ENGINEERING SUCCESS
Questions which are important to local people
Staphylococcus aureus (S. aureus) is a common pathogen that the enterotoxins released by it is a sever pathogenic factor of food poisoning cases. S. aureus doesn’t need restricted conditions to grow; It has great resistance to arid, heated and saliferous conditions, therefore spreading and surviving widely in the environment around people. S. aureus is ubiquitous in nature which can be found in air, water, dust and human and animal’s waste. As a result, People can easily be exposed to S. aureus and get infected. Nowadays, S. aureus has become a worldly health problem. Reported by the U.S. Centers of Disease Control, about 25% food poisoning cases are caused by S. aureus infection in China, about 33% in the U.S., and even about 45% in Canada. As people infected by S. aureus, it will cause detrimental diseases as pneumonia, pericarditis, sepsis and death. In the hospital or communities, people catch S. aureus through contact infection and the morbidity rate in a developed country is between 100,000 to 300,000 people each year. According to data from the Emerging Infections program (EIP) and Cerner Electronic Health Record databases, 19,832 associated deaths occurred in an estimated 119,247 S. aureus bloodstream infections in 2017. To understand the spreading range of S. aureus, we construct the following experiments.
Primers:
Escherichia coli (E.coli) primers:
F1: GACGTTACAGCTGCCGGT
R1: TCCCGTGGTTCTGAC
S. aureus primers:
F2: TCCTTACTTACACGA
R2: TTATTTCAGGATAT
Materials
1. Genome DNA isolation: Kit from Beyotime biotechnology
2. PCR reaction: Phusion enzymes (M0530S)
3. DNA electrophoresis： 100V run for 1-2 hours with 5 ul ER staining for 100 ml 1% agarose gel.
Experiment Design：
Results：
Fig.1 DNA electrophoresis from PCR.
According to the results above, we can conclude that in soil, river water and fish, they contain both E.coli and S. aureus’s DNA; In tap water, it contains considerable amount of S. aureus’s DNA. To conclude, the S. aureus infection is a adverse factor that threaten people’s health and it is very common in our daily life, driving us to inhibit the growth of S. aureus in food.
The model of part 1
Fig.1.1 Schematic model of Part1 system.
If there is no IPTG TurboID will not be activated. If we add IPTG, it will activate TurboID and eventually complete the hole process.
The model of part 2
Fig.2.1 Schematic model of Part2 system.
If there is no IPTG, Dethiobiotin synthase will not be activated.
Fig.2.2 Schematic model of Part2 system.
If we add IPTG, it will activate Dethiobiotin and eventually complete the hole process.
Materials in the part 1
1. TurboID-AgrD particles
2. ATP powder
3. Biotin powder
4. Engineering Bacteria
5. Strepavdin-568
Materials in the part 2
1. Dethiobiotin synthase particles
2. ATP powder
3. Biotin powder
4. Engineering Bacteria
5. Strepavdin-568
Procedures in part 1:
1. Expression of TurboID-AgrD protein in E. coli.
2. Staining Staphylococcus aureus (S. aureus) by using strepavdin-568.
3. Tracking the growth of S. aureus.
Procedures in part 2:
1. Expression of Dethiobiotin synthase in E. coli.
2. Staining S. aureus by using strepavdin-568.
3. Tracking the growth of S. aureus.
Fig.2.3 TurboID expression in E. coli induced by IPTG
Results of part part1:
Fig.1.2 TurboID-AgrD stained S. aureus.
We can clearly know that the one, which TurboID is activated, streptavidin is successfully labeled on the bacteria’s DNA. The one, which TurboID is not activated, nothing happened.
Fig.1.3 The growth curve of TurboID-AgrD stained S. aureus.
As we can see from the data, the growth of TurboID-AgrD stained S. aureus is inhibited in growth.
Fig.1.4 The growth rate of TurboID-AgrD stained S. aureus is 2 folded slower than unstained S.
Fig.2.4 TurboID-AgrD stained S. aureus.
We can clearly know that the one, which part 1 and part 2combine together, generate a greater effect.
Fig.2.5 The growth curve of TurboID-AgrD stained S. aureus.
As we can see from the data, the growth of TurboID-AgrD stained S. aureus is inhibited in growth.
Fig.2.6 The growth rate of TurboID-AgrD stained S. aureus is 5 folded slower than unstained S. aureus.
The engineering success since when we compare the result is more significant when Part 1 and Part 2 combine.
Testing TurboID-AIP toxicity to human cells
Purpose:
The purpose of the experiment is to evaluate the damage of S. aureus enterotoxin toward human cells, and the amount of enterotoxin produced by Biotinylated S. aureus. We intended to use the apoptosis rate of epithelial cell to reflect the production of enterotoxin.
Design:
We divided S. aureus into two groups. One is the control group with lysate extracted only from S. aureus. Another is a lysate extracted from TurboID-AIP and biotin blocked S.aureus.
Materials:
1. Inner lining epithelial cell sample in human mouth
2. Culture media containing bovine serum
3. S. aureus DNA
4. Biotin powder
5. S. aureus Lysate
6. Biotin labeled S aureus Lysate
S. aureus Lysate preparation:
1. Extract 1ml of S. aureus (OD0.3)
2. S. aureus in bovine serum lysed via supersonic schizolysis method.
Biotin labeled S. aureus Lysate preparation:
3. Let Biotin reacts with S.aureus.
4. Collect 1ml of S. aureus (OD0.3) after centrifugation.
5. S. aureus in bovine serum lysed via supersonic schizolysis method.
Fluid preparation for flow cytometry:
1. Scrape epithelial cells with a dentiscalprum.
2. Separate the sample into two parts.
3. Mix the samples with S. aureus Lysate and Biotin labeled S. aureus lysate respectively (10 minutes).
4. Centrifugation.
5. Discard the supernatant.
6. Resuspend the cell pellet for 15 minutes.
7. Collect precipitated cells (1400rpm) after centrifugation.
8. Analyze the fluid with flow cytometry.
Result:
It is presented that in figure 3, 5, 7, the enterotoxin damages the epithelial cells. In figure 4, 6, 8, the apoptosis rate of cells declined indicating that the damage of enterotoxin decreased. Thus, we can conclude that our project can successfully block the production of enterotoxin in S. aureus.
Fig.3 Analysis of epithelial cell of subject 1 with S. aureus lysate from (A) to (C);
(A) size of the cells. (B) width of cells. It eliminates the duplicates which are useless to the Analysis. (C) The apoptosis rate of cells.
Fig.4 Analysis of epithelial cell of subject 1 with biotin labeled S. aureus lysate from (A) to (C);
(A) size of the cells. (B) width of cells. It eliminates the duplicates which are useless to the Analysis. (C) The apoptosis rate of cells.
Fig.5 Analysis of epithelial cell of subject 2 with S. aureus lysate from (A) to (C);
(A) size of the cells. (B) width of cells. It eliminates the duplicates which are useless to the Analysis. (C) The apoptosis rate of cells.
Fig.6 Analysis of epithelial cell of subject 2 with biotin labeled S. aureus lysate from (A) to (C);
(A) size of the cells. (B) width of cells. It eliminates the duplicates which are useless to the Analysis. (C) The apoptosis rate of cells.
Fig.7 Analysis of epithelial cell of subject 3 with S. aureus lysate from (A) to (C);
(A) size of the cells. (B) width of cells. It eliminates the duplicates which are useless to the Analysis. (C) The apoptosis rate of cells.
Fig.8 Analysis of epithelial cell of subject 3 with biotin labeled S. aureus lysate from (A) to (C);
(A) size of the cells. (B) width of cells. It eliminates the duplicates which are useless to the Analysis. (C) The apoptosis rate of cells
Protecting food from S. aureus contamination
Description: This experiment is conducted to testify the effectiveness of our products to inhibit the growth of S. aureus. Our samples have been made by mixing TurboID-biotin fusion protein with food DNA extracting solutions. After that, we use PCR to amplify DNA of our samples and confirm the existence of S. aureus and E. coli in electrophoresis process.
Design:
Procedure：
1. Genome DNA isolation: Beyotime
a. Lysozyme solution was prepared: 20 mM Tris, pH 8.0, 2 mM EDTA, 1.2% Triton x-100, 20 mg/ml lysozyme. Lysozyme was added before use. Centrifugation will collect up to 2 billion bacteria and discard the supernatant.
b. Bacteria were fully resuspended with 180 microliters of lysozyme solution. Incubate at 37 ° C for 30 min to lyse bacteria. Centrifuge as the same speed to remove the supernatant. Add 200 microliters of absolute ethanol and Vortex mix.
c. (Ethanol must be thoroughly mixed after addition, otherwise it will seriously affect the extraction effect. White precipitate may be produced after adding ethanol, which is a normal phenomenon. In the following steps, the white precipitate and solution must be transferred to the purification column).
d. Add the mixture from step c to the DNA purification column. Centrifuge at least 6000g (Approximately ≥ 8000rpm) for 1 minute. Discard the liquid in the waste liquid collection tube. Add 500 microliters of washing liquid I, centrifuge at least 6000g (about ≥ 8000rpm) for 1 minute. Discard the liquid in the waste liquid collection tube. Add 600 microliters of washing solution II, centrifuge at least 18000g (about ≥ 12000 rpm) for 1 minute. Discard the liquid in the waste liquid collection tube. Then centrifuge at least 18000g (about ≥12000rpm) for 1 min to remove the residual ethanol.
e. The DNA purification column was placed on a clean 1.5-ml centrifuge tube and 50-200 microliters of eluate were added. Leave at room temperature for 1-3 minutes. Centrifuge at ≥12000rpm for 1 min. The resulting liquid is the purified total DNA.
PCR reaction: Phusion emzymes
Solution:20ul reaction:10 uM forward primer 1ul, 10uM reverse primer 1ul, taq enzyme mix 8ul, template DNA 2ul,Nuclease-free water 8ul; for experimental group, 10 uM forward primer 1ul, 10uM reverse primer 1ul, taq enzyme mix 8ul, template DNA 2ul, TurboID-Biotin fusion protein 1 ul, Nuclease-free water 8ul
Condition:initial denaturation:98 30seconds,30 cycles 98 10seconds,68 30seconds,72 30seconds,final extension 72 10seconds,hold 4
DNA electrophoresis：200V run for 30-60min, solution:5 ul ER staining for 100 ml 1% agarose gel.
Results:
Fig.9 DNA electrophoresis of control groups and experimental groups from PCR
Conclusion:
The results shown above reveal that the amount of S. aureus contained in samples treated with TurboID-biotin is less than that of the groups without any treatment. For example, we can clearly notice that the group of shrimp DNA with TurboID-biotin mixture reacted for 60 minutes have more vague figure of stripes ,the one contains same base pairs with S. aureus, compared with the group includes sorely shrimp DNA. We can conclude that our products have effects on preventing S. aureus growth.