Description

An aptamer-based biosensor for colorific detection of pathogens in refrigerator.

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One day in May this year, my grandma was urgently sent to ICU for treatment due to a brain abscess caused by Listeria infection after eating watermelon put in the refrigerator for several days. This aroused my great curiosity. Why did the food in fridge cause disease?

After searching website, I discovered many similar cases (Fig.1). For example, a pregnant woman in Wenzhou city caused sepsis by drinking fresh milk that had been opened in refrigerator, resulting in premature birth and septic shock of the fetus. Mr. Zheng, aged 62, was sent to the hospital due to a high fever of 39 C° and 30 consecutive episodes of diarrhea. The doctor found that his body was severely infected with bacteria, leading to sepsis. The day before the onset of his illness, Mr. Zheng ate red chili stew that had been stored in refrigerator for many days.

When I went home on vacation and opened the refrigerator to get some foods, I also found a serious odor in the refrigerator. Is it caused by the proliferation of bacteria in the refrigerator? If I eat these foods, is it also cause poisoning or illness?

 

Fig.1 people suffered from food stored in refrigerator.

 

Since then, I began to pay attention to the problem of refrigerator bacteria causing illness. In this iGEM competition season, I wanted to do something using synthetic biology to solve this problem, helping some people who suffering from bacteria of refrigerator. So, we discussed this topic in the brainstorm before we started our project. After several times of discussion, we determined this topic.

 

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To comprehensively understand the problem of food contamination in refrigerators, we interviewed Dr. Fan who works in gastroenterology of Shandong Provincial Qianfoshan Hospital (Fig.2).

From the interview we knew that cases caused by fridge bacteria account for 20%-30% in all clinic patients every year, usually occur in spring or summer. The symptoms of refrigerator bacterial poisoning are diverse, but usually mainly gastrointestinal symptoms such as nausea, vomiting, abdominal pain, diarrhea, etc. In severe cases, symptoms such as dehydration and fever may occur. For specific bacterial infections such as Listeria, symptoms like upper respiratory tract infections (such as chills, fever, headache, muscle pain) and central nervous system infections (such as meningeal irritation, ataxia, etc.) may also occur.

 

Fig.2 We interviewed with Dr. Fan who works in Shandong Provincial Qianfoshan Hospital.
He was so busy that the interview activity was conducted in the doctor's duty room.

 

Bacteria that may breed in refrigerators, such as Shigella, Salmonella, Listeria, E. coli etc., are common foodborne pathogens. Bacteria in the refrigerator may not only contaminate food, but also spread to family members through air, hand contact, and other means.

These bacteria can cause food poisoning symptoms in patients by contaminating food, especially for elderly, children, or chronic disease patients with weak immune system who are more susceptible to infection and disease caused by these bacteria. In severe cases, it may even endanger life.

If we develop a method for detecting bacteria in refrigerator, some injuries would be avoided. So, we were going to focus on developing a biosensor for detecting bacteria in fridge in our iGEM project.

 

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It is an opportunity and challenge. How do we develop a method for detecting bacteria in refrigerator to remind people cleaning the fridge? To solve this problem, we searched and read lots of literatures, as well as consulting professor Liu of Shandong University (Fig.3).

 

Fig.3 We consulted Prof. Liu of Shandong University in TenCent Meeting.

 

Prof. Liu told us there are 3 common bacterial detection methods based on molecular recognition: recognition of nucleic acid probe, antigen antibody immune, and nucleic acid aptamer ligand, which are showed in Fig.4. And she introduced the mechanism of these 3 bacteria detection methods.

 

Fig.4 bacterial detection methods based on molecular recognition.

 

After several rounds of discussion, we determined to develop a method for detecting the bacteria of fridge based on the recognition of nucleic acid aptamer ligand. We chose 3 RNA aptamers which can bind specifically to Shigella, Salmonella and E. coli, respectively, to design the RNA Toehold Switch for detecting these 3 bacteria. The mechanism of RNA Toehold switch is showed as follows (Fig.5).

 

Fig.5 Bacteria are detected by the RNA Toehold Switch.

 

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The RNA Toehold Switches are constructed and transcribed in E. coli, then the cell free systems containing RNA Toehold Switches are prepared for making hydrogel, which is used to make a biosensor. The biosensor is connected to a RGB sensor and a pilot lamp, which constitute a detector, showing in the Fig.6. The detail of the detector is showed in HARDWARE page (please refer to Hardware section for details).

 

Fig.6 The detector is composed of biosensor, RGB sensor and a pilot lamp.

 

Put the hardware into the fridge for detecting the pathogenic bacteria in refrigerator. When the bacteria fall to the hydrogel in fridge, it will switch on the RNA Toehold to express lacZ gene and produce β-galactosidase, catalyzing the blue product which are detected by the Biosensor. This information is scanned and transported by RGB sensor to the pilot lamp, making the red light lighting up on the screen of fridge, reminding people to clean the fridge (Fig.7).

 

Fig.7 The Biosensor put in the fridge is used for detecting bacteria.
When the red light lights up on the screen of fridge, it means it’s time to clean the fridge.

 

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We intended to develop a convenient hardware that is put in refrigerator, monitoring the bacteria in fridge. Since this bacteria detector is mainly used at home, we designed a question “How much price increase can you accept a refrigerator with a bacterial detector?” in the survey. The result showed that most people can accept 10% price increase, and the others can accept less than 30% price increase (Fig.8).

After budget, the price of our bacteria detector is estimated about 10 dollars which is lower than 10% price of most refrigerator in market, which means that all interviewers in the survey can accept our bacteria detector in terms of price. So, our product has broad market prospects.

 

Fig.8 The survey result of question “How much price increase can you accept a refrigerator with a bacterial detector?”.
(a), less than 10% price increase; (b), less than 20% price increase; (c), less than 30% price increase; (d), less than 40% price increase.

 

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References

 

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