production, testing, and applications outside the laboratory, extensive testing should be performed to ensure that our engineered bacteria are safe for humans and the environment.
    Besides, China passed a new biosecurity law and it has come into effect on April 15, 2021. The law establishes systems for biosecurity risk prevention and control, including risk monitoring and early warning, risk investigation and assessment, and information sharing. It also has provisions to prevent and respond to specific biosecurity risks, including major emerging infectious diseases, epidemics, and sudden outbreaks, and biotechnology research, development, and application. To keep our activities legal, applying for permits and approvals is necessary if we execute our proposed implementation.

Reference

      1. M. L. Situmorang et al., Supplementation of ex situ produced bioflocs improves immune response against AHPND in Pacific whiteleg shrimp (Litopenaeus vannamei) postlarvae. Appl Microbiol Biotechnol 106, 3751-3764 (2022).