Our team focus on inventing possible helicase inhibitor and aim to design an efficient drug screening kit to test whether a substance has the function of an inhibitor or not. Therefore, We added new parts based on the pet28a: pET28a-NS3 HEL/pET28a-NS3 ZIKA (Part: BBa_K4862002/BBa_K4862003). The new parts successfully break the double strands of the ZIKA and Hel genes into a single strand with a stem-loop structure. The fluorescence group reacts with the BHQ quencher, which results in a decrease in the fluorescence value. This suggests that NS3 enzymes can be used as targets for the search for dengue inhibitors.

The pET series is a widely used vector of protein expression and reproduction; the main reason we chose the pET28a plasmid as the vector of the experiment is because of the 6*his tag, the Kanamycin Resistance Gene of the plasmid, and the T7 tag the plasmid is carrying.

As the graph shows, the residue of histidine (His) is part of the plasmid pET28a. HIS has an imidazole group, which could bind itself to transition metal ions, for example, Ni2+, and form a coordination bond with the metal ions. Therefore, proteins with HIS tag could selectively bind to the medium while passing the chromatographic medium assembled with metal ions; the impurity proteins would not bind or only weakly bind. The protein that carries the HIS tag, which is attached to the medium, could be eluted by increasing the imidazole concentration in the buffer solution, as the HIS tag could also be attracted by imidazole. [1]

Kanamycin Resistance Gene: Resistance of the growth of necessary bacteria While increasing the bacteria we need, we must resist the development of the ones we do not need. As the plasmid pET28a is carrying KanR, adding a few Kanamycin microliters could hinder other bacteria's growth. Proliferation occurs after heat shock is processed, which means the plasmid is already inserted into the bacteria. T7-promoter and T7 terminator

T7 RNA polymerase has high promoter specificity and only transcribes DNA or T7 DNA copies located downstream of the T7 promoter in T7 bacteriophages. In the vector pET28a, the T7 promoter controls gene expression. The lac operator sequence is added downstream of the promoter to reduce basal expression. The word of the vector needs IPTG induction to relieve inhibition of the lacUV5 promoter and the lac operator. Word. We regulate the T7 RNA polymerase expression in E. coli BL21 (DE3) to provide more host options for recombinant protein production.

Both pET28a-NS3 HEL/pET28a-NS3 ZIKA are the final plasmid constructions. The function of them is to express proteins.

RNA helicase is responsible for almost all metabolism of RNA; in the Dengue virus, NS3 protein contains the activity of multiple enzymes, including helicase. Insert NS3 sequence is for expressing the protein we need and testing whether the medicine finally injected would be effective. The other structure of the pET28a plasmid would not be changed. [2]

While these two plasmids are constructed, they would be transplanted into BL21 bacteria, which can express the NS3 protein we need. After growing for a few hours, the solution (these two plasmids and the liquid medium) would be ultrasonically crushed, destroying the bacteria's basic structure. The answer, consisting of the proteins and bacteria fragments, would be purified for extracting the NS3 proteins.

The NS3 proteins have the ability as a Helicase, which would activate the helicase to work. The protein would be added to a fluorescence DNA vector to observe whether the helicase is working. If so, the fluorescence would disappear; if not, the fluorescence would be observed, which also means the medicine for restraining the replication of DNA is working. As NS3 proteins are the main protein active helicase in the Dengue virus, if the ability is restricted, the duplication of the RNA of the bacteria would be stopped, which means the spread of the virus would be halted.

[1] Malhotra,Arun.[methods in enzymology] guide to protein purification, 2nd edition volume 463 || chapter 16 tagging for protein expression[J]. 2009:239-258.DOI:10.1016/S0076-6879(09)63016-0.

[2] Jain, R., Coloma, J., García-Sastre, A., & Aggarwal, A. K. (2016). Structure of the NS3 helicase from Zika virus. Nature Structural & Molecular Biology, 23(8), 752-754. doi:10.1038/nsmb.3258