iGEM 2023 — Team bzk-sh

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Project NoteBook

Week 1

Week 2

Week 3

Week 4

Week 5

Week 1

Event: Ice breaking games and team discussions

Agenda: Self-introduction, introduction of the program and rules, brainstorm of
team name, logo, slogan, and the team uniform.

logo and name:

![](https://static.igem.wiki/teams/4862/wiki/notebook/notebook-1.png)
![](https://static.igem.wiki/teams/4862/wiki/notebook/notebook-2.png)

Week 2

Event:  Lab day 1

Agenda: Laboratory safety training, use of pipette gun, allocation of reagents,
manipulation and use of precision instruments, allocation of LB medium, PCR manipulation and use, shaken
plasmid, agarose gel electrophoresis.

Purpose:  Amplified NS3-Hel and NS3-ZIKA genes together with pET28a and
constructed the plasmids by instrumentation.

![](https://static.igem.wiki/teams/4862/wiki/notebook/notebook-3.png)
![](https://static.igem.wiki/teams/4862/wiki/notebook/notebook-4.png)

Event: Lab day 2

Agenda: Plasmid extraction and concentration determination, DNA tool enzyme
digestion, ligand, transformation of DH5α cells, plate coating screening.

Purpose: After enzyme digestion, the target fragment of NS3-Hel/NS3-ZIKA was
obtained by PCR, and the recombinant plasmid was obtained by enzyme-linking.

![](https://static.igem.wiki/teams/4862/wiki/notebook/notebook-5.png)
![](https://static.igem.wiki/teams/4862/wiki/notebook/notebook-6.png)
![](https://static.igem.wiki/teams/4862/wiki/notebook/notebook-7.png)
![](https://static.igem.wiki/teams/4862/wiki/notebook/notebook-8.png)

Event: Lab day 3-4

Agenda: The transformers were identified by PCR, the monoclonal seed solution
was
selected, and the plasmids pET28a-NS3hel and ET28a-NS3-ZIKA were extracted and transformed into BL21(DE3).

Purpose: Prove that the plasmid solution contain NS3-Hel and NS3-ZIKA by
comparing the magnitude of the stripes, and transfer the DNA recombinant plasmid into BL21(DE3).

![](https://static.igem.wiki/teams/4862/wiki/notebook/notebook-9.png)
![](https://static.igem.wiki/teams/4862/wiki/notebook/notebook-10.png)

Week 3

Event: Lab day 5

Agenda: Isolation of single bacteria, PCR identification of inverters, agarose
gel electrophoresis verification of target bands, isolation of monoclonal culture seed solution,
preparation
of protein purification buffer.

Purpose: The purpose of running a gel and performing PCR validation after
amplification is to verify the presence of target bands for NS3-Hel and NS3-ZIKA, and to inoculate and
validate the correct colonies, and to perform pre-culture of the bacterial suspension for subsequent
experiments.

![](https://static.igem.wiki/teams/4862/wiki/notebook/notebook-11.png)
![](https://static.igem.wiki/teams/4862/wiki/notebook/notebook-12.png)
![](https://static.igem.wiki/teams/4862/wiki/notebook/notebook-13.png)

Event:  Lab day 6-7

Agenda: Expanded culture seed solution, IPTG induced expression, SDS-PAGE
protein
glue preparation.

Purpose:  To trigger the expression of the target gene or protein in the
transformed cells.

![](https://static.igem.wiki/teams/4862/wiki/notebook/notebook-14.png)
![](https://static.igem.wiki/teams/4862/wiki/notebook/notebook-15.png)

Event: Lab day 8-9

Agenda: Ultrasonic crushing, nickel column affinity purified protein, SDS-PAGE
protein electrophoresis.

Purpose:  Filter or purify the target protein from other protein.

![](https://static.igem.wiki/teams/4862/wiki/notebook/notebook-16.png)
![](https://static.igem.wiki/teams/4862/wiki/notebook/notebook-17.png)

Event: Lab day 10-11

Agenda: SDS-PAGE protein electrophoresis, protein glue staining and
decolorization

Purpose:  Validation protein expression.

![](https://static.igem.wiki/teams/4862/wiki/notebook/notebook-18.png)
![](https://static.igem.wiki/teams/4862/wiki/notebook/notebook-19.png)

Week 4

Event: Lab day 12-13

Agenda: Functional test - enzyme marker test

Purpose:  Test the function.

![](https://static.igem.wiki/teams/4862/wiki/notebook/notebook-20.png)
![](https://static.igem.wiki/teams/4862/wiki/notebook/notebook-21.png)

Week 5

Event: Lab day 15

Agenda: Functional test II - enzyme marker test

Purpose:  Duplicate the function test by adding controls.

Event:  Lab day 16

Agenda: WIKI&PARTS key points interpretation and task division, organization of
writing WIKI, organization of uploading PARTS

![](https://static.igem.wiki/teams/4862/wiki/notebook/notebook-22.png)

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![](https://static.igem.wiki/teams/4862/wiki/engineering.png)

Project Engineering

Overview

Cycle 1

Cycle 2

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.

![](https://static.igem.wiki/teams/4862/wiki/engineering/1.png)

Cycle 1 [pET-NS3-ZIKA]

· Design stage

![](https://static.igem.wiki/teams/4862/wiki/engineering/2.png)

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

![](https://static.igem.wiki/teams/4862/wiki/engineering/3.png)

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.

![](https://static.igem.wiki/teams/4862/wiki/engineering/4.png)

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.

![](https://static.igem.wiki/teams/4862/wiki/engineering/5.png)

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

![](https://static.igem.wiki/teams/4862/wiki/engineering/6.png)

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

![](https://static.igem.wiki/teams/4862/wiki/engineering/7.png)

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.

![](https://static.igem.wiki/teams/4862/wiki/engineering/8.png)

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.

![](https://static.igem.wiki/teams/4862/wiki/engineering/9.png)

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.](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..

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Project Experiments

PCR amplification and Plasmid construction

Expression and purification of NS3 protein

Functional verification

1. PCR amplification and Plasmid construction

A.LB medium (g/L)

|  |  |
| --- | --- |
| Reagent | Dosage (g) |
| Tryptone | 10 |
| Yeast Extract | 5 |
| NaCl | 10 |

Add ddH2O to 1 L, sterilization at 121℃ for 20 min.

Solid plus 3 g agar per 100 mL.

B. Kanamycin (kan+) (50 mg/mL)

|  |  |
| --- | --- |
| Reagent | Dosage |
| kan | 500 mg |
| ddH2O | 10 mL |

Filter and remove bacteria.

Store in 1 mL package.

C. Inoculation: Take 5 μL of cryopreserved glycerol bacteria pET28a and add
to
5 mL liquid LB medium in test tube, and then add the final working concentration of kanamycin is 50 μg/mL,
culture at 37℃, 220 rpm in constant temperature shaker for 12~18h.

D. After the configured LB solid plate is lowered to room temperature, add
the
final concentration of 50 μg/mL kan+ and mix well, and then invert on the super clean table.)

E. PCR(μL)

|  |  |
| --- | --- |
| Reagent | Dosage (μL) |
| 2\*mix | 25 |
| NS3-HeL/ZIKA-F | 2.5 |
| NS3-HeL/ZIKA-R | 2.5 |
| DNA | 2 |
| ddH2O | 18 |
| Total | 50 |

PCR procedure (NS3-HeL-1344bp / NS3-ZIKA-1854bp)

|  |  |  |
| --- | --- | --- |
| Step | Temperature (℃) | Time |
| 1 | 95 | 5 min |
| 2 | 95 | 30 sec |
| 3 | 55 | 30 sec |
| 4 | 72 | 2 min |
| 5 | 72 | 10 min |
| 6 | 4 | forever |

PS: Steps 2 to 4 cycle 30 times.

F. Agarose gel

|  |  |
| --- | --- |
| Reagent | Dosage |
| 1\*TAE | 50 mL |
| Agarose | 0.5 g |

High heat for 2 min to melt. Add 5 μL Nucleic acid dye.

Install the gel plate and insert the electrophoresis comb. Pour the prepared agarose gel into the gel
platen at a height of 3~4 mm (avoid bubbles), and keep at room temperature to solidification. When fully
set, pull out the electrophoresis comb and transfer the solid gel to the electrophoresis tank, and then we
add 1\*TAE buffer and completely immerse the gel. Put them into the spot hole of the agarose gel, final add
10 μL DNA Maker. Cover the electrophoresis tank, Loading hole Located at the electric field negative,
power on, 150 V, 20 min. Turn off the power, remove the gel, and observe under UV light illuminator.

G. Gel extraction

Cut the gel which have destination fragment. Then weigh, add a certain proportion of gel melting agent
to melt agarose gel, mix well and heat in water bath at about 50℃ until the gel is completely melted. Add
binding fluid to allow the DNA to selectively bind to the DNA preparation membrane. Add DNA washing
solution for cleaning, and then get purified DNA by elution.

H. Plasmid extraction

Take bacterial solution centrifuge at 12000 rpm for 1 min and discard the supernatant, collect bacteria.
Add 250 μL buffer S1 to suspend the bacteria. Add 250 μL Buffer S2, gently and fully turn up and down 4 to
6 times and mix well to fully crack the bacteria until the clear solution is formed. Add 350 μL Buffer S3,
gently and fully turn up and down 6 to 8 times, centrifuge at 12000 rpm for 1 min, and discard the
filtrate. Absorb the supernatant and transfer it to the preparation tube, centrifuge at 12000 rpm for 1
min, and discard the filtrate. Add 500 μL Buffer W1, centrifuge at 12000 rpm for 1 min, and discard the
filtrate. Add 700 μL Buffer W2, centrifuge at 12000 rpm for 1 min, discard filtrate, and then use the same
way again. Place the preparation tube back into the 2 mL centrifuge tube, centrifuge at 12000 rpm for 1
min. Place the preparation tube back into the 1.5 mL centrifuge tube, let the ethanol in W2 evaporate for
1 to 2 min, eluent or ddH2O preheated at 60-65℃ is added with 50 μL eluent in the center of the
preparation membrane, and then water bath at 60-65℃ for 1 to 2 min, centrifuge at 12000 rpm for 1 min. Get
plasmid DNA.

I. Digestion plasmid pET28a

|  |  |
| --- | --- |
| Reagent | Dosage (μL) |
| pET28a Plasmid (<=1ug)< /td> 10 | |
| BamHI | 1 |
| XhoI | 1 |
| Cutsmart | 5 |
| ddH2O | 13 |
| Total | 50 |

PCR Recovered product

|  |  |
| --- | --- |
| Reagent | Dosage (μL) |
| NS3-Hel/ZIKA | 10 |
| BamHI | 1 |
| XhoI | 1 |
| Cutsmart | 2 |
| ddH2O | 6 |
| Total | 20 |

Reaction at 37℃ for 30 min.

Agarose gel electrophoresis (Same method as above)

Gel extraction and plasmid extraction (Same method as above)

J. Ligation

|  |  |
| --- | --- |
| Reagent | Dosage |
| pET28a after digestion | 7 |
| NS3-HeL/ZIKA after digestion | 10 |
| T4 DNA Ligase | 1 |
| 10\*T4 Ligase DNA buffer | 2 |
| Total | 20 |

Reaction at 16℃ for 30 min.

K. Heat shock transformation

Place centrifuge tube containing DH5α on ice and add 10 μL of the recombinant product, treated with ice
bath for 30 minutes, treated with 42℃ hot water bath for 45 s, and then immediately treated with ice bath
for 2 minutes. Add 900 μL LB liquid medium, gently blow and mix, and place the centrifuge tube in a
shaking table at 37℃ and culture at 200 rpm for 1 hour. And then centrifuge at 5000 rpm for 5 min, leave a
little supernatant to reinsert the cells, and coat them on LB plate medium containing kan+, and culture at
37℃ overnight.

L. Verified the transformant DH5α and inoculated DH5α

PCR

|  |  |
| --- | --- |
| Reagent | Dosage (μL) |
| 2\*mix | 10 |
| Primer F | 1 |
| Primer R | 1 |
| ddH2O | 8 |
| Total | 20 |

PS: DNA plate: DH5α

Transferred the constructed recombinant plasmid to BL21(DE3) and plate coated for screening. (Same method
as above)

2. Expression and purification of NS3 protein

Expanded the culture seed solution and performed IPTG induced expression BL21(DE3). Expanded the culture
seed solution: 1% Inoculated volume is transferred to 100 mL LB Liquid medium, and OD600 is grown at 37°C
to 0.4 to 0.6 (about 3 to 4 h).The final concentration of 0.4 mM IPTG was added to induce expression, and
overnight culture was performed at 14°C.

A.Configuration buffer

a.His buffer A (0.5 L)

|  |  |
| --- | --- |
| Reagent | Dosage (g) |
| 20 mM Na2HPO4·2H2O | 1.78 |
| 500 mM NaCl | 14.6 |
| 20 mM imidazole | 1.02 |

PS: Adjust pH to 7.4 with HCl (6 M) and set the volume to 0.5 L.

b.His buffer B (0.25 L)

|  |  |
| --- | --- |
| Reagent | Dosage (g) |
| 20 mM Na2HPO4·2H2O | 0.89 |
| 500 mM NaCl | 7.3 |
| 20 mM imidazole | 8.5 |

PS: Adjust pH to 7.4 with HCl(6M), set the volume to 0.25 L and 0.45 mm filter membrane filtration
sterilization.

c.10 mM MOPS buffer (50 mL): 0.1 g powder MOPS, 50 mL ddH2O, pH6.5

d.10 mM Tris-HCl buffer (50 mL): 0.02 g powder Tris-HCl, 50 mL ddH2O, pH6.5

e.2 mM MgCl2 buffer (50 mL): 0.02 g powder MgCl2, 50 mL ddH2O

B.Ultrasonic cell fragmentation

a.Centrifuge to collect bacteria, add 15 mL His buffer A, fully suspended, place it in a 50 mL centrifuge
tube, ultrasonic crushing on ice, power 500 w, open 3 s off 3 s, total duration 10 to 20 min.

b.Centrifuge the collected supernatant, 4℃ , 12000 rpm, 30 min, collect the supernatant.

C.Nickel column affinity purified protein

Remove the pre-loaded nickel column and wash 10 column volumes with ddH2O, then wash 10 column volumes
with His buffer A. The filtered cel supernatant is Loaded into the nickel column at a flow rate of 1
mL/min. Wash the sampled nickel column with His buffer A. The recombinant protein is eluted with His
buffer B. Wash the column with His buffer B, His buffer A and water in turn. Wash the column with 5 mL 20%
ethanol.

D.D.Configured SDS-PAGE protein glue

Assemble the glue mold, configure a 1.00 mm thick layer of glue. Configure a 1.00 mm thick layer of glue.
Stand for 10 to 15 minutes, wait for the upper layer of glue to solidify, carefully pull out the comb, use
the gun head or syringe to absorb the electrophoresis buffer and rinse the sample hole to perform SDS-PAGE
electrophoresis.

E.SDS-PAGE protein electrophoresis

Add 40 μL crude protein solution into 6 μL loading buffer, boil at 100℃ for 10 min, centrifuge at 4℃,
take 10 μL treated supernatant sample and add into sample hole.
Cover the top, connect the electrophoresis device, and control the voltage of the sample before gluing at
100-200 V, about 15-20 min.When the bromophenol blue indicator reaches the separation glue, the voltage
rises to 200 V, and the electrophoresis process keeps the voltage stable. Electrophoresis can be stopped
when the bromophenol blue indicator reaches 1-2 cm away from the front, about 0.5-1 h.

F.Configure dyeing solution and decolorizing solution

Formula of dyeing solution (1 L) : 400 mL ethanol, 500 mL ddH2O, 100 mL acetic acid, 0.1% R250, filter
with filter paper after dissolution.

Decolorizing solution formula: 40% ethanol, 10% acetic acid , 50% ddH2O.

G.Dyeing and decolorization of protein glue

Dyeing: After electrophoresis, turn off the power, take out the glass plate, separate the rubber surface
from the glass plate, and then Light R250 for dyeing, about 0.5-1 h.
Decolorization: Discard the dyeing solution, wash the rubber surface 2-3 times with ddH2O, and then add
the decolorization solution for diffusion decolorization, and change the decolorization solution every 30
min until the protein bands are clear.

Configuration of SDS-PAGE protein glue : Separation glue/Concentrate glue

6% PAGE jelly: 70~300 KD

8% PAGE jelly: 30~200 KD

10% PAGE jelly: 20~80 KD

12% PAGE jelly: 15~60 KD

15% PAGE jelly: 10~45 KD

3. Functional verification

A.Synthetic DNA substrate: Helicase substrate is synthesized by combining
a
1:1 molar ratio with a single strand with a final concentration of 20 μM in 10 mM Tris hydrochloric acid
pH 8.5, 95°C 5 min, 75°C 5 min, 55°C 5 min, 35°C 5 min, at room temperature 25°C 1 h.

B. NS3-HEL/NS3-ZIKA: 10 mM MOPS (pH 6.5), 2 mM MgCl2, 2.5 nM-labeled
double-stranded substrate, and 9.75 nM NS3 enzyme are mixed, ATP is added until the final concentration
is 3 mM, and the test is performed 15 min Later.
The reaction is performed at 100 μL and repeated three times. At the same time, blank (distilled water),
negative (only NS3 enzyme is added) and positive are set up, and different concentration gradients are
set. After reaction for 15 min, fluorescence detection is carried out with enzyme labeling instrument,
with excitation wavelength of 620 nm and emission wavelength of 685 nm.

Overall system

|  |  |
| --- | --- |
| Reagent | Dosage (μL) |
| 10 mM MOPS | 10 |
| 2 mM MgCl2 | 10 |
| 2.5 nM Double stranded substrate | 1 |
| 9.75 nM NS3 protease | 4 |
| 3 mM ATP | 3 |

PS: Supplement with ddH2O to 100 μL

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![](https://static.igem.wiki/teams/4862/wiki/results.png)

Project Results

Overview

PCR amplification and plasmid construction

Protein Expression and Purification

Function Validation---FRET

Future Plan

Overview

The incidence of dengue has grown dramatically worldwide in recent decades,[1] making dengue fever a
significant emerging public health problem for which no effective treatment exists. RNA helicase is
responsible for almost all RNA metabolism, and helicase plays an essential role in virus replication. We
propose to analyze the helicase sequence and obtain the NS3 protease with high activity by molecular
cloning and protein purification. Finally, we aim to construct a high-throughput screening method in vitro
that can be applied to the rapid discovery of helicase inhibitors.

We obtained the target fragment of NS3-HEL/NS3-ZIKA by PCR and the recombinant plasmid by enzyme
ligation. The recombinant plasmids, pET-28a-NS3-Hel and pET-28a-NS3-ZIKA, were then transferred to BL21
for inducible gene expression, and the resulting protein was verified for function, as shown in Figure 1.

![](https://static.igem.wiki/teams/4862/wiki/results/1.png)

PCR amplification and plasmid construction

We designed two genes, NS3-Hel and NS3-ZIKA, and obtained a plasmid pET28a from the laboratory. We then
amplified NS3-Hel and NS3-ZIKA genes with pET28a and constructed the plasmids by instrumentation. The
target
DNA was obtained through PCR. Then, enzyme digestion was performed, and the plasmid was converted into
BL21(DE3) by heat shock. Finally, we transferred the plasmid into the BL21(DE3). As shown in Figure 2,
NS3-Hel (1344bp) & NS3-ZIKA (1854bp), we obtained the specific plasmids we wanted. We verified that we got
the correct gene by confirming the bp length as well as the sequencing results. We can use this as a
precondition for further operations.To ensure that the plasmid construction is 100% correct, we sequenced
the target genes that NS3-Hel and NS3-ZIKA.

![](https://static.igem.wiki/teams/4862/wiki/results/n-2.png)

Figure 2. A: Amplification of NS3-Hel/NS3-ZIKA; B: pET-NS3-Hel/pET-NS3-ZIKA in DH5ɑ. C. pET-28a-NS3-Hel
plasmid; D. pET-28a-NS3-ZIKA plasmid; E. Sequencing results of NS3-Hel and NS3-ZIKA.

Protein Expression and Purification

We validated the recombinant plasmid by inserting DNA fragments into a plasmid (pET-28a), as shown in
Figure 3a. The data confirms that the fragments are in the correct position. After transferring the
recombinant plasmid into BL21 to induce its expression by IPTG, we purified the two proteins to obtain
two
pure proteins by His-Tag.

![](https://static.igem.wiki/teams/4862/wiki/results/3.png)

Figure 3. A. pET-NS3-Hel/pET-NS3-ZIKA in BL21(DE3); B. SDS-PAGE.

As shown in Figure 3B, the size of the two recombinant proteins NS3-Hel / NS3-ZIKA is round 49 kDa as
expected. Therefore, we can see that the proteins were successfully induced to express.

Function Validation---FRET

The Fluorescence Resonance Energy Transfer (FRET) is based on the resonance energy transfer of
fluorescent proteins to study the structure and dynamics of proteins and nucleic acids, in the detection
and visualization of intermolecular association and in the development of intermolecular binding
assays.[2] In this project, the FRET method was used to test the activity of NS3 protein, where an FRET
pair consisting of a donor and an acceptor fluorophore is attached to the ends of a double-stranded DNA
or
RNA substrate. When the helicase, NS3 protein unwinds the substrate, the donor and acceptor fluorophores
move apart which will result in their distance reaching a critical range (typically 1-10 nanometers),
and
then FRET (a measurable decrease in fluorescence) occurs.

After purifying the protein, we reacted with ATP, MOPS Buffer, MgCl2, and double-chain substrate for 15
mins. We then measured the fluorescence. As the concentration of our protein increased, the fluorescence
gradually decreased. As shown in Figure 4, our fluorescence tended to decline as our protein gradually
increased since the NS3 protein . Therefore, we can prove that the NS3 protein possesses helicase
activity.

![](https://static.igem.wiki/teams/4862/wiki/results/4.png)

Figure 4. Fluorescence Value in Different Protein Concentration.
\*Our team measured the fluorescence by using fluorometers with an excitation of 620 nm and an emission
wavelength of 685 nm.

Firstly, we successfully designed and validated two genes, NS3-Hel and NS3-ZIKA, and obtained a plasmid
pET28a from the laboratory. We then transformed pET28a into DH5a through enzymatic cleavage,
enzyme-linked
construction of the plasmid, and heat shock. The construction of target strains was successfully
obtained
per the verification of electrophoresis gel and sequencing.

After transferring the recombinant plasmid into BL21 to induce its expression by IPTG, we purified the
two proteins to obtain two pure proteins by His-Tag which were validated by SDS-PAGE.

We then reacted the purified protein with ATP, MOPS Buffer, MgCl2, and double-chain substrate for 15
minutes and measured the fluorescence. The FRET result confirmed our proteins functioned well that
possesses helicase activity, the fluorescence tended to decline as the protein concentration gradually
increased.

Overall, we successfully obtained the engineered strains and NS3 proteins with good activity which
means
we made some progress in our research goal to develop a drug screening kit against the dengue virus.

Future Plan

Dengue is an acute febrile disease caused by mosquito-borne dengue viruses (DENVs), consisting of four
serotypes (DENV 1 to 4). In our experiment, we only used one serotype. Thus, in the future, we could use
more serotypes to improve our test kit. We can determine the optimal inhibitory concentration to
optimize
the conditions for sensitivity and specificity. We can maximize the engagement of different NS3 proteins
and the helicase inhibitor compounds. By doing so, we can improve the effectiveness of our test kit.
Additionally, we could try different combinations to verify our results and find a more efficient one.
These optimizations will help us improve the accuracy and efficiency of our test kit, making it a more
effective tool for diagnosing dengue.

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]. Hussain S A .An Introduction to Fluorescence Resonance Energy Transfer
(FRET).2009[2023-09-19].DOI:doi:http://dx.doi.org/.

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