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This year, our team has developed an effective platform for the detection of drug-resistant bacteria in the ocean that is amplification-free, fast and accurate, and we have completed proof-of-concept for the project in both wet and dry labs. In addition, we have provided subsequent igem teams with a crispr/cas solution that is accurate to single base identification and does not require amplification, which we hope will help subsequent teams to better complete their projects.

I. Current status of the farming industry

Aquaculture is a major way to increase the global supply of aquatic products. The UN Food and Agriculture Organization (FAO) released a report on June 29, 2022, stating that global fisheries and aquaculture production is at record highs, accompanied by significant growth in aquaculture, and that aquatic foods are making a significant contribution to securing food nutrition and security in the 21st century. The State of World Fisheries and Aquaculture 2022 states that total fisheries and aquaculture production rises to an all-time high of 214 million tons in 2020, driven by global growth in aquaculture, particularly in Asia, of which 178 million tons are aquatic animals and 36 million tons are algae. As the global population grows, the demand for fish and other aquatic products is increasing, and global food supply security issues are driving the aquaculture market, mariculture-based "blue agriculture" is playing an important role in alleviating the pressure of population growth on land-based agriculture and promoting economic development in coastal areas.

II. the causes of microbial drug resistance

In recent years, the frequent occurrence of microbial epidemics and outbreaks of diseases in mariculture worldwide has seriously restricted the healthy development of blue agriculture. Since Fleming extracted penicillin in 1929, the research of antibiotics has made great progress and is widely used in the research and treatment of biological diseases. However, with the development of aquaculture, fisheries and other industries, the abuse of antibiotics continues to occur. According to statistics, in recent years, the annual use of antibiotics in the world has reached 100,000~200,000 tons.

Currently, the main means of controlling bacterial diseases in aquaculture animals is the use of antimicrobial drugs. A large number of antibiotics are used in various stages of the aquaculture process. Due to incomplete understanding of etiology and pharmacology, there is a mixture of multiple drugs in large doses during the aquaculture process. The widespread use of antibiotics allows bacteria to retain the most resistant strains under extensive selection pressure. Bacterial resistance can be transmitted not only at the genetic level between bacteria of the same or different species, but also globally through structurally intact drug-resistant strains. This is because bacteria have the ability to mutate and develop resistance rapidly and can be transferred.

The large-scale use of antibiotics has created a serious microbial resistance problem. In combination with various human activities, drug-resistant microorganisms and antibiotic resistance genes are transmitted across species and habitats at the "human marine animal environment" interface. Antibiotics and their metabolites are enriched in the environment and then transmitted to humans through marine animals and animal products. It has serious implications for human health. Of particular concern is the rapid spread of multi-drug resistant and pan-drug resistant bacteria (also known as "superbugs") around the world, which is causing huge economic losses to countries and is one of the major threats to sustainable development worldwide.

III.why is it difficult to detect drug-resistant microorganisms in the ocean

There are two reasons why drug-resistant microorganisms in the ocean are difficult to detect

1. Bacterial resistance is the result of evolutionary selection of bacteria, and the misuse of antibiotics has exacerbated the development of bacterial resistance. Bacteria produce mutations in resistance genes during growth and reproduction, and under the selection pressure of using antibiotics, resistant bacteria are screened out and multiply dominantly. With the massive human use of antibiotics in marine organisms worldwide, marine microorganisms have had to mutate genes in different directions in order for populations to persist, which increases the difficulty of detection.

2. One of the characteristics of drug-resistant bacteria is that they are susceptible to base mutations compared to normal bacteria, so that there are a large number of non-target nucleic acids that are similar to the target nucleic acid in the detection system. The traditional detection technology is not accurate to single base identification, when the detection of the target solution contains similarities to the target or the target mutants (one or two base mutations) will be mismatched, resulting in false fluorescence signal, which will greatly affect the reliability of the test results. Therefore, a simple method to enable Cas13 and Cas14 to achieve single-base detection is one of the directions we are seeking.

IV. Solution ideas

1. Simple and efficient CRISPR tools still have shortcomings

CRISPR (clustered regularly interspaced short palindromic repeats), known as clustered regularly interspaced short palindromic repeats, is an immune system used by bacteria to protect themselves from viral infections, and it is widely used today in gene editing technologies. When exogenous DNA invades a bacterium, its CRISPR system specifically captures a segment of exogenous DNA sequence called proto-spacers inserted between the leading sequence and the spacer repeat sequence, accompanied by a single replication of the repetitive fragment, thus forming a new segment of repetitive units that are the structural basis of bacterial-specific immunity. Knockout cell lines can be established simply by artificially designing crRNA, inserting it into a suitable plasmid, and cotransfecting cells with plasmids expressing tracrRNA and Cas protein, respectively, or injecting it into specific cells after transcription into RNA in vitro. In recent years, this technique is being widely used for nucleic acid detection through improvement.

However, the tolerance of the Cas protein in the CRISPR system to 1-2 base nucleotide polymorphisms in the target sequence leads to a significant reduction in the efficiency of the cleavage of the Cas protein in the detection system, and incorrect recognition and cleavage will also lead to "false positives" in the detection. Thus, we can assume that if the detection system contains a certain degree of target sequence SNPS, it will be difficult to distinguish whether the detection signal comes from the target fragment we want to track, which is extremely unfavorable for research projects that require accurate single-base identification detection.

2.Cas13a1 in series with csm6 idea

We were inspired by the study of Jennifer et al. When cas13a and csm6 are used in tandem, the sgRNA on cas13a recognizes the target sequence and activates the activator on csm6 to cut off A4-U6 to release coA. coA binds to the CARF structural domain and activates the HEPN structural domain for non-specific cleavage, cutting off the pre-designed fluorescent motif-fluorescent burst cut off and release fluorescent signal. It is able to increase the detection efficiency up to 100-fold without the need for pcr amplification.

3. Advantages of PNA

Peptide nucleic acids (PNAs), a class of DNA analogs that replace the sugar phosphate backbone with a peptide backbone, are a new class of nucleic acid sequence-specific reagents that began to be investigated in the 1980s.

Since PNA is not negatively charged, there is no electrostatic repulsion between PNA and DNA and RNA, thus the stability and specificity of binding are greatly improved. Moreover, unlike DNA or hybridization between DNA and RNA, the hybridization of PNA with DNA or RNA is almost not affected by the salt concentration of the hybridization system, and the hybridization ability with DNA or RNA molecules is much better than DNA/DNA or DNA/RNA, as shown by very high hybridization stability, excellent specific sequence recognition ability, and not hydrolyzed by nucleases and proteases.

Therefore, in the CRISPR/Cas system we use the pre-designed primers PNA and mutant strand binding, which greatly improves the stability of the binding compound and enables the CAS protein to precisely and efficiently target the target gene for recognition and cleavage, thus achieving high precision and high efficiency detection.

4. A light weight, small size, human-machine interface friendly enzyme labeling instrument

We hope to achieve rapid detection of drug-resistant bacteria in the ocean, but this technology is limited by the traditional enzyme markers are large, heavy, need to plug into the power supply is not conducive to carry the problem, can not really achieve rapid, in situ detection and other shortcomings. Therefore, we make a breakthrough innovation for the shortcomings of these technologies. We use special heat-conducting aluminum, heating film and temperature sensor, stm32f103c8t6 master control chip and pid algorithm to achieve precise control of temperature, to achieve faster results than some traditional enzyme labeling preheating and reach a stable temperature time. We use the "green light source" led beads in the light circuit to achieve the goal of small size, low power consumption, high luminous efficiency and fast response time under the same brightness. At the same time, we have developed a software interface based on the ready-made Android screen for users to use, to achieve the expected friendly human-machine interface.