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Description

BACKGROUND

It was estimated that each year 31 major pathogens have caused 9.4 million episodes of food-borne illness by bacteria and viruses, 55,961 hospitalizations, and 1,351 deaths in the United States ¹. Key facts by the world health organization (WHO): An estimated 600 million fall ill after eating contaminated food and 420,000 die every year, resulting in the loss of 33 million healthy life years. Children under 5 years of age carry 40% of the food-borne disease burden, with 125 000 deaths every year. US$110 billion is lost each year in productivity and medical expenses resulting from unsafe food in low- and middle-income countries ².

THEORY

Microorganisms often persist in their natural niches by attaching to surfaces and forming complex, sessile microbial communities known as biofilms. P. aeruginosa is one of the most common food pathogens which is able to form biofilms either independently by itself or jointly with other microorganisms. When the concentration of quorum sensing (QS) molecule, AHL, secreted by pathogens in the environment is above the threshold, P. aeruginosa will be coordinated by QS signal to construct the biofilms.

Biofilms exist everywhere in our daily life while its cleaning is still a difficult issue to be solved. We propose an innovative strategy to control unwanted P. aeruginosa biofilm growth through the development and application of an engineered E. coli with QS disabling function. This synthetic biology work will eventually provide the market with effective, stable, and non-toxic alternatives for controlling food contamination caused by P. aeruginosa and other pathogens.

DESIGN

P. aeruginosa is one of the most common food pathogens which is able to form biofilms either independently by itself or jointly with other microorganisms. How close are we to pathogenic microorganism? We utilize PCR system to verify that P. aeruginosa is in high concentration in the environment even on leaves and in soils.

Fig.1 Results of concentration of P. aeruginosa through PCR system. Group1 is 5000bp marker, group 2 and 3 are P. aeruginosa, group 4 and 5 are PCR results of soil samples, and group 6 and 7 are PCR results of leaves samples.

Form 1. PCR Primer

Our E. bsuahlscout bio-brick contains 3 key elements including LuxR, QS promoter, and mCherry. LUXR is a protein coded by LuxR gene. The LuxR gene is regulated by T7 promoter, Lac operator and T7 terminator. The LUXR protein can combine the AHL to formate a LUXR-AHL complex which can bind to the QS promoter and recruit RNA polymerase to start the down-stream gene expression. QS promoter is a DNA element to react with LUXR-AHL complex. The mCherry gene is down-stream report gene of QS promoter and codes a red fluorescence protein. As shown in figure2, we originally decided to make bio-brick consist of T7 promoter, Lac operator, LuxR, QS promoter, mCherry, QS promoter, PVDQ, and GFP.

Fig.2 Original design of the construction of engineering bacteria (created with BioRender.com)

T7 promoter serves as starting the whole synthetic gene. Lac operator is used for releasing LuxR. LuxR functions as receiving AHL signal molecules, and activates mCherry red fluorescence gene. QS promoter is the place that LuxR can recruit RNA polymerase. mCherry is a red fluorescence reporting gene. PVDQ protein functions as restraining P. aeruginosa from growing and activating GFP gene. GFP is a green fluorescence reporting gene.
Through our interview with a branch store manager of Yonghui superstore, we found that the market wishes to use E. bsuahlscout to identify the freshness of food they stock. More essentially, they require PVDQ protein in E. bsuahlterminator being able to extend the food shelf-life. As a result, we solely create E. bsuahlscout bacteria.
The bio-brick for E. bsuahlscout, which is synthesized by Beijing Genomics Institution, aims at quantize the degree of food pollution.

Fig.3 Construction of E. bsuahlscout (Created with BioRender.com)

To affirm that the plasmid is effective, we transformed it into E. coli BL21, which is a competent cell. E. bsuahlscout was induced by IPTG to express LuxR protein, and was mixed with P. aeruginosa supernatant, which made it red, and use fluorescence microscope to observe it. After we confirmed that E. bsuahlscout is effective, we used N-Acyl Homoserine Lactone (AHL) of different concentration to induce E. bsuahlscout to generate red fluorescence protein, so as to draw the standard curve of AHL concentration versus red fluorescence intensity.

Fig.4 standard curve of AHL concentration versus red fluorescence intensity

Similarly, we help our cooperation team NEU_CHINA to measure in 1-16 hours, their engineering bacteria could generate how much AHL, by using E. bsuahlscout to detect the red fluorescence intensity. Besides, we request our cooperation team Jilin_China to use micro-reader to caculate the mCherry intensity which shows the lowest concentration of AHL and draw the standard curve.
We used P. aeurginosa to induce E. bsuahlscout to produce red fluorescence protein, and prepared 4 samples, which are without supernatant and IPTG, without supernatant with IPTG, with LB medium with IPTG, and with NB with IPTG. we concluded that as the concentration of P. aeruginosa increases, the quantity of AHL would increase, leading to that more E. bsuahlscout is induced and more red fluorescence proteins express.

Fig.5 The bio-brick in E. bsuahlscout is activated by the AHL released from P. aeruginosa.

We made a kit, a product of E. bsuahlscout , which is user-friendly, especially for middle-aged and elder people, to quickly detect AHL on food surface.

Fig.6 Product of E. bsuahlscout

The bio-brick of E. bsuahlterminator, synthesized by Beijing Genomics institution, is a fused protein.

Fig.7 Construction of E. bsuahlterminator (Created with BioRender.com)

The advantages of it is that it is more stable than PVDQ protein itself, and fully exert enzyme activity as well. Besides, it also has a reporting system GFP protein, which can indicate the amount it generated. To affirm that the plasmid is effective, we transformed it into E. coli BL21, which is a competent cell. E. bsuahlterminator was induced by IPTG to express LuxR protein, and was mixed with P. aeruginosa supernatant, which made it green, and use fluorescence microscope to observe it. We concluded that E. bsuahlterminator can restrain the generation of bio-toxin.
We used PVDQ protein of different concentration, which are E1, E5, E8, to restrain P. aeruginosa, and drew the growth curve of P. aeruginoisa in each group.

Fig.8 Growth curves of P. aeruginosa with no PVDQ, with E1 PVDQ (High concentration of PVDQ), with E5 PVDQ (low concentration of PVDQ), and with E8 PVDQ (low concentration of PVDQ)

We used Histag kit to make small amounts of purification first, and large amounts of purification secondly. Then, we used sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) and Commassie blue staining solution to demonstrate that PVDQ is biodegradable in human stomach, which is simulated by 37 Celsius degree and hydrochloric acid.

Fig.9 pictured by glue illuminator (group1 is control group, while group2 is the group reacting with acetic acid, and group3 is the group reacting with hydrochloric acid)

Through this experiment, we summarized that PVDQ protein is biodegradable. We also imitated the process of PVDQ protecting food. We found it can shield food from toxin generated by bacteria.

Fig.10 PVDQ protein is inhibiting the growth of P. aeruginosa

We fabricated a mist spray product, containing PVDQ protein solution, and we sprayed it onto fish, shrimp, and meat, proving it effectively protect food, through PCR system.

Fig.11 Product of E. bsuahlterminator

Form 2. PCR Primer ³

Fig.12 PVDQ protein is protecting the fish, shrimp, and meat from P. aeruginosa contamination

We make E. bsuahlscout and E. bsuahlterminator apart, which is definitely an improvement, since it is easier to purify, and it can produce more PVDQ protein. We purify PVDQ protein from E. bsuahlterminator, which is different from bacteria. According to our interview, the market request an extending-shelf-life product which is able to spray onto food surface. That means it must be not harmful to human health after people eat food, which results in making PVDQ protein supernatant since E. coli is conditioned pathogen which is possibly detrimental to human. Additionally, we use hydrochloric acid to simulate gastric acid to ensure that PVDQ protein is able to be decomposed by human stomach.
We did all our experiments in Professor Fu’s laboratory, which strictly control biology safety. During our experiments, we all followed laboratory safety rules.

REFERENCE

1. Scallan, E, Hoekstra, RM, Angulo, FJ, Tauxe, RV, Widdowson, MA, Roy, SL, Jones JL and PM Griffin, 2011. Foodborne Illness Acquired in the United States—Major Pathogens. Emerging Infectious Diseases, 17(1), 7-15. https://doi.org/10.3201/eid1701.p11101
2. WHO Food safety https://www.who.int/news-room/fact-sheets/detail/food-safety
3. Gholami, A. , et al. "PCR-based assay for the rapid and precise distinction of Pseudomonas aeruginosa from other Pseudomonas species recovered from burns patients." Journal of Preventive Medicine & Hygiene 57.2(2016):E81-E85.