Team:BS United China/Results

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Result

Protein purification

In order to get protein products and prepare for the subsequent experiment: Evaluate the influence of PVDQ protein on the growth of P. aeruginosa, we purified PVDQ protein from E. bsuahlterminator by conjugating PVDQ protein with His-tag Purification Resin and mearsure concentrations of protein.

Fig.1 Construction of E. bsuahlterminator(Created with BioRender.com)

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 His-tag Purification Resin to bind with PVDQ. The resin can be used to purify His tag protein under non-denaturing conditions. When the protein sample solution passes through His-Tag purification Resin, the histidine residue on His tag of recombinant protein could specifically bind to His-Tag Resin. After washing, His-tag protein can be eluted under non-denaturing conditions and thus isolated and purified.

Our experimental plan was divide into three parts: firstly, purify small amount of protein to get familiar with the operation. Secondly, purify large amount of protein. Thirdly, use SDS-PAGE to run electrophoresis for the protein in order to make sure the activity of PVDQ protein.

Small amount of protein purification

In order to test expression quantity of PVDQ protein and make sure PVDQ can be purified, we first did small amount of protein purification.

First, we centrifuged and collected 1 ml of bacterial precipitate into a EP tube, and added 100 ml of non-denatured lysate with a slight vortex. Then, 1 mg/ml lysozyme was added, and the tube was placed on ice for 30 min and then vortex. Next, centrifugation（15000 g×10 min）was performed and supernatant was collected into a new tube; then 20 ml His-Tag Purification Resin was added, and the tube was cultured in 4℃ shaking table for 30 min. The tube was centrifuged（1000 g×10 s） to precipitate gel. 100 ml non-denaturing solution was then added into the second tube and the gel was resuspended, and the tube was centrifuged; this process was repeated for 3 times. In order to separate Resin and the protein, we then added 20 ml non-denaturing eluent. The gel was resuspended and supernatant was collected.

Fig.2 Blue gel from small amount of protein purification

Large amount of protein purification

To get more pure and more concentrated protein, and to prepare for the subsequent experiment: Evaluate the influence of PVDQ protein on the growth of P. aeruginosa, we did large amount of protein purification.

There were two differences of operation between large amount of protein purification and small amount of protein purification.

Fig.3 Mass bacterial culture

The first one was that after adding lysozyme, we used ultrasonic cracking (200-300 W 10 s) 6 times, and each interval is 10 seconds. This step was for a more complete lysis of cells. Because the amount of cells was large, we need make sure every single cell was disrupted completely. The second difference is that we did washing and eluting in a special instrument--affinity chromatography column.

Fig.4 Affinity chromatography column

The affinity chromatography column is made of high purified polypropylene, with a height of 66 mm and an inner diameter of 12.7 mm. The sieve plate is made of high purified ultra-high molecular weight polyethylene (UHWMPE); it has low adsorption, great hydrophilicity, and uniform aperture(about 50 microns). This tube can be used to fill Ni-NTA, and it is suitable for the separation and purification of proteins with His tag.

The appropriate aperture of the sieve plate on the affinity chromatography column ensures a flow rate of approximately 1-2 ml/min under gravity. When we added gel into this column, protein would pass though the sieve plate and others would attach on it, so we can make sure the protein was separate completely with other thing we did not need.

SDS-PAGE

In order to make sure the success of protein purification and detect concentrations of PVDQ protein, we used SDS-PAGE to run electrophoresis for the protein.

Form.1 Frequently-used SDS-PAGE gel composition

Fig.5 SDS-PAGE result of target proteins

E1：Marker

E2: PVDQ protein processed by PVS

E3: PVDQ protein processed by 17% acetic acid

E4: PVDQ protein processed by hydrochloric acid

The SDS-PAGE result showed that the target protein was not lost activity. The normal length of PVDQ protein is between 45 kDa to 70 kDa. The lengths of target proteins are 50 kDa and 45 kDa.

Fig. 6 Protein after the eight elution under fluorescence microscope

We also used fluorescence microscope to check the activity of PVDQ protein. If there was appearance of fluorescence, it meant that the protein clump together and some may lose activity. But our result was ideal: we were basically invisible to fluorescent under microscope.

Measurement of growth curve of P. aeruginosa

In order to demonstrate the inhibitory effect of PVDQ protein on P. aeruginosa growth, we first cultivate P. aeruginosa for 7 hours; second, we set up 4 groups: one controlled group and three experimental groups with 15 ml P. aeruginosa solution respectively. We purified protein and chose three groups of protein with different elution times and measured their concentrations.

Next, we added three group of protein into three conical flasks containing P. aeruginosa solution and measured growth curve of P. aeruginosa.

Fig. 7 Growth curve of the P.aeruginosa

Apparently, the higher the PVDQ protein concentration, the better the bacteria growth.

LB solid medium simulation

To further testify the inhibitory effect of PVDQ protein on P. aeruginosa, we also used LB solid medium to simulate food and cultured P.aeruginosa on LB plate. There are two groups: controlled group with only P. aeruginosa and experimental group with P. aeruginosa and PVDQ protein; each group has three plates.

Fig.8 LB solid medium culture result

Form.2 Average number of P. aeruginosa

According to the table, the number of strains grown in the experimental group was much smaller than that in the control group, so PVDQ protein has an inhibitory effect on P. aeruginosa.

Fresh food test

To test the effect of PVDQ protein on P. aeruginosa in an actual environment, we brought fresh food to do this experiment. We have two groups: experimental group and control group with identical amounts of fish and shrimp respectively. Then we spayed 0.62 mg/ml PVDQ protein onto fresh food of the experimental group while did nothing to the control group. After putting those samples in a 4℃ refrigerator for six hours, we did agarose gel electrophoresis to test the amount of P. aeruginosa in each sample.

Fig.9 AGE result of fresh food samples

The result shows that E1 and E2 are lighter than E3 and E4 while E5 and E6 are lighter than E7 and E8. The figure indicates that the number of P. aeruginosa in the experimental group, which has PVDQ protein, is less than the control group, so PVDQ protein has an inhibitory effect on P. aeruginosa.

Summary

In order to test the effect of PVDQ protein on P. aeruginosa, we purified PVDQ protein than added it into P. aeruginosa solution, measured the growth curve of P. aeruginosa; we also use LB solid medium and fresh food to simulate real environment. The conclusion is: PVDQ protein does has inhibitory effect on P. aeruginosa. However, there are more we need to consider: whether PVDQ protein can inhibit other bacteria, how long dose PVDQ protein can protect food, and how can we lower the cost so that more people can benefit... We will continue to explore.