Team:GreatBay United/Proof Of Concept

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• Artificial Limulus amebocyte lysate
• Construction of HCV
• Enzyme Activity Test
• Testing agglutination system with HCV
• Conclusion

Artificial Limulus amebocyte lysate

Artificial Limulus amebocyte lysate is aimed to detect gram positive bacteria by detecting the LPS (lipopolysaccharide) part of bacterial endotoxin. By giving visual test results, Artificial Limulus amebocyte lysate have a wide range of applications in daily lives as no professional equipment is needed. As it replaced the function of limulus amebocyte lysate whose main component is tachypleus amebocyte lysate blood, it can contribute to the protection of tachypleus amebocyte lysate who is already endangered.

In artificial Limulus amebocyte lysate, the agglutination system plays an important role as it is the key to giving the visual result by agglutination reaction. By using yeast surface display, we designed inhibited lectin(GFP) on Yeast A which can be activated by using HCV protease to cut the substrate between lectin and inhibitor, allowing lectin(GFP) to bind with agglutinogen (anti-GFP) on Yeast B and trigger yeast agglutination between these two kinds of yeasts. In this way, HCV acts as a signal from the detection and cascade system to make the agglutination system show a positive result.

Construction of HCV

We used pEB28b vector to construct the plasmid for expression of HCV. The sequences code for HCV is inserted into the plasmid, and then the plasmid is transformed into two types of protein expressing strain: E.coil BL21 and Roseeta.

We used pEB28b vector to construct the plasmid for expression of HCV. The sequences code for HCV is inserted into the plasmid, and then the plasmid is transformed into two types of protein expressing strain: E.coil BL21 and Roseeta.
In order to induce the protein expression, we transformed the bacteria fluid into a test tube and cultured the bacteria at 37℃, 220rpm overnight. Then we transformed it again into the conical flask with inoculation with a cultural medium by 1:200 and cultured it again at 37℃, 220rpm. Until the OD600 reaches 500-600，cooled down at 16℃,220 rpm overnight.
The proteins expressed are then purified and underwent rotein electrophoresis for checking the size of proteins.

Enzyme activity test

Analysis and dealing of experimental data

Experimentally, we tested the dynamic behavior of the enzymatic system, recording time-course data of fluorescence intensity, in conditions of HCV protease volume.The results showed that the reaction trends of different groups is the same, and the fluorescence signal intensity is positively correlated with the enzyme amount at the initial stage. When the substrate is completely reacted, the fluorescence signal intensity tends to be consistent.

We also tested the dynamic behavior of the enzymatic system, recording time-course data of fluorescence intensity, in conditions of substrate concentrations.The results showed that the fluorescence signal intensity of the reaction has the same change trend. When the substrate is completely reacted, the higher the substrate concentration, the stronger the fluorescence signal intensity.

In this experiment, fluorescence modified peptide substrate was successfully used to test the enzyme activity of HCV, and Mie's equation was calculated by mathematical modeling analysis.

Testing agglutination system with HCV

We used HCV to verify our construction of agglutination system. In our design,after react Yeast A and HCV together, the substrate and inhibitor anti-GFP should be cut and the GFP should be activated.

We constructed a Yeast C which express GFP alone to simulate the form of Yeast A after the reaction, and react it with Yeast B. We gained a series of agglutination phenomenons. Then we mixed Yeast A and Yeast B with the addition of HCV protease.By comparing the agglutination results gained this time with the one after Yeast B and Yeast C react, we find that the agglutination phenomenon is similar. This means that HCV successfully cut the substrate and activate GFP to allow agglutination happen as we designed.

We also tested the dynamic behavior of the enzymatic system, recording time-course data of fluorescence intensity, in conditions of substrate concentrations.The results showed that the fluorescence signal intensity of the reaction has the same change trend. When the substrate is completely reacted, the higher the substrate concentration, the stronger the fluorescence signal intensity.
The test verified that our agglutination system is constructed successfully.

Conclusion

The HCV protease works as an important part in our experiment, it connects the cascade system and agglutination system. During the proving of concept, we ran the protein assay to test the concentration of HCV and also used the HCV protease to test the agglutination system. Both experiments’ results showed that our plasmid of HCV was successfully constructed and the activity of the HCV protease was high. Hence, our agglutination system was constructed successfully.