iGEM 2023 — Team tsb-center

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![](https://static.igem.wiki/teams/4843/wiki/notebook.png)

\*Last update of this notebook happened on 2023/08/10 and the detailed protocol can be found on the Experiment page.

0729

Team online meeting: experiment design and confirm intensive laboratory schedule.

0729

Team offline meeting: team logo design and teamwork allocation.

Order primers by the instructor.

0730

Laboratory safety training and basic instrument training.

Preparation of LB medium and E. coli cultivation.

[0730.pdf](https://static.igem.wiki/teams/4843/wiki/notebook/0730.pdf)

0731

PCR for target genes and recycle.

Plasmid extraction for pET28a vector.

[0731.pdf](https://static.igem.wiki/teams/4843/wiki/notebook/0731.pdf)

0801

Double enzyme digestion, plasmid extraction and ligation.

PCR and electrophoresis gel for validation.

[0801.pdf](https://static.igem.wiki/teams/4843/wiki/notebook/0801.pdf)

0802

Repeat Double enzyme digestion, plasmid extraction and ligation.

BL21 transformation.

[0802.pdf](https://static.igem.wiki/teams/4843/wiki/notebook/0802.pdf)

0803

PCR and electrophoresis gel for validation.

LB medium preparation and E. coli cultivation.

[0803.pdf](https://static.igem.wiki/teams/4843/wiki/notebook/0803.pdf)

0804

OD detection and IPTG induced expression.

[0804.pdf](https://static.igem.wiki/teams/4843/wiki/notebook/0804.pdf)

0805

Coomassie brilliant blue dye and run SDS-PAGE gel.

[0805.pdf](https://static.igem.wiki/teams/4843/wiki/notebook/0805.pdf)

0806

Repeat Coomassie brilliant blue dye and run SDS-PAGE gel.

0807

Break day.

0808

Function test---Fret by the microplate reader.

[0808.pdf](https://static.igem.wiki/teams/4843/wiki/notebook/0808.pdf)

0809

Fret Results analysis.

0810

Sequencing result analysis.

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![](https://static.igem.wiki/teams/4843/wiki/engineering.png)

Overview

We aim to develop a method for screening other proteins or potential compounds that might possess the
ability to inhibit the monkeypox virus.

![](https://static.igem.wiki/teams/4843/wiki/engineering/1.png)

First, we optimized the monkeypox virus I7L protein sequence, constructed a cloning recombinant vector, and
transferred it into E. coli for expression. We isolated the I7L-His tag protein in vitro and finally
verified its activity through the principle of fluorescence quenching.

Design

![](https://static.igem.wiki/teams/4843/wiki/engineering/2-1.png)
![](https://static.igem.wiki/teams/4843/wiki/engineering/2-2.png)

DNA map of pET-28a-I7L and pET-28a-ASFV-I7L

By considering that monkeypox virus protease I7L is also recognized as an ideal target against monkeypox
for inhibiting virus replication. A recombinant plasmid containing I7L sequence was planned to be structured
to purify I7L protein. The correction and biological activity of our purified protein would be confirmed and
the protein I7L could be used to discovery the novel lead compounds with inhibitory activity against I7L
which has the therapeutic potential against monkeypox.

Build

1.Recombinant plasmid construction

(1)Target genes I7L and ASFV-I7L extraction

(2)Vector plasmid PET28a extraction

(3)Double enzyme cleavage by Nhel+Xhol

(4)Target DNA fragments recycle

(5)Ligation by T4 ligase

![](https://static.igem.wiki/teams/4843/wiki/engineering/3.png)

Electrophoresis gel results after PCR

2.Preparation of protein expression engineering bacteria

(1)Recombinant plasmid verification by DNA agarose gel electrophoresis

(2)Transformation of recombinant plasmid into Escherichia coli

(3)Achievement of monoclonal colony expressing I7L and ASFV-I7L

(4)Amplification of Escherichia coli expressing I7L and ASFV-I7L

![](https://static.igem.wiki/teams/4843/wiki/engineering/4.png)

Electrophoresis gel validation results: 1: I7L; 2: I7L digested fragments; 3: ASFV-I7L; 4:
ASFV-I7L digested fragments; 5: pET28a; 6: pET28a digested fragments

3.Plasmid sequencing

No mutations were found in the sequencing files and the sequences were consistent with the sequences of
Monkeypox virus (I7L) and African Swine Fever (ASFV) by NCBI blast comparison. Thus we could confirm the
plamid construction is successful.

![](https://static.igem.wiki/teams/4843/wiki/engineering/5.png)

Sequencing results

Test

1.Purification of target protein

(1)Protein expression induced by IPTG

(2)fragmentation and lysis of Escherichia coli

(3)Purification of protein by nickel column

(4)Collect the target protein by elution

2.Protein Validation - Western Blot

Two pure proteins (I7L and ASFV-I7L proteins) with correct molecular weight (47kD) were observed which
confirmed the successful engineering of these two plasmids in E. coli BL21.

![](https://static.igem.wiki/teams/4843/wiki/engineering/6.png)

Western Blot results

3.Biological Activity Validation - FRET

(1)Construction of reaction system

Detection of fluorescence intensity

The linear curves show that the fluorescence values of the two groups of target proteins decreased with the
increase of time, and the control protein (BSA protein) remained unchanged with the increase of time,
demonstrating that I7L and ASFV-I7L were biologically active.

![](https://static.igem.wiki/teams/4843/wiki/engineering/7.png)

\*Our team measured fluorescence using an enzyme labeler with an excitation wavelength of 320 nm and an
emission wavelength of 380 nm3.

Learn

We have successfully obtained the target proteins in the experiment, and in the future, we can use our
proteins to screen some compounds that may have potential therapeutic effects on monkeypox according to the
existing literature. and we can also optimize our protein and try different preparation processes to reduce
the cost of our kit, and improve the accuracy and efficiency of our kits by trying different compounds to
find more efficient and accurate ones.
All in all, I think that we achieved success on various different levels. The most obvious one is being able
to test the protein and gain positive results. But far from that, I think that during this project, the
success that we have gained is that we united together as a team, solved one problem after another, and
gained friendship through the entire process. The success itself has elevated the platform and taken a step
closer to biological engineering for the cure of monkeypox. I’m extremely glad that I participated in this
iGEM competition, because it not only made me less narrow-minded and broadened my horizon, but all the same,
it has opened up an irresistible world to me that I’ve never dabbled in.

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![](https://static.igem.wiki/teams/4843/wiki/experiments.png)

LB culture mediums preparation

Goal: LB culture mediums were prepared to culture bacteria especially for Escherichia coli, which will be
needed in latter experiment.

1. Materials

a. Reagent

Tryptone: 5g

Yeast extract: 2.5g

NaCl: 5g

Deionized Water: 500ml

b. Apparatus

balance

digital scale

autoclave sterilizer

beaker

test tubes

2.Procedure

1. A digital balance was used to get the exact amount of reagents that we need (2
g yeast extract, 4 g Tryptone, 4 g NaCl and 400 mL deionized water).

2. Placed all the material in a beaker.

3. Shook all reagents and let them mix completely.

4. Separated the solution into small test tubes.

5. Autoclaved EP tubes in a temperature of 121 ℃.

Making LB agar plate

Goal: LB agarose plate was used to get monoclonal Escherichia coli.

1. Materials

A. Reagent

LB: 100 mL

agar: 1.5 g

antibiotic (Kana+): 100µg/mL

B. Apparatus

balance

beaker

2. Procedure

·A digital balance was used to get the exact amount of reagent that we need (1.5 g agar).

·Added 1.5 g agar into the LB

·Autoclaved LB (with agar) in a temperature of 121 ℃.

·Let the mixture cooled down until its temperature at roughly 50 ℃.

·Add antibiotic (100 µg Kana+) into the mixture and let them mixed completely.

·placed the solution into the plate and waited the agarose to solidify.

Making a bacteria liquid PCR

Goal: The aim of this experiment is to enlarge DNA fragments of I7L and ASFV-I7L to insert the plasmids.

1. Material

A. Reagent

Primer Front: 6.5 µL

Primer reverse: 6.5 µL

2x Heiff master mix: 130 µL

deionized water: 104 µL

plasmid/bacteria:13 µL

B. Apparatus

pipette

pipette tips

EP tubes

PCR thermo cycle:95 °C 5 min; 95 °C 30s, 55 °C 30s,72 °C 30, Cycles; 72 °C 10 min
4 °C

2. Procedure

• Choose a correct pipette and correct pipette.

• In the process of adding reagents (forward primer, reversed primer, 2x Heiff
master mix, deionized water, plasmid/bacteria:), we followed an order that adding the one with the highest
volume to lowest volume.L

• Remember to replace pipette tip after adding a reagent.

• Put test tube in PCR thermo cycle.

• Kept the DNA fragments enlarged production in -20 ℃ refrigerator.

Making 1% agarose gel

1. Material

A. Reagent

Agarose: 1 g

deionized water: 100 mL

tae: 100 mL

nuclei acid dye: 5 mL

B. Apparatus

balance

pipette

mold

beaker

digital scale

microwave

1. Material

• A digital balance was used to get the exact amount of reagent that we need.

• Added agarose into a beaker with 100 mL tae buffer.

• Heated the beaker unit the reagents in the beaker turn clear.

• Placed the beaker under water and slowly shook it so that the mixture cooled
down evenly.

• Add 5 mL nuclei acid dye into the reagents (be careful as it is poisonous).

• Put the solution into the mold and wait it to solidify.

Recycle the gel

1. Goal: recycle the target DNA sequence that we needed for latter experiment.

2. Material

a. Reagent

agarose gel

DNA Gel Extraction Kit

deionized water: 20 µL

wash solution:500 µL

b. Apparatus

pipette

pipette tips

metal bath instrument

3, Procedures

• Cut the gel block with the target segment from the agarose gel

• Add about three times buffer 2 respectively

• Placed it in a metal bath instrument for 10 min

• Add 20 µL of ddH2O between the adsorption membranes

• 2 rounds of wash solution and centrifugation.

PCR of bacteria solution

1. Goal: Identify whether the target DNA fragments I7L/ASFV-I7L in E. coli are correct

2. Material

A. Reagent

seed liquid: 1µL

gel: 50 mL

B. Apparatus

pipette

pipette tips

EP tubes

3. Procedures

• Divided it into small tubes

• Added seed liquid using pipette and pipette tips.

• After inject the seed liquid, the pipette tips should be replaced.

• Remember the seed liquid has to be injected by insert the tips into the gel so
that people could be sure that the seed liquid has been injected correctly.

Plasmid extraction

1.Goal: Plasmids were extracted and transfected into BL21 and DH5α E. coli cells

2. Material

a. Reagent

Buffer SP1: 250 µL

bacteria liquid:

Buffer S: 500 µL

Buffer SP2: 250 µL

Buffer SP3: 350 µL

Buffer DW1: 500 µL

Wash Solution: 500 µL

Elution Buffer: 50 µL

b. Apparatus

centrifuge

EP tube

pipette

pipette tips

3. Procedure

1. Added Buffer S, centrifuge 12,000 xg for 1min, drain the waste liquid in the
collection tube, and put the adsorption column back into the collection tube.

2. Toke bacterial solution, centrifuge 8,000 xg for 2 min to collect bacteria,
and discarded the medium.

3. Added Buffer SP1 to the precipitate and completely suspended the bacteria.

4. Added Buffer SP2 and immediately gently reversed the EP tube for 5-10 times
for mixing well. Let stand at room temperature for 2-4 min.

5. Added Buffer SP3 and immediately gently reversed the EP tube for 5-10 times to
mix.

6. Centrifugation (12,000 xg) for 5-10 min. Transfered the supernatant into the
adsorption column, centrifuged at 8,000xg for 30 s, and drain the liquid in the collection tube.

7. Added Buffer DW1, centrifuged at 9,000 xg for 30 s, and drain the liquid in
the collection tube.

8. Added Wash Solution, centrifuged at 9,000 xg for 30 s, and drain the liquid in
the collection tube.

9. Repeat Step 8.

10. Centrifuged the empty adsorption column at 9,000 xg for 1 min.

11. Put the adsorption column into a clean 1.5 mL centrifuge tube, added Elution
Buffer in the center of the adsorption film, stood at room temperature for 1 min, and centrifuged at 9,000
xg for 1 min. Kept the DNA solution in the tube.

Shake seed liquid

Aim: to cultivate more bacteria for protein extraction

Transformation of plasmid into DH5o and BL21(DE3) cells

1. Aim: Transformation of plasmids containing I7L and ASFV-I7L fragments into DH5o and BL21 engineering
bacteria, to preserve the plasmids and express the target proteins.

2. Material

a. Reagent

DH5o receptor cells: 200 µL

BL21 (DE3) receptor cells: 200µL

LB (no antibiotic): 1 mL

seed liquid: 100 µL, 5 µL

b. Apparatus

pipette

pipette tips

ice

water bath instrument

centrifuge

LB (with antibiotic)

3. Procedures

1. Warmed the water bath at 42 ℃ in advance

2. 200 μL DH5p cells was taken from the -80℃ refrigerator and thawed on ice. (100 µL per gene)

3. After thawing the receptive state, added the target DNA 1-10 ng, volume 《10 µL into the receptive cell
suspension, gently rotated the centrifuge tube to mix the contents, and took an ice bath for 30 min.

4. Incubated in water bath at 42 ℃ for 90 s, and quickly put on ice to cool for 3min.

5. Added l mL LB liquid medium (no antibiotics!!) to the tube After mixing and oscillating culture 1h
at 37℃, the bacteria were recovered.

6. After shaking the above bacterial solution, took 100 μL (can be concentrated if the receptive state
efficiency is low) and smeared it on the screening plate containing K+, placed it face up for 30 min,
and after the bacterial solution was completely absorbed by the medium, placed the petri dish at 37℃ for
16h - 24h.

\* 2 controls simultaneously preparation

Negative control: DNA solution was replaced with equal volume of sterile double steaming water, and
only 5 μL bacterial solution was taken and coated on LB plate containing antibiotics. There should be no
colonies in this group under normal conditions.
Positive control: A certain amount of known concentration of control DNA was added to the same amount of
receptive cells, and other operations were the same as adding the connection system. Under normal
circumstances, this control should have a relatively large number of colonies on the antibiotic
containing LB plate.

IPTG induced protein expression in shaker for 10 h at 14℃

1. Material

a. Reagent

IPTG:0.238 g

deionized water: 10 mL

b. Apparatus

pipette

pipette tips

balance

2. Procedures

• A digital balance was used to get the exact amount of reagent that we need

• Dissolved IPTG with deionized water

• Add IPTG solution to 100 mL I7L-BL21 and ASFV-I7L-BL21 bacterial fluid with the final concentration of
0.4 mM IPTG, and incubated for 10h at 14℃

Double enzyme cleavage by Nhel+Xhol

1. Aim: The DNA double strand was broken and cut by “Nhel”, and “Xhol” enzyme to ensure that the I7L and
ASFV-I7L sequence can be inserted into the plasmid in the correct orientation to prevent self-association.

2. Material

a. Reagent

![](https://static.igem.wiki/teams/4843/wiki/description/ff1.png)

b. Apparatus

test tubes

pipette

pipette tips

PCR thermal cycle

3. Procedures

• Operation on ice

• Digestion the I7L, ASFV-I7L and PET28a plasmid by NheI+XhoI, with in 2.1 cutsmart NEB buffer by the
table.

• Double cleavage plasmid vector PET-28a and target gene PCR product by using NheI and XhoI for 30min at
37℃.

Ligation

1. Aim: To ligate the I7L or ASFV-I7L cleavage fragments with plasmids by T4 ligase

2. Material

a. Reagent

plasmid: 20 ng

segment of the DNA (I7L and ASFV): 20 ng

10x T4 ligase buffer: 2 µL

T4 ligase Buffer: 0.2 µL

water: 20 µL

b. Apparatus

centrifuge tube

centrifuge

pipette

pipette tips

3. Procedures

1. The DNA was transferred to the sterile centrifuge tube, at least 1 molar amount of foreign DNA fragments
was added.

2. Then 10 × T4 ligase buffer and T4 ligase buffer were added.

3. Sterile ultra-pure water was added to the volume of 10 μL,

4. Collected all the liquid at the bottom of the tube by microcentrifuge after full mixing. Connected at 16
°C for 1h

5. The DNA concentration of recombinant plasmid was measured after connection.

Pick up monoclonal colony

1. Aim: To obtain the monoclonal Escherichia coil which had been transformed into recombinant plasmid
(PET-28a-I7L and ASFV-I7L-ASFV)

2. Material

a. Reagent

LB

b. Apparatus

shaker tubes

culture dishes

refrigerator

fire

EP tube

pipette

pipette tips

shaker

3. Procedures

• Took out some culture dishes and placed the shaker tubes near the fire for a few second in every open or
close process in order to maintain the pure environment of the shaker tubes and to make sure that the tubes
were not infected or polluted by anything

• Placed a pipette tip on fire for a few second to make sure a pure environment but also helped us to
collect bacteria easier. Picked up bacteria with a with a pipette tip and transferred into shaker tubes with
5 mL LB

• Labeled the shaker tubes by the type of bacteria that we used

• Lastly, placed those shaker tubes into a Shaker and shake tubes for 5 hours.

Ultrasonic fragmentation of Escherichia coli

1. Aim: To get the protein of I7L and ASFV-I7L

2. Material

a. Reagent

ultrasonic cell disruptor

DNA fragments of I7L and ASFV-I7L

3. Procedures

1.The Escherichia coli was collected by centrifugation at 3,000 xg for 15 min

2. Added lysis solution into the tube with Escherichia coli and Placed the tube under the ultrasonic cell
disruptor

3. The ultrasonic cell disruptor have to stop in every 10 s due to the temperature of the ultrasonic cell
disruptor could get very high jurying the process. Without stop for a minute, it is relatively easy for cell
to denature.

4. Ultrasonic cell disruptor have to attach to the lysate and the lysate has to be straight up.

Polyacrylamide electrophoresis gel for SDS-PAGE

Procedure:

1.Took the same volume of the lower rubber solution and the lower rubber buffer, 2.0/2.7/4.0 mL each, and
mix well

2. Added 40/60/80 μL of modified coagulant to the mixture in Step 1 and mixed well

3. Injected the mixed solution of step 2 into the plastic glass plate so that the distance between the
liquid level and the upper edge of the short glass plate is 0.5 cm longer than the comb length (note:
This solution is excessive, do not inject all, can leave a little in the glue cup, to judge the glue setting
condition), added appropriate amount of water or alcohol (such as isopropyl alcohol, positive

4. After the lower layer of glue solidifies (about 15 min), poured the upper layer of water or alcohol;
Note: When there is a broken ray between water (alcohol) and glue, the gelatin has solidified.

5.Took the same volume of upper adhesive solution and color upper adhesive buffer solution, 0.5/0.75/1.0 mL
each, and mixed well;
Note: Due to the special physical and chemical properties of the dye, please shake well before use.

6.Add 10/15/201 L of modified coagulant to the mixture of step 5 and mixed well;

7.Injected the mixture of step 6 into the plastic glass plate and inserted the comb;

8.After the upper layer of glue solidifies (about 15 min), the comb teeth can be removed for
electrophoresis.

Nickel column affinity purification: I7L and ASFV-I7L protein

1. Material

a. Reagent

b. Apparatus

2. Procedure

1. CL retention sample 20 μL

2. Took 1 mL of uniformly mixed 50% BeyoGold™ His-tag Purification Resin (reducation-resistant chelating
type), centrifuged at 4℃ (1,000×g, 10 s) and discarded the storage solution. Added 0.5 mL of non-denaturing
lytic solution to the gel and mix well to balance the gel. Centrifugation at 4℃ (1,000×g, 10 s) to discard
the liquid, and then repeated the balance twice to discard the liquid.
About 4 mL of the supernatant of the bacterial lysate was added into the mixture, and the supernatant was
slowly shaken at 4℃ on a side shaking table or a horizontal shaking table for 60 min.

3. The mixture of the lysate and BeyoGold™ His-tag Purification Resin (reduction-resistant chelating type)
was loaded into the empty affinity column tube provided with the kit.
h. Opened the lid at the bottom of the purification column, let the liquid in the column flow out under the
action of gravity, and collected about 20 μL of the draining liquid for subsequent analysis.

4. Washed the column three times, added 1 mL of non-denatured washing liquid each time, and collected about
20 μL of washing liquid through the column for subsequent analysis and detection.

5. Eluted the target protein 4 times with 0.5 mL of non-denaturing eluent. Each eluent was collected
separately into a different centrifuge tube. The eluent obtained from the collection is the purified His
label protein sample.

Enzyme Labeling Instrument (ELI) test: Functional validation

Material:

a. Reagent

b. Apparatus

Procedure:

1.100 nM I7L/ASFV-I7L/Control protein (BSA) protease, 0.4 mM EDTA, 4 mM DTT and 20% glycerol were added to
a
reaction buffer containing 20 mM HEPES, pH 6.5, 120 mM NaCl of the test compounds for 30 min at 30 °C.

2.Set up different concentrations of protein sample from 0.01,0.02,0.05,0.1mg/ml of protease and set up
three parallel groups for each concentration group to reduce the error. At the time of statistical data, the
average value of parallel groups was calculated as the reference fluorescence intensity.

3.Detection the baseline under a 320/380 nm filter.

4.Detection was carried out by adding 10 μM FRET- peptide substrate (MCA-T-N-A-G-T-C-TV-Lys(dnp)-K-K-NH2))
for a total of 1 h of incubation followed by detection under a 320/380 nm filter.

5.Analyzed the fluorescence intensity change and protein concentration with a linear regression analysis.

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![](https://static.igem.wiki/teams/4843/wiki/results.png)

Overview

The number of monkeypox infections worldwide has increased significantly during the COVID-19 epidemic1. Due
to there has been no effective treatment for monkeypox, we think it could help if we could develop a kit
that can effectively screen for effective compounds against monkeypox.

According to recent research, it is found that I7L and ASFV-I7L are highly conserved in Orthopoxvirus and
play a key role in virus assembly23. Therefore, drug design and development targeting I7L and ASFV-I7L are
of great value for controlling monkeypox virus infection.

In this project, we plan to obtain and purify I7L protein and ASFV-I7L protein by the construction of
recombinant E. coli and then further develop a kit for screening potential monkeypox inhibitors.

![](https://static.igem.wiki/teams/4843/wiki/results/1.png)

PCR amplification and plasmid construction

We designed two genes, I7L and ASFV-I7L, and extracted the plasmid pET28a from Escherichia coli.We then
amplified the I7L and ASFV-I7L genes and the target DNA was obtained by PCR (figure 3). The I7L and ASFV-I7L
genes are inserted into pET28a to get a recombinant plasmid. Then, the I7L and ASFV-I7L-pET28a plasmid were
transformed into BL21 (DE3) and DH5α by heat shock method. .

![](https://static.igem.wiki/teams/4843/wiki/results/2.png)

Experiment Design Draft

![](https://static.igem.wiki/teams/4843/wiki/results/3.png)

Electrophoresis gel results after PCR

As shown in Figure 4, I7L and ASFV-I7L obtained the correct genes we needed by confirming the length of bp
which is 1269bp and 723bp around , respectively. So we can make this a prerequisite for furtheraction.

![](https://static.igem.wiki/teams/4843/wiki/results/4.png)

Electrophoresis gel validation results: 1: I7L; 2: I7L digested fragments; 3: ASFV-I7L; 4:
ASFV-I7L digested fragments; 5: pET28a; 6: pET28a digested fragments

Plasmid sequencing

To insure over I7L and ASFV-I7L DNA fragment have inset into the plasmid in a right direction, and there is
no mutation during the heat shock process, we sent the plasmids constructed successfully in PCR
identification for sequencing, and sequencing using template primers showed that the I7L and ASFV fragments
were inserted into the plasmid in the correct orientation. No mutations were found in the sequencing files
(figure 5), and the sequences were consistent with the sequences of Monkeypox virus (I7L) and African Swine
Fever (ASFV) by NCBI blast comparison2.

![](https://static.igem.wiki/teams/4843/wiki/results/5.png)

Sequencing results

Protein expression and Purification

We validated protein purification production from the recombinant plasmid by Western Blot. As shown in
Figure 4, two pure proteins (I7L and ASFV-I7L proteins) with correct molecular weight (47kD) were obtained
by purification of the two proteins with His-Tag. The protein markers used were Beyotime 14.4-116 protein
markers.

![](https://static.igem.wiki/teams/4843/wiki/results/6.png)

Western Blot results

Function Validation

In order to evaluate the activity of our purified proteins, we applied the FRET assay for function
validation. After the purified protein was configured, 0.4 mM EDTA,4 mM DTT and 20% glycerol were added into
the reaction buffer containing 20 mM HEPES,120 mM NaCl and pH 6.5, and then 100 nM I7L and ASFV-I7L were
added into two parts, respectively. Finally, the blank control group was set up with BSA protein.

Table 1. FRET data of I7L and I7L-ASAV against the control group (BSA protein)

![](https://static.igem.wiki/teams/4843/wiki/results/ff2.png)

The linear curves in figure 7 show that the fluorescence values of the two groups of target proteins
decreased with the increase of time, and the control protein (BSA protein) remained unchanged with the
increase of time, demonstrating that I7L and ASFV-I7L were biologically active3.

![](https://static.igem.wiki/teams/4843/wiki/results/7.png)

\*Our team measured fluorescence using an enzyme labeler with an excitation wavelength of 320 nm and an
emission wavelength of 380 nm3.

In summary, we successfully designed and validated I7L and ASFV-I7L dna parts, and inserted these two genes
into plasmid pET28a from Escherichia coli by enzyme digestion, enzyme-linked plasmid construction and
thermal excitation. We then transferred recombinant plasmids I7L and ASFV-I7L-pET28a into BL21, and the
protein expression of the recombinant plasmid was induced by 0.4 mM IPTG, and the two target proteins were
purified by His-Tag to obtain two pure proteins. Finally, the I7L and ASFV-I7L biological activity were
confirmed by fluorescence assay.

Future Plan

We have successfully obtained the target proteins in the experiment, and in the future, we can use our
proteins to screen some compounds that may have potential therapeutic effects on monkeypox. In addition, we
are still looking for positive controls that could supplement our kit. We will also optimize our protein and
try different preparation processes to reduce the cost of our kit, improve the accuracy and efficiency of
our kits by trying different compounds to find more efficient and accurate ones.

References

1. Lum FM, Torres-Ruesta A, Tay MZ, et al. Monkeypox: disease epidemiology, host immunity and clinical
interventions. Nat Rev Immunol. Oct 2022;22(10):597-613. doi:10.1038/s41577-022-00775-4

2. Aleshin AE, Drag M, Gombosuren N, et al. Activity, specificity, and probe design for the smallpox virus
protease K7L. J Biol Chem. Nov 16 2012;287(47):39470-9. doi:10.1074/jbc.M112.388678

3. Katritch V, Byrd CM, Tseitin V, et al. Discovery of small molecule inhibitors of ubiquitin-like poxvirus
proteinase I7L using homology modeling and covalent docking approaches. J Comput Aided Mol Des. Oct-Nov
2007;21(10-11):549-58. doi:10.1007/s10822-007-9138-7

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