iGEM 2022 — Team shanghai-hs

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
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# Notebook

## Day1 2022/08/18

**Theme:** Team icebreaker

**Time:** 9:00-16:30, August 18, 2022

**Location:** Road Show Hall, 2F, Block A, 58 Xiangke Road, Pudong New Area,
Shanghai

**Experiments:**

Introduction to the mentor team and team members

○ Ice Breaker (game)

○ Electing candidates for team captains of the wet and dry teams

Team discussion: team name, team LOGO, merchandise design, etc.

**Meaning:**

Bis-Vacc

The direction of this experiment is to make a safe oral vaccine for the infant and young children without
endotoxin. Bis originally means "dual". The research on this vaccine can solve the problems caused by EV71 and
RV viruses comprehensively, which is more economic and time-saving. The research subject is vaccine, so the
suffix “Vacc” is added to “Bis”. The team’s name is based on the research direction and the basic word root.
On the premise of not deviating from the topic, the team carries out certain innovation, which is novel and
closely related to the theme.

![](https://static.igem.wiki/teams/4276/wiki/notebook/t-shanghai-hs-notebook-01.jpg)

## Day2 2022/08/19

**Theme:** LB nutrient solution configuration, PCR amplification

**Time:** 9:00-16:30, August 19, 2022

**Place:** Dart Laboratory, No. 1077 Zhangheng Road, Pudong New Area,
Shanghai

**Experiments:**

● Laboratory safety training

● LB culture medium

● PCR amplification of vp1, vp7, IL12, GFP and GLBP genes

● Gel extraction of vp1, vp7, IL12, GFP and GLBP genes

● Overlap extension PCR of vp7 and vp1

● Overlap extension PCR of IL12 and GFP

● Culturing E.coli

**Progress of the team:**

Before the experiment in the morning, the coach of the wet team led the students to conduct laboratory safety
training. After that, PCR amplification of *vp1*, *vp7*, *IL12*, *GFP* and *GLBP*
genes was completed, and LB medium was
prepared at the same time.

In the afternoon, students performed agarose gel electrophoresis and image analysis, and completed the DNA gel
extraction. Finally, *E. coli* was incubated.

![](https://static.igem.wiki/teams/4276/wiki/notebook/t-shanghai-hs-notebook-02.jpg)

## Day3 2022/08/20

**Theme:** extraction of pET28a plasmid, gel extraction, enzyme digestion,
ligation, and heat shock transformation

**Time:** 9:00-16:30, August 20, 2022

**Place:** Dart Laboratory, No. 1077 Zhangheng Road, Pudong New Area,
Shanghai

**Experiments:**

● Plasmid pET28a was extracted

● Gel extracted of VP1-VP7 gene

● Enzyme digestion, ligation and transformation

● Plate coating screening

● Overlap extension PCR of *GLBP* and *vp7-vp1*

**Progress of the team:**

The students of the wet team extracted the plasmid pET28a in the morning, determined its concentration, and
then used the agarose gel method to separate the sizes of *vp1-vp7* DNA fragment, and then completed the
DNA
extraction.

In the afternoon, the plasmid pET28a and *vp1-vp7* DNA fragments were digested with restriction enzymes
EcoRI
and XhoI, and then ligate them with T4 Ligase. Finally, the ligations were transformed into *E.coli* DH5α

![](https://static.igem.wiki/teams/4276/wiki/notebook/t-shanghai-hs-notebook-03.jpg)

## Day4 2022/08/21

**Theme:** Identification of transformants by colony PCR, selection of
monoclonal culture seed liquid, and extraction of plasmids

**Time:** 9:00-16:30, August 21, 2022

**Place:** Dart Laboratory, No. 1077 Zhangheng Road, Pudong New Area,
Shanghai

**Experiments:**

● Identification of transformants by PCR

● Select monoclonal culture

● Plasmid pET28A-vp7-vp1 was extracted and transformed into BL21(DE3)

● Culturing bacteria

● Overlap extension PCR of *GLBP-vp7-vp1* and *IL12-GFP*

● Overlap extension PCR of *GLBP-vp7-vp1* and *GFP*

● Enzyme digestion, ligation and transformation

● Plate coating screening

**Progress of the team:**

In the morning, the coach and the students of the wet team selected the *E. coli* colony PET28A-VP1-VP7,
and
then identified whether the colonies were transformed PET28A-VP1-VP7 by PCR. At the same time, we amplified
*GLBP-vp7-vp1-IL12-GFP*, and *GLBP-vp7-vp1-GFP* by overlap PCR, respectively.

In the afternoon, the students transformed plasmid pET28A-vp7-vp1 into *E.coli* BL21(DE3). Then, plasmid
pET28a,
*GLBP-vp7-vp1- IL12-GFP* DNA fragments, and *GLBP-vp7-vp1-GFP* DNA fragments were digested with
restriction
enzymes EcoRI and XhoI, and then ligate them with T4 Ligase. Finally, the ligations were transformed into
*E.coli* DH5α.

![](https://static.igem.wiki/teams/4276/wiki/notebook/t-shanghai-hs-notebook-04.jpg)

## Day5 2022/08/22

**Topics:** *E. coli* transformation, learning plasmid profile, learning
and configuration solutions

**Time:** 9:00-16:30, August 22, 2022

**Place:** Dart Laboratory, No. 1077 Zhangheng Road, Pudong New Area,
Shanghai

**Experiments:**

● Identification of transformants by PCR

● Select monoclonal culture

● Plasmids were extracted and transformed into BL21(DE3)

● Proliferating bacteria

● Preparation of protein expression reagent

**Progress of the team:**

In the morning, the coach and the students of the wet team transformed the plasmid PET28A-VP1-VP7 into E.coli
BL21(DE3), and coated the plate overnight for culture. Next, we selected the *E. coli* colony
pET28A-*GLBP-vp7-vp1-IL12-GFP* and pET28A-*GLBP-vp7-vp1-GFP*, and then identified whether the
colonies were
correct.

In the afternoon, the students transformed plasmid pET28A-*GLBP-vp7-vp1-IL12-GFP* and pET28A-*GLBP-vp7-vp1-GFP*
into *E.coli* BL21(DE3). The instructor taught the students how to prepare the buffer including HisA
buffer,
HisB buffer, Tris-HCl buffer and electrophoretic decolorization solution in groups, and then learned to adjust
the buffer pH with the pH meter, in preparation for the later purification protein experiment.

![](https://static.igem.wiki/teams/4276/wiki/notebook/t-shanghai-hs-notebook-05.jpg)

## Day6 2022/08/23

**Theme:** IPTG induction

**Time:** 9:00-16:30, August 23, 2022

**Place:** Dart Laboratory, No. 1077 Zhangheng Road, Pudong New Area,
Shanghai

**Experiments:**

● Select *E. coli* colonies and run PCR test

● Agarose gel electrophoresis

● Analysis of electrophoresis images and results

● IPTG induced protein expression

**Progress of the team:**

In the morning, the students and the coach of the wet team picked up the *E. coli* colonies in the
sterile
ultra-clean table, prepared the colony PCR system, and amplified them with PCR instrument. Then nucleic acid
gel was prepared, agarose gel electrophoresis was performed, and the results and images were analyzed.

In the afternoon, the correct colony was induced the expression of with IPTG, and cultured in the shaker
overnight. Then the coach introduced the principle and operation steps of SDS-PAGE.

![](https://static.igem.wiki/teams/4276/wiki/notebook/t-shanghai-hs-notebook-06.jpg)

## Day7 2022/08/24

**Topics:** ultrasonic cell crushing, nickel column affinity chromatography

**Time:** 9:00-16:30, August 24, 2022

**Place:** Dart Laboratory, No. 1077 Zhangheng Road, Pudong New Area,
Shanghai

**Experiments:**

● Collect bacteria

● Ultrasonic cell crushing

● Nickel column affinity purification

● SDS-PAGE

● Proliferating bacteria

**Progress of the team:**

In the morning, the students of the wet team collected the *E. coli* induced by IPTG by centrifugation,
and then
the cells of the bacterial solution were broken by ultrasound for 15 minutes. It was observed that the
bacterial solution became clear.

In the afternoon, the students collected the supernatant of the cells for affinity purification with a nickel
column. Then the coach taught the students the principle of SDS-PAGE and the configuration of Buffer. Finally,
the supernatant and protein samples were loaded into wells of SDS-PAGE and the experimental data were
analyzed. Last but not least, the bacteria pET28A-*GLBP-vp7-vp1-GFP/E.coli BL21(DE3)* were cultured in
the
LB medium.

![](https://static.igem.wiki/teams/4276/wiki/notebook/t-shanghai-hs-notebook-07.jpg)

## Day8 2022/08/25

**Topics:** SDS-PAGE protein electrophoresis, sorting experimental results
and data

**Time:** 9:00-16:30, August 25, 2022

**Place:** Dart Laboratory, No. 1077 Zhangheng Road, Pudong New Area,
Shanghai

**Experiments:**

● SDS-PAGE protein electrophoresis

● Detection of GFP fluorescence signal

● Organize the electrophoresis results and GFP data

**Progress of the team:**

The experiment has come to an end. The students of the wet team cultured the pET28A-*GLBP-vp7-vp1-GFP/E.coli
BL21(DE3)* with IPTG, and detected GFP fluorescence signal every hour. Then, we carried out SDS-PAGE
electrophoresis on the protein and analyzed the electrophoresis experiment results and related data.

In the afternoon, students continue to detect GFP fluorescence signals every hour and took profile photos, and
the coach explained the experimental principle, precautions, and operation steps of Western Blot. The SDS-PAGE
gel was stained and destained. Finally, the laboratory was cleaned up and the experimental journey was
successfully completed.

![](https://static.igem.wiki/teams/4276/wiki/notebook/t-shanghai-hs-notebook-08.jpg)

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{% block title %}Engineering{% endblock %}
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# Engineering

## INTRODUTION：

EV71 is a neurotropic virus that often causes clinical symptoms such as fever, hand-foot-mouth disease, and
herpetic angina in children. Severe cases can lead to neurological diseases and heart disease, and even death.
RV mainly causes viral diarrhea in children. Every season, infants and young children are vulnerable to
infectious diseases caused by these two viruses. At present, the coverage coverage of EV71 vaccine is
relatively low in the market, and the number of vaccinations is relatively limited. It still takes some time
to evaluate the safety and immune persistence of this kind of vaccine. At present, there is no effective
treatment for diarrhea caused by rotavirus. The rotavirus vaccines on the market are attenuated rotavirus
vaccines, which play a certain role in preventing RV infection, but there are also problems such as
intussusception, virulence reversion, and contamination by exogenous factors. Studies have shown many
similarities between EV71 and RV viruses. Therefore, we expressed the antigen vp7 of RV virus in tandem with
the antigen vp1 of EV71 virus to effectively protect the health of infants and young children. More
importantly, compared with injection, oral vaccination is much more convenient, faster to popularize, and does
not require professionals to vaccinate, and consumes less social resources.

## DESIGN：

VP1 is the major antigenic gene of the EV71 virus, which encodes coat protein and promotes infection of host
cells by viral particles. Therefore, VP1 was identified as the target gene for the development of an EV71
vaccine. VP7 encodes an important protein of the RV viral coat (the protein of the glycoprotein), which is the
primary antigen-activated by antibodies and plays an important role in vaccine development. Therefore, we want
to fuse VP1 and VP7 and design the plasmid map of pET28a-VP7-VP1.

![](https://static.igem.wiki/teams/4276/wiki/engineering/t-shanghai-hs-engineering-01.jpg)

Figure 1. Plasmid map of pET28a-vp7-vp1

## BUILD：

### 1. Overlapping amplification of VP7-VP1 sequences

#### 1.1 PCR cloning VP1 and VP7 genes

To build the plasmid, we let the synthetic company synthesize the DNA fragment of VP7 and VP1, and
integrate it into the pUC57 vector. Then, we did PCR to amplify the DNA fragment of VP7 and VP1.

![](https://static.igem.wiki/teams/4276/wiki/engineering/t-shanghai-hs-engineering-02.jpg)

Figure 2. The PCR result of VP7 and VP1.
Line 1, 3, 5, 7 demonstrates VP1 genes
Line 2, 4, 6, 8 demonstrates VP7 genes

The DNA gel electrophoresis image above showed the bands of VP 1 and VP 7 genes. In theory, the VP1 gene
is 891bp in length, and the VP7 gene is 843bp. The strongly visible bands of cloned VP1 and VP7 genes
located in the correct range within 500 and 1000 bp. Figure 2 proved that we are obtained fragments VP1
and VP7 successfully.

#### 1.2 Overlap PCR VP7-VP1 sequences

To link the genes of two different viruses vp1 and vp7 together, we connected them by overlap PCR.

![](https://static.igem.wiki/teams/4276/wiki/engineering/t-shanghai-hs-engineering-03.jpg)

Figure 3. The PCR result of VP7-VP1
Line 1-6 are all VP7-VP1 (length: 1791bp).

The length of VP7-VP1 gene is 1734bp. The bands showed the length of the cloned genes 1, 2, 3, 4, 7, and 8
are in the right location relative to the marker. The result showed that even though it was unsuccessful
in two scenarios, the rest produced the desired result of linked vp7 and vp1 genes. The results showed
that vp1-vp7 sequences were successfully fused (Figure 3).

### 2. Transform into E.coli DH5α

The fusion fragment VP7-VP1 was inserted into the pET28a vector digested with EcoRI and XhoI and transformed
it into *E. coli* DH5α.

![](https://static.igem.wiki/teams/4276/wiki/engineering/t-shanghai-hs-engineering-04.jpg)

Figure 4: The colony PCR identification result of pET28a-VP7-VP1/DH5α.

The two lines to the right side of the marker exhibit the bands of the vp7-vp1 in pET28a. The result of the
electrophoresis shows a visible band at approximately 1700bp.The PCR identification results showed that the
transformation of plasmids pET28a-vp7-vp1were successful (Figure 4).

### 3. Sanger sequencing of pET28a- VP7-VP1 plasmid

We send the plasmid pET28a-vp7-vp1 to biological company for sequencing. The returned sequencing comparison
results showed that there were no mutations in the ORF region(Figure 5)

![](https://static.igem.wiki/teams/4276/wiki/engineering/t-shanghai-hs-engineering-05.jpg)

Figure 5. Sanger sequencing of pET28a-vp7-vp1 plasmid

### 4. Transform into *E.coil* BL21(DE3)

Next, the plasmid pET28a-vp7-vp1 was extracted from *E.coil* DH5α, then transformed into *E.coil*
BL21(DE3) to
conduct IPTG induced protein expression.

![](https://static.igem.wiki/teams/4276/wiki/engineering/t-shanghai-hs-engineering-06.jpg)

Figure 6: The PCR identification result of pET28a-vp7-vp1 in *E.coil* BL21 (DE3)

Out of the 8 line, only 6 and 8 are successful since only their bands are located at around 1700bps. It
means that only samples from colonies line 6 and 8 contain proper vp7-vp1 gene fragments. Figure 6 showed
that the plasmid pET28a-vp7-vp1 was successful conversion.

## TEST

### Protein expression and purification

We added IPTG to induce protein expression when the OD600 reached 0.3-0.5. After overnight induction and
culture, we collected the cells and ultrasonic fragmentation of cells to release the intracellular proteins
vp7-vp1. Next, we used nickel column purification to purify the protein vp7-vp1 we wanted.

![](https://static.igem.wiki/teams/4276/wiki/engineering/t-shanghai-hs-engineering-07.jpg)

Figure 7. The SDS-PAGE result of the protein vp7-vp1

Referring to the marker in Figure 7, we found the proteins vp7-vp1 in unpurified protein samples
(supernatant) and purified protein samples, indicating that proteins were successfully expressed in *E.
coli*
BL21 (DE3).

## LEARN:

Our experimental results showed that the pET28a-vp7-vp1 plasmid was successfully constructed, the protein was
successfully expressed. It proves that it is feasible to express VR-vp7 in tandem with EV71-vp1. We fused two
kinds of VR-vp7 and EV71-vp1 expression, not only can avoid the infant by two kinds of virus damage, but also
can improve the infant vaccination rate, because oral way was more convenient. In the future, we will fuse the
membrane-bound protein GLBP to display on the surface of *Bifidobacterium*, express the vaccine adjuvant
IL-12,
and finally, add the green fluorescent protein (GFP) tag to facilitate the detection of vp7-vp1 on
*Bifidobacterium*. Eventually, *Bifidobacterium* was transferred to make a vaccine that can be taken
orally. This
method can not only improve the vaccination rate but also facilitate vaccination. In the future, we hope that
it can be truly put into use to effectively protect the health of infants and young children.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
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# Experiments

{% include 'experiments/section-1.html' %}
{% include 'experiments/section-2.html' %}
{% include 'experiments/section-3.html' %}
{% include 'experiments/section-4.html' %}

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
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# Results

## Overview

Our experiments could be grouped into two categories based on their purposes: plasmid construction and protein
expression. Since we are designing a bivalent vaccine, the most important goal is to be able to express
proteins of both VP7 and VP1 on one plasmid. By constructing a plasmid with vp7-vp1, *GLBP-vp7-vp1-GFP*,
and
*GLBP-vp7-vp1-IL12-GFP* genes, we obtained that these genes linked together on a vector. And by showing
that
proteins are successfully expressed, we are able to prove that the proteins of the genes can be simultaneously
coded in a strand of DNA, therefore, confirming that there is potential that a bivalent vaccine can be
developed for EV71 and RV.

## 1. Plasmid Construction

### 1.1 PCR cloning VP1 and VP7 genes

![](https://static.igem.wiki/teams/4276/wiki/results/t-shanghai-hs-results-01.jpg)

Figure 1: Gel electrophoresis result showed size of *vp1* and *vp7* gene fragments.
Line 1, 3, 5, 7 demonstrates vp 1 genes (length: 891bp).
Line 2, 4, 6, 8 demonstrates vp 7 genes (length: 843bp).

The DNA gel electrophoresis image above showed the bands of vp1 and vp7 genes. In theory, the vp1 gene is
891bp in length, and the vp7 gene is 843bp. The strongly visible bands of cloned vp1 and vp7 genes are
located in the correct range within 500 and 1000 bp. The gel electrophoresis helps to examine that we have
cloned our genes without error.

### 1.2 PCR cloning vp1, vp7, GLBP, GFP and IL-12 DNA fragments

![](https://static.igem.wiki/teams/4276/wiki/results/t-shanghai-hs-results-02.jpg)

Figure 2: Gel electrophoresis result showed vp1, vp7, GLBP, GFP and IL-12 fragments
M: DNA marker

The vp1, vp7, GLBP, GFP and IL-12 DNA fragments PCR result shown in Figure 2. Compared to DNA maker, the PCR
products exhibits the correct bands.

### 1.3 Overlap extension PCR

![](https://static.igem.wiki/teams/4276/wiki/results/t-shanghai-hs-results-03.jpg)

Figure 3: Gel electrophoresis result showed length of combined vp7-vp1.
Line 1-6 are all vp7-vp1(length: 1791bp).

This image demonstrated the result of the overlap PCR, in which the purpose is to link the genes of two
different viruses together. The length of vp7-vp1 gene is 1734bp. The bands showed the length of the cloned
genes 1, 2, 3, 4, 7, and 8 are in the right location relative to the marker. The result showed that even
though it was unsuccessful in two scenarios, the rest produced the desired result of linked vp7 and vp1
genes.

### 1.4 Overlap extension PCR cloning GLBP-VP7-VP1-GFP and GLBP-VP7-VP1-IL12-GFP

![](https://static.igem.wiki/teams/4276/wiki/results/t-shanghai-hs-results-04.jpg)

Figure 4: Gel electrophoresis result showed overlap extension PCR.
M: DNA marker

Due to large GLBP-VP7-VP1-GFP DNA fragment, we used overlap extension PCR in two steps. Firstly, we splice
GLBP and VP7, VP1 and GFP, respectively (shown as left panel). Then, combine two fragments *GLBP-VP7* with
*VP1-GFP* to the final DNA fragment (shown as middle panel). The right panel showed the PCR-amplified
GLBP-VP7-VP1-IL12-GFP.

### 1.5 Restriction Enzyme Digestion

![](https://static.igem.wiki/teams/4276/wiki/results/t-shanghai-hs-results-05.jpg)

Figure 5: Gel electrophoresis result exhibited difference between undigested pET28a and digested pET28a.

Line 1 is undigested supercoiled pET28a.
Line 2-4 are digested linear pET28a.

To obtain recombinant plasmid, we used restriction enzymes EcoRI and XhoI to digest the gene fragments and
plasmid to make double digestion samples, then used DNA ligase to join pET28a vector and vp7-vp1,
GLBP-VP7-VP1-GFP, and GLBP-VP7-VP1-IL12-GFP DNA fragment, respectively. After the pET28a plasmids were
digested, we used electrophoresis to confirm whether the cleavage was thorough. *In vivo*, linear (cleaved)
plasmids travel a shorter distance compared to circular plasmids due to the more friction. Thus, the
digested plasmids in wells 2-4 ran slower than the supercoiled pET28a plasmid in the first well. This result
showed that the plasmids were successfully cleaved by restriction enzymes.

### 1.6 *E.coli* colony PCR

The fusion fragment VP7-VP1 was inserted into the pET28a vector digested with EcoRI and XhoI and transformed
it into *E. coli* DH5α.

![](https://static.igem.wiki/teams/4276/wiki/results/t-shanghai-hs-results-06.jpg)

Figure 6. The colony PCR identification result of pET28a-VP7-VP1/DH5α.

The two lines to the right side of the marker exhibit the bands of the vp7-vp1 in pET28a. The result of the
electrophoresis shows a visible band at approximately 1700bp.The PCR identification results showed that the
transformation of plasmids pET28a- VP7-VP1 were successful (Figure 6). Thus, we picked up positive colonies
to sequence.

![](https://static.igem.wiki/teams/4276/wiki/results/t-shanghai-hs-results-07.jpg)

Figure 7: Gel electrophoresis result demonstrated the length of pET28a-GLBP-VP7-VP1 -GFP in *E.Coli* DH10B.

Line 1-10 colony PCR of pET28a-GLBP-VP7-VP1 -GFP colonies.

We picked up 10 colonies to verify whether the colony containing the recombinant plasmid pET28a-GLBP-
VP7-VP1 -GFP. The result shown as figure 7, and the transformants 2, 4, 7, and 9 have right size. Thus, we
picked up positive colony 2 to sequence.

![](https://static.igem.wiki/teams/4276/wiki/results/t-shanghai-hs-results-08.jpg)

Figure 8: Gel electrophoresis result demonstrated the length of pET28a-GLBP-VP7-VP1-IL12-GFP in *E.Coli*
DH10B.

Line 1-10 colony PCR of pET28a-GLBP- VP7-VP1 -IL12-GFP colonies.

We picked up 4 colonies to verify whether the colony containing the recombinant plasmid pET28a-GLBP- VP7-VP1
-IL12-GFP. The result shown as figure 8 and the transformants 1, 3, and 4 have right size. Thus, we picked
up positive colony 3 to sequence.

### 1.7 DNA sequencing

![](https://static.igem.wiki/teams/4276/wiki/results/t-shanghai-hs-results-09.jpg)

Figure 9. Sanger sequencing of recombinant plasmid
A. pET28a-vp7-vp1 plasmid
B. pET28a-GLBP-vp7-vp1-GFP plasmid
C. pET28a-GLBP-RV-EV-GFP plasmid

The sanger sequencing results showed as Figure 9 and the sequence well matched with the template. Thus, we
used this plasmid to perform the subsequent experiment.

## 2. Protein Expression

### 2.1 Transform BL21(DE3)

BL21 (DE3) is a suitable bacterium to conduct IPTG induced protein expression, so in order for the protein
to be successfully expresses in the subsequent experiments, we extracted the recombinant plasmids
pET28a-vp7-vp1, pET28a-GLBP-VP7-VP1-GFP, and pET28a-GLBP-RV-EV-GFP from DH5α or DH10B, and transferred them
into BL21 (DE3) , added IPTG to induce protein expression, repectively.

![](https://static.igem.wiki/teams/4276/wiki/results/t-shanghai-hs-results-10.jpg)

Figure 10: Photo of BL21(DE3) colonies grown containing the pET28a vp7-vp1 plasmid.

### 2.2 SDS-PAGE

![](https://static.igem.wiki/teams/4276/wiki/results/t-shanghai-hs-results-11.jpg)

Figure 11: SDS-PAGE gel demonstrated the size of the protein samples.
P: precipitant
S: supernatant
E: elution

The negative control is pET28a plasmid without exogenous gene. The expression of VP7-VP1, GLBP-VP7-VP1-GFP,
and GLBP-VP7-VP1-IL12-GFP were confirmed by SDS-PAGE, as shown in Figure 11.

![](https://static.igem.wiki/teams/4276/wiki/results/t-shanghai-hs-results-12.jpg)

Figure 12: SDS-PAGE gel electrophoresis diagram demonstrating the length of the VP7-VP1 protein samples.

Line 1-7 are unpurified protein samples (supernatant).
Line 8-13 are purified protein samples.

We have gained protein from the different concentration of IPTG induced vp7-vp1 BL21 (DE3) bacteria through
centrifuge, cell crushing, and purification. The six IPTG concentration as follows: 0.1 mM,0.25 mM, 0.5 mM,
1 mM, 5 mM and 10 mM. In the SDS-PAGE experiment, we took a sample from each of the 6 unpurified proteins
(supernatant) and 6 purified proteins. It can be observed that proteins are successfully expressed despite
the impurities in the samples.

### 2.3 Green Fluorescent Signal

This image showed the GLBP-VP7-VP1 fused GFP expressed in the bacteria.

![](https://static.igem.wiki/teams/4276/wiki/results/t-shanghai-hs-results-13.jpg)

Figure13. The comparison of supernatant of GLBP-VP7-VP1 fused GFP and without GFP

The left tube is bacteria expressing pET28a-VP7-VP1 without GFP as the negative control. The right tube is
bacteria expressing pET28a-GLBP-VP7-VP1-GFP. The lift panel showed the supernatant is under visible light,
and the right panel showed the supernatant is under blue light.

![](https://static.igem.wiki/teams/4276/wiki/results/t-shanghai-hs-results-14.jpg)

Figure 14. Fluorescence microscopy of GLBP-VP7-VP1-GFP.

It’s obvious that the supernatant contains lots of protein tagged GFP after ultrasonic crushing bacteria
expressing pET28a-GLBP-VP7-VP1-GFP, which indicating the VP7 and VP1 overexpressed in the bacteria and bond
to the cell surface.

![](https://static.igem.wiki/teams/4276/wiki/results/t-shanghai-hs-results-15.jpg)

Figure 15. The GFP intensity indicated protein expression

At the same time, we monitored the bacteria expressed the VP1 and VP7 for 0 to 8 hours using GFP intensity.
as shown in figure 15, the result exhibited the relationship of protein expression with time and the effect
of IPTG on fluorescence intensity. The longer induction time is beneficial for protein expression, but
according to the result, 3 hours is enough for VP1 and VP7 expression. In addition, the IPTG concentration
has a significant influence on the GFP intensity during the cultivation. Concentration of IPTG in 0.25 to 10
mM has a little effect on the protein expression. However, higher concentration of IPTG (10 mM) increased
the GFP intensity.

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