Overview

Dengue is a highly transmissible disease. The incidence of Dengue has grown dramatically around the world in recent decades. According to WHO or the World Health Organization, the reported cases increased from 505430 points in 2000 to 5.2 million in 2019.[1]

The world is in desperate need of medical treatment targeted to cure Dengue. Since helicase plays an essential role in virus replication and is a very promising target for the development of specific antiviral inhibitors,[2] 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.

Figure 1. Experiment Flowchart

Cycle 1 [pET-NS3-ZIKA]

· Design stage

Figure 2. Construction design of plasmid pET-NS3-ZIKA

We designed to use enzyme digestion to connect the ZIKA gene with the PET28a plasmid. To do that, we first obtain the NS3-ZIKA through PCR cloning. Then, we use double enzyme digestion with BamHI, XhoI, CutSmart, and ddH2O to connect the ZIKA gene with PET28a.

· Build stage

Figure 3. Electrophoresis gel and sequencing of pET-NS3-ZIKA

Per the construction design, we obtain the plasmid pET-NS3-ZIKA and In order to express the DNA as protein, we use heat shock conversion that inserts PET28a-NS3-ZIKA into BL21(DE3), a type of Escherichia coli. After this, we transplant the bacteria by touching the single colony linings of PET-NS3-ZIKA with the pipette gun and leaving the pipette in the LB broth for the 16-hour incubation.

Then, we amplifed our target ZIKA DNA sequence by PCR and then extracted the plasmid with buffer SP1, SP2, SP3, washing solution, and water. Electrophoresis gel was conducted to valid the successful result as shown in figure 3, the strip sizes of PET-NS3-ZIKA were detected in 1854 bp. In addition, the sequencing result also supported this conclusion.

· Test stage

Since we have obtained the BL21 pET-NS3-ZIKA, firstly we need to check if this part pET-NS3-ZIKA could work as expected to express the protein.

Figure 4. SDS-PAGE gel indicating NS3-ZIKA’s kDa value

We use IPTG to express the ZIKA gene through BL21(DE3) while preparing the 15% SDS-PAGE gel. We break the bacteria with an ultrasonic cell breaker device. We collected the unpurified ZIKA protein solution by adding HIs buffer A to the centrifuge. To purify the protein solution, we utilize Ni-NTA His Tag Purification. Per the SDS-PAGE shown in figure 4, the protein NS3-ZKA was detected in 49 kDA around.

Figure 5. FRET curves of pET-28a-NS3-ZIKA and pET-28a
*Our team measured the fluorescence using fluorometers with an excitation of 620 nm and an emission wavelength of 685 nm.

Secondly, we further sued FRET method to check if the protein NS3-ZKA possess helicase activity. We added fluorescence and ATP into the NS3-ZIKA pure protein solution to test whether the helicase is inactive. According to the pET-28a-NS3-ZIKAcurve comparing to the control pET-28a in Figure5，the fluorescence value decreases, whereas protein concentration increases. Therefore, we can prove that the NS3 protein possesses helicase activity.

· Learn stage

After through the plasmid design, build, and test, we could conclude that the whole engineering of the part pET-NS3-ZIKA is successful. Besides, we will also apply the FRET data to build a model to evaluate the unspinning ability of the protein pET-NS3-ZIKA as a helicase.

In addition, we also noticed that the SDS-PAGE is not very clear and we will further work on this improvement of the protein purification as the next move to continue this cycle.

Cycle 2[ pET-NS3-Hel ]

· Design stage

Figure 6. Construction design of plasmid pET-NS3-Hel

We designed to use enzyme digestion to connect the Hel gene with the PET28a plasmid. To do that, we first obtain the NS3-Hel through PCR cloning. Then, we use double enzyme digestion with BamHI, XhoI, CutSmart, and ddH2O to connect the Hel gene with PET28a.

· Build stage

Figure 7. Electrophoresis gel and sequencing of pET-NS3-Hel

Per the construction design, we obtain the plasmid pET-NS3-Hel and In order to express the DNA as a protein, we use heat shock conversion that inserts PET28a-NS3-Hel into BL21(DE3), a type of Escherichia coli. After this, we transplant the bacteria by touching the single colony linings of PET-NS3-Hel with the pipette gun and leaving the pipette in the LB broth for the 16-hour incubation.

Then, we amplifed our target Hel DNA sequence by PCR and then extracted the plasmid with buffer SP1, SP2, SP3, washing solution, and water. Electrophoresis gel was conducted to valid the successful result as shown in figure 7, the strip sizes of PET-NS3-Hel were detected in 1344bp round. In addition, the sequencing result also supported this conclusion.

· Test stage

Since we have obtained the BL21 pET-NS3-Hel, firstly we need to check if this part pET-NS3-Hel could work as expected to express the protein.

Figure 8. SDS-PAGE gel indicating NS3-Hel’s kDa value

We use IPTG to express the Hel gene through BL21(DE3) while preparing the 15% SDS-PAGE gel. We break the bacteria with an ultrasonic cell breaker device. We collect the unpurified Hel protein solution by adding HIS Buffer A to the centrifuge. To purify the protein solution, we utilize Ni-NTA His Tag Purification. Per the SDS-PAGE shown in figure 8, the protein NS3-Hel was detected in 49 kDA around.

Figure 9. FRET curves of pET-28a-NS3-Hel and pET-28a
*Our team measured the fluorescence using fluorometers with an excitation of 620 nm and an emission wavelength of 685 nm.

Secondly, we further sued FRET method to check if the protein NS3-Hel possess helicase activity. We added fluorescence and ATP into the NS3-Hel pure protein solution to test whether the helicase is inactive. According to the pET-28a-NS3-Hel curve comparing to the control pET-28a in Figure 9，the fluorescence value decreases, whereas protein concentration increases. Therefore, we can prove that the NS3 protein possesses helicase activity

· Learn stage

After through the plasmid design, build, and test, we could conclude that the whole engineering of the part pET-NS3-Hel is successful. Besides, we will also apply the FRET data to build a model to evaluate the unspinning ability of the protein pET-NS3-Hel as a helicase.

In addition, we also noticed that the SDS-PAGE is not very clear and we will further work on this improvement of the protein purification as the next move to continue this cycle.

References:

[1]World Health Organization: WHO & World Health Organization: WHO. (2023). Dengue and severe dengue. www.who.int. https://www.who.int/news-room/fact-sheets/detail/dengue-and-severe-dengue.

[2]Belon C A , Frick D N .Monitoring helicase activity with molecular beacons[J].Biotechniques, 2008, 45(4):433-440.DOI:10.2144/000112834..