iGEM 2023 — Team canton-hs

Introduction:

We would like to provide a detailed explanation of our project timeline. In order to do so, we will regularly review and update our progress to ensure that we are on track to meet our project goals. By closely monitoring our timeline and progress, we can make any necessary adjustments and ensure that we deliver a high-quality final product on time and within budget.

Day 1:

-Team meeting and design logo, slogan

![](https://static.igem.wiki/teams/4872/wiki/notebook432.png)![](https://static.igem.wiki/teams/4872/wiki/notebook433.png)

Day 2:

-Expert interviews

-Questionnaire survey

- Laboratory Safety Training

- Configuring LB media

- Autoclave, pour plate

- PCR amplification of GII.4-VP1, GII.17-VP1, and RV VP7 genes

- Inoculate with pGEX-4T-1 strain

Participation list:

Arron/Naomi/Hedy/Candy/Tom/John/Belinda/Leah/Fiona/Yoyo/Ben

![](https://static.igem.wiki/teams/4872/wiki/notebook742.png)![](https://static.igem.wiki/teams/4872/wiki/notebook743.png)

Day 3:

-Expert interviews

-Product research

- Electrophoresis GII.4-VP1, GII.17-VP1 and RV VP7 gene

- Gel purification

- Plasmid extraction pGEX-4T-1

Participation list:

Arron/Naomi/Hedy/Candy/Tom/John/Belinda/Leah/Fiona/Yoyo/Ben

![](https://static.igem.wiki/teams/4872/wiki/notebook984.png)![](https://static.igem.wiki/teams/4872/wiki/notebook985.png)

Day 4:

-Interview Summary

- Electrophoresis of plasmid pGEX-4T-1

- Gel extraction of the separated target DNA

- Homologous Recombination of pGEX-4T-1 and RV VP7

- Transformation-Heat shock of DH5 alpha into the plasmid

- Digestion of plasmid pGEX-4T-1

- Digestion of DNA VP1/VP7

- Ligation of pGEX-4T-1 and VP1

- PCR purification

Participation list: Arron/Naomi/Hedy/Candy/Tom/John

![](https://static.igem.wiki/teams/4872/wiki/notebook1374.png)![](https://static.igem.wiki/teams/4872/wiki/notebook1375.png)

Day 5:

- Wiki Writing

- Survey questionnaire analysis

- PCR identification of transformants

- Pick monoclonal culture seed solution

- Plasmid extraction pGEX-GII.4-VP1, pGEX-GII.17-VP1, pGEX-VP7-GII.4-VP1 and pGEX-VP7-GII.17-VP1 were transformed into BL21(DE3) and Nissle 1917 cells - Spread plate screening

Participation list: Belinda/Leah/Fiona/Yoyo/Ben

![](https://static.igem.wiki/teams/4872/wiki/notebook1740.png)![](https://static.igem.wiki/teams/4872/wiki/notebook1741.png)

Day 6:

- Business Plan

- Wiki Writing

- PCR for plasmid DNA

- Electrophoresis of plasmid DNA

- Extracting plasmid from *E.coli*

- Select monoclonal culture seed solution

- Sent plasmids for sequencing

Participation list: Arron/Naomi/Hedy/Candy/Tom/John

![](https://static.igem.wiki/teams/4872/wiki/notebook2002.png)![](https://static.igem.wiki/teams/4872/wiki/notebook2003.png)

Day 7:

- Education Workshop

- Expand the volume of cultivated bacteria

- Electrophoresis

- IPTG protein induce

- DNA gel recovery

- Homologous recombination

Participation list: Belinda/Leah/Fiona/Yoyo/Ben

![](https://static.igem.wiki/teams/4872/wiki/notebook2220.png)![](https://static.igem.wiki/teams/4872/wiki/notebook2221.png)

Day 8:

- Science Education

- Ultrasonication

- GST protein purification

- SDS-PAGE modified acrylamide color gel

Participation list: Belinda/Leah/Fiona/Yoyo/Ben

![](https://static.igem.wiki/teams/4872/wiki/notebook2391.png)![](https://static.igem.wiki/teams/4872/wiki/notebook2392.png)

Day 9

- Pitch Financing Roadshow

- Enterprise Visit

- Denaturation of the protein

- Preparation of SDS-PAGE gel

- SDS-PAGE

- Dyeing and decolorization of protein gel

Participation list: Arron/Naomi/Hedy/Candy/Tom/John

![](https://static.igem.wiki/teams/4872/wiki/notebook2619.png)![](https://static.igem.wiki/teams/4872/wiki/notebook2620.png)

Day 10

 - Repeat construction procedure of plasmid pGEX-RV-GII.17-VP1

 - Wiki documentation

Participation list: Belinda/Leah/Fiona/Yoyo/Ben

![](https://static.igem.wiki/teams/4872/wiki/notebook2765.png)![](https://static.igem.wiki/teams/4872/wiki/notebook2766.png)

Day 11

- Pick monoclonal culture seed solution

- Plasmid extraction pGEX-VP7-GII.17-VP1 were transformed into BL21(DE3) and Nissle 1917 cells

- Spread plate screening

Participation list: Belinda/Leah/Fiona/Yoyo/Ben

![](https://static.igem.wiki/teams/4872/wiki/notebook2989.png)![](https://static.igem.wiki/teams/4872/wiki/notebook2990.png)

Day 12

- Expand the volume of cultivated bacteria

- Electrophoresis

- IPTG protein induce

- DNA gel recovery

- Sent for sequencing

Participation list: Arron/Naomi/Hedy/Candy/Tom/John

![](https://static.igem.wiki/teams/4872/wiki/notebook3182.png)![](https://static.igem.wiki/teams/4872/wiki/notebook3183.png)

Day13

- Preparation of SDS-PAGE gel

- SDS-PAGE

- Dyeing and decolorization of protein gel

Participation list: Arron/Naomi/Hedy/Candy/Tom/John

![](https://static.igem.wiki/teams/4872/wiki/notebook3332.png)![](https://static.igem.wiki/teams/4872/wiki/notebook3333.png)

Day14

- IPTG protein induce under different conditions

- Wiki documentation

Participation list: Belinda/Leah/Fiona/Yoyo/Ben

![](https://static.igem.wiki/teams/4872/wiki/notebook3462.png)![](https://static.igem.wiki/teams/4872/wiki/notebook3463.png)

Day 15

- Preparation of SDS-PAGE gel

- SDS-PAGE

- Dyeing and decolorization of protein gel

- Wiki documentation

Participation list: Belinda/Leah/Fiona/Yoyo/Ben

![](https://static.igem.wiki/teams/4872/wiki/notebook3630.png)![](https://static.igem.wiki/teams/4872/wiki/notebook3631.png)

**Introduction:**

Norovirus is characterized by high infectivity, diverse modes of transmission,
and easy mutation of the pathogen, and is the main pathogen of sporadic and fulminant acute gastroenteritis
in the population, which causes a huge disease burden to the society, and there is no vaccine for norovirus
in the market at present
1-3
. Therefore, our team is attempting to produce a vaccine for norovirus, and
meantime
we
also
tried to produce bivalent vaccines against norovirus and rotavirus .

In this process, we first genetically engineered the major antigenic genes of
norovirus or (and) rotavirus into vector plasmids and transformed them into
*E. coli* BL21(DE3) or
Nissle 1917
, and then induced the plasmids to express the proteins upon culture. Finally,
we purify and analyze the proteins.

**Cycle I****:**[**BBa\_K4872010**](http://parts.igem.org/Part%3ABBa_K4872010)**(****pGEX-GII.****4-****VP1)**

**Design:**

We used the Tac promoter, which is suitable for
*E. coli*
protein production, for transcription of the expression frame. At the
C-terminus of target gene GII.4-VP1, we fused a GST tag, which facilitates soluble expression of the protein
and can be used to purify the protein (Figure 1). After obtaining this recombinant plasmid, we transformed
it into
*E. coli*
BL21(DE3).

Finally, we used IPTG to induce the expression of the target protein
GII.4-VP1.

![](https://static.igem.wiki/teams/4872/wiki/engineering-success1356.png)

**Figure 1 Plasmid design diagram****of pGEX-GII.4-VP1**

**Build:**

Firstly,
 we
amplified
 the antigen gene GII.4-VP1 us
ing
 the PCR amplifier

(
Figure 2A
). Then,

the GII.4-VP1 fragment and the pGEX-4T-1 plasmid vector were digested with
restriction endonucleases
*Eco*
RI and
*Xho*
I, followed by the ligation using T4 DNA ligase to obtain the recombinant
plasmid pGEX-GII.4-VP1
(Figure 2B)
. As shown in Figure 2C-D, the colony PCR and sequencing results confirmed the
successful construction of the plasmid.

![](https://static.igem.wiki/teams/4872/wiki/engineering-success1849.png)

**Figure 2 Construction of plasmid pGEX-GII.4-VP1**

**Test:**

We inoculated the positive transformant and induced protein expression by
IPTG. After obtaining protein lysate, we purified the target protein using GST tags and verified the protein
expression and purification results by SDS-PAGE. The results are shown in Figure 3, we successfully
expressed GII.4-VP1 protein
 (60 KDa)
, but the purified protein was barely visible, which might be due to the low
expression of protein.

![](https://static.igem.wiki/teams/4872/wiki/engineering-success2337.png)

**Figure 3****SDS-PAGE results of pGEX-GII.4-VP1****protein****expression**

**(P represents****" Precipitation" ,****S represents****" Supernatant" ,****Trepresents****" Flow through" ,****Wrepresents****" Wash solution" and****Erepresents****" Eluent****"****)**

**Learn:**

Since the low expression of the protein was not successfully purified, we need
to explore the optimal expression conditions for the purification of this protein to increase the expression
level of the protein.
 After we solve this problem,
we could further step to continue the construction of recombinant plasmid pGEX-RV VP7-GII.4-VP1.

**Cycle2:**[**BBa\_K4872011**](http://parts.igem.org/Part%3ABBa_K4872011)**(****pGEX-GII.17-VP1****)**

**Design:**

Following the same idea as the previous round, we used the Tac promoter to
transcribe the plasmid A GST tag was fused to the C-terminus of the target gene GII.17-VP1 (Figure 4). After
obtaining the recombinant plasmid, we transformed it into
*E. coli* BL21(DE3). Finally, we used
IPTG to induce the expression of the target protein GII.17-VP1.

![](https://static.igem.wiki/teams/4872/wiki/engineering-success3338.png)

**Figure 4 Plasmid design diagram of pGEX-GII.17-VP1**

**Build:**

W
e
 first
amplified
 the antigen gene GII.17-VP1
us
ing
 the PCR amplifier (
Figure
5
A
). Then,

the GII.17-VP1 fragment and the pGEX-4T-1 plasmid vector were digested with
restriction endonucleases
*Eco*
RI and
*Xho*
I, followed by the ligation using T4 DNA ligase to obtain the recombinant
plasmid pGEX-GII.4-VP1
(Figure
5
B)
. As shown in Figure 5C-D, the colony PCR and sequencing results confirmed the
successful construction of the plasmid.

![](https://static.igem.wiki/teams/4872/wiki/engineering-success3831.png)

**Figure 5 Construction of plasmid pGEX-GII.17-VP1**

**Test:**

We inoculated the positive transformant and induced protein expression by
IPTG. After obtaining protein lysate, we purified the target protein using GST tags and verified the protein
expression and purification results by SDS-PAGE. As shown in Figure 6, we successfully expressed and
purified GII.17-VP1 protein (60 kDa).

![](https://static.igem.wiki/teams/4872/wiki/engineering-success4226.png)

**Figure 6 SDS-PAGE results of pGEX-GII.17-VP1 protein expression**

**(Prepresents " Precipitation" , Srepresents " Supernatant" , Trepresents
" Flow through" , Wrepresents " Wash solution" and Erepresents " Eluent" )**

**Learn:**

Unlike the expression and purification of GII.4-VP1 protein, the GII.17-VP1
protein was successfully expressed and purified in this round of experiments, which is expected to be
prepared as a norovirus vaccine.
 In order to develop this
norovirus vaccine, the next plan will be focused on the research of the
GII.17-VP1
expression in E. coli Nissle 1917 and more evaluation tests shall be made
before the implementation. Meantime, we can continue to construct the recombinant plasmid pGEX-RV-GII.17-VP1
to investigate the potential of the
bivalent
vaccine against norovirus and rotavirus.

**Cycle III:**[**BBa\_K4872013**](http://parts.igem.org/Part%3ABBa_K4872013)**(****pGEX-RV-GII.17-VP1****)**

**Design:**

To obtain a bivalent vaccine against norovirus and rotavirus, we used a linker
that connects the GII.17-VP1 antigenic gene of norovirus to RV VP7 of rotavirus. As in the previous two
rounds of experiments, we used the Tac promoter and added a GST tag to the C-terminus of the fusion gene for
subsequent purification (Figure
 7
). After obtaining the recombinant plasmid, we transformed it into
*E. coli* BL21(DE3) and
Nissle 1917
, respectively. Finally, we used IPTG to induce the expression of target
proteins and optimized their expression conditions.

![](https://static.igem.wiki/teams/4872/wiki/engineering-success5694.png)

**Figure 7 Plasmid design diagram of pGEX-RV-GII.17-VP1**

**Build:**

W
e
 first
amplified
 the antigen gene
 RV VP7 and GII.17-VP1 us
ing
 the PCR amplifier (
Figure
8
A
). Then,

the
RV VP7
 and GII.17-VP1 fragments
underwent homologous recombination with the pGEX-4T-1 plasmid vector to obtain the recombinant plasmid
pGEX-GII.4-VP1
(Figure
8
B)
. As shown in Figure 8C-D, the colony PCR and sequencing results confirmed the
successful construction of the plasmid.

![](https://static.igem.wiki/teams/4872/wiki/engineering-success6144.png)

**Figure 8 Construction of plasmid pGEX-RV-GII.17-VP1**

**Test:**

We inoculated the transformant containing pGEX-RV-GII.17-VP1, induced its
expression, and explored its optimal expression conditions.

To find the optimal conditions for the highest protein expression, we chose
different concentrations (OD
600
=0.3/0.6/0.8/1) of the bacterial solution and different IPTG induction times
(0 h/4 h/8 h/16 h). After obtaining each protein lysate, we measured the total protein amount (A280) under
different induction conditions (
Figure
10A
). In addition, we examined the expression of the target proteins
 (119 KDa) using SDS-PAGE (
Figure
9
) and used the ImageJ software to quantify the target bands on the SDS-PAGE
gel, collected and organized the data, and plotted a line graph with OD
600 as the x-axis and gray value
as the y-axis (Figure 10B).

As shown in Figure 10, the protein concentration roughly tended to increase
with increasing bacterial concentration at the same IPTG induction time. When the bacterial concentration
was 0.8 and the induction time was 8 hours, the best protein expression level was achieved, indicating that
this condition was more suitable for expressing more target proteins.

![](https://static.igem.wiki/teams/4872/wiki/engineering-success7341.png)

**Figure 9 SDS-PAGE results of RV-GII.17-VP1 protein under different
expression conditions**

![](https://static.igem.wiki/teams/4872/wiki/engineering-success7433.png)

**Figure 10 Effect of IPTG induction time and bacterial concentration on
protein concentration**

Finally,
we
transformed
 plasmid
pGEX-
RV-
GII.17-VP1
 into *E. coli*
Nissle 1917. Through the SDS-PAGE gel map, it was found that we have weak
bands within the 90

kD
a
 range, which means that RV-
GII.17-VP1
 protein
(119 KDa)

expression level is weak in
*E. coli*
Nissle 1917
 (Figure 11)
.
 This initially confirmed that
RV-GII.17-VP1 could be successfully expressed in Nissle 1917, but the expression conditions need to be
optimized.

![](https://static.igem.wiki/teams/4872/wiki/engineering-success7952.png)

**Figure 11 SDS-PAGE results of pGEX-RV-GII.17-VP1 protein expression in E.
coli Nissle 1917**

**Learn:**

The expression of RV-GII.17-VP1 in
*E. coli*
Nissle 1917 was low, so we need to improve and optimize the protein expression
method to achieve large-scale expression of RV-GII.17-VP1.
 But we did see a potential of
this
bivalent vaccine against norovirus and rotavirus,
 no doubt this research will
be continually developed and is a promising product in the vaccine market for society.

**References:**

1.

Hallowell, B. D., Parashar, U. D., & Hall, A. J. (2019). Epidemiologic
challenges in norovirus vaccine development. Human vaccines & immunotherapeutics, 15(6),
1279-C1283.

2.

Esposito S, Principi N.
[Norovirus Vaccine: Priorities for Future Research and Development.](https://pubmed.ncbi.nlm.nih.gov/32733458/) Front Immunol. 2020 Jul
7;11:1383.

3.

Offit Paul A. Challenges to Developing a Rotavirus Vaccine.[J]. Viral
immunology,2018,31(2).

**CONTENTS**

1.
Preparation of liquid LB
medium

2.

PCR-
Amplifying virus gene

3.
Agarose gel Electrophoresis

4.
Inoculation

5.
Plasmid Extraction

6.
DNA Gel Extraction

7.
Double enzyme digestion and homologous
recombination

8.
Ligation and homologous
recombination

9.
Transformation-Heat Shock

10.
IPTG-induced protein
expression

11.
Ultrasonication and
protein
purification

12.
SDS-PAGE protein
electrophoresis

**1. Preparation of liquid LB
medium**

Material:

|  |  |
| --- | --- |
| **Reagent** | **Mass (g)** |
| Tryptone | 10 |
| Yeast extract | 5 |
| NaCl | 10 |

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

Solid plus 1.5% agar.

Apparatus:

|  |  |
| --- | --- |
| Test tube | 1 |
| triangular flask | 1 |
| measuring cylinder (0-500 mL) | 1 |
| ultra-clean bench | 1 |
| media dispenser | 1 |
| balance | 1 |
| Pipette (100-1000 μL) | 1 |
| Petri dishes | 8 |
| sealing strips | 8 |

Procedure: On this basis, add 100 μL of antibiotics (diluted by 1000
times), prepare the plate after the temperature is not hot, write the relevant information on the bottom
with a marker, and then pour it into the plate while the medium is not solidified, wait for solidification
and then send to the refrigerator (during the period, disinfect the bottleneck, do not take your hands out
of the workbench).

2.
**PCR-****Amplifying virus gene (GII.4-VP1 + GII.17-VP1 +
RV VP7 gene)**

|  |  |
| --- | --- |
| **Reagent** | **Volume** **(μL)** |
| Mix | 25 |
| Template | 1 |
| Forward primer | 1 |
| Reverse primer | 1 |
| ddH2O | 22 |
| Total | 50 |

**PCR Procedures:**

|  |  |  |
| --- | --- | --- |
| Step | Temperature ( ℃) | Time |
| 1 | 98 | 3 min |
| 2 | 98 | 30 sec |
| 3 | 60 | 30 sec |
| 4 | 72 | 2 min |
| 5 | 72 | 10 min |
| 6 | 4 | forever |

PS: Steps 2 to 4 cycle 30 times.

Apparatus:

|  |  |
| --- | --- |
| PCR tubes | 10 |
| pipettes (range: 2  μL-20   μL) | 2 |
| pipettes (range: 20  μL-20 0  μL) | 2 |
| PCR Thermal Cycler | 1 |

3.
**Agarose gel Electrophoresis**

(1)
Preparation of TAE

Material:

|  |  |
| --- | --- |
| **Reagent** | **Volume/mass** |
| TAE buffer powder | 1 g |
| ddH₂O | 500 mL |

(2)
Preparation of Agarose gel

|  |  |
| --- | --- |
| **Reagent** | **Volume/mass** |
| Agarose | 1 g |
| 1x TAE(buffer) | 100 mL(1%) |
| GelRed | 2-3 mL |
| Marker | 6 μL |

Apparatus:

|  |  |
| --- | --- |
| Electrophoresis instrument | 1 |
| UV analyzer | 1 |
| measuring cylinder | 1 |
| balance | 1 |
| Erlenmeyer flask | 1 |
| Mold (base plates and tooth combs) | 1 |
| Pipettes (2-20μL) | 2 |

Procedure:

a. Weigh out 1 g of agarose.

b. Pour 100 mL TAE into a measuring cylinder.

c. Mix them into an Erlenmeyer flask and put in the powder before
the liquid.

d. Microwave the mixture on high heat for 2 min.

e. Prepare the mold, put in the base plate and tooth comb, and
then add the mixture. Wait for it to solidify.

f. Add
the Marker into the first well.

g. After that, close the lid, connect the positive and negative
poles, and let stand for 20 min.

h. Remove the prepared gel and place it in the UV
analyzer.

i. The Marker position on the left corresponds from bottom to top
to a DNA length.

**4.** **Inoculation:**

Procedure**:**

1. Sterilize both hands and equipment brought into the clean
bench.

2. Label an EP tube with the correct data, subject, and
experimenter name.

3. Disinfecting tubes and equipment using an alcohol
burner.

4. Add 3 mL LB liquid medium into the labeled tube.

5. Add 20 μL glycerol bacteria (pGEX-4T-1. etc.) into the
labeled tube.

6. Add 3
μ
L Ampicillin (100 mg/mL) into the labeled tube.

7. Close the labeled tube with its cap.

8. Sterilized all the equipment.

9. Wash both hands with proper detergents.

10.culture at 37 ℃, 220 rpm in constant temperature shaker for
12~18h.

**5. Plasmid Extraction**

Material:

|  |  |
| --- | --- |
| reagent | Volume/mass |
| overnight cultured bacterial solution | 1.5-5 mL |
| Buffer SP1 | 250 μL |
| Buffer SP2 | 250 μL |
| Buffer SP3 | 350 μL |
| Buffer DW1 | 500 μL |
| Wash solution | 500 μL |
| Elution Buffer | 50-100 μL |

Apparatus:

|  |  |
| --- | --- |
| centrifuge | 1 |
| pellets | 6 |
| adsorption columns | 6 |
| collection tubes | 12 |
| Pipette (range: 0.5 μL ~ 10 μL) | 2 |
| Pipette (range: 20  μL ~  200  μL ) | 2 |
| Pipette (range: 100  μL ~ 10 00  μL ) | 2 |

Procedure:

a. Take 1.5-5 mL of overnight cultured bacterial solution,
centrifuge at 8000 x*g* for 2 min to collect bacteria, and discard the
medium.

b. Add 250 μL of Buffer SP1 to the pellet and suspend the
bacteria thoroughly.

c. Add 250 μL of Buffer SP2 and immediately mix and invert
the centrifuge tube 5-10 times gently and let stand at room temperature for 2-4 min (lysing the
bacteria).

d. Add 350 μL of Buffer SP3 and immediately mix and invert
the tube 5-10 times gently (Neutralization, Plasmids are preferentially adsorbed on the
membrane).

e. Centrifuge at 12000 x*g* for 5-10 min, pipette the supernatant into the adsorption
column, centrifuge at 8,000 x*g* for 30 s
ec
, and drain the liquid in the collection tube.

f. Add 500 μL Buffer DW1, centrifuge at 9000 x*g* for 30 s
ec
, and drain the liquid from the collection tube.

g. Add 500 μL Wash solution, centrifuge at 9000 x*g* for 30 sec, and drain the liquid from the collection tube
(Repeat once) (wash away the unwanted substances).

h. Centrifuged the empty adsorption column at 9000 x*g* for 1 min (Drain the cleaning solution).

i. Place the adsorption column into a 1.5 mL centrifuge tube and
add 50-100 μL Elution Buffer in the center of the adsorption membrane, let it stand at room temperature
for 1 min followed by centrifugation for 1 min, then preserve the plasmid liquid in the collection
tube.

**6. DNA Gel Extraction**

Material:

|  |  |
| --- | --- |
| reagent | Volume/mass |
| DNA | 40 μL |
| Buffer B2 | 300 μL |
| Wash solution | 500 μL |
| Elution Buffer | 50-100 μL |

Apparatus:

|  |  |
| --- | --- |
| centrifuge | 1 |
| adsorption columns | 6 |
| collection tubes | 6 |
| Pipette (range: 20  μL ~  200  μL ) | 2 |
| Pipette (range: 100  μL ~ 10 00  μL ) | 2 |
| 1.5 mL centrifuge tubes | 6 |

Procedure
:

a.
Add 300 μL of buffer B2 into the centrifuge tubes with the
solution that finished PCR.

b.
The liquid was moved into the adsorption columns and centrifuged
at 8000 x*g* for 30 sec to dump the liquid from the collection
tubes.

c.
Add 500 μL Wash solution, centrifuge at 9000 x*g* for 30 sec, and drain the liquid from the collection tubes
(Repeat once).

d.
Centrifuged the empty adsorption column at 9000 x*g* for 1 min.

e.
Place the adsorption columns into 1.5 mL centrifuge tubes and add
50-100 μL Elution Buffer in the center of the adsorption membrane, let it stand at room temperature for
1 min followed by centrifugation for 1 min, then preserve the plasmid liquid in the collection
tubes.

**7. Double enzyme digestion**

|  |  |
| --- | --- |
| **Reagent** | **Volume(μL)** |
| Plasmid  ( pGEX-4T-1 ) | 30 |
| *Eco*RI | 1 |
| *Xho*I | 1 |
| Buffer (Cutsmart) | 5 |
| ddH2O | 13 |
| Total | 50 |

|  |  |
| --- | --- |
| **Reagent** | **Volume(μL)** |
| DNA (GII.4- VP1 /GII.17- VP1 ) | 20 |
| *Eco*RI | 1 |
| *Xho*I | 1 |
| buffer(Cutsmart) | 5 |
| ddH2O | 23 |
| Total | 50 |

Procedure:

1. Add all of the elements in the form into a 1.5 mL tube using
pipettes.

2. Heat in the water bath at 37 ℃ for 30 min.

 Apparatus:

|  |  |
| --- | --- |
| pipette (range: 100  μL- 1000  μL) | 1 |
| Water bath | 1 |

8.
**Ligation****and** **homologous recombination**

I.
Ligation

Apparatus:

|  |  |
| --- | --- |
| PCR tubes | 4 |
| pipette (range: 2  μL- 20  μL) | 1 |
| electrophoresis instrument | 1 |
| UV analyzer | 1 |
| PCR Thermal Cycler | 1 |

**Materials:**

|  |  |
| --- | --- |
| **Reagent** | **Volume****(****μL****)** |
| pGEX-4T-1 | 4 |
| GII.4- VP1 /GII.17- VP1 | 13 |
| T4 ligase | 1 |
| T4 ligase buffer | 2 |
| Total | 20 |

Procedure:

a.
Add all of the elements in the form into PCR tubes using
pipettes.

b.
Adjust the PCR profile to 16 °C for 30 min with 65 °C
for
 5 min and start running it.

c.
After the gel is set, demould, pour TAE that can overwhelm the
gel into the electrophoresis instrument, inject the prepared DNA solution into the wells one by one with a
pipette (except the first well), and then inject DNA Marker into the first well.

d.
After that, close the lid, connect the positive and negative
poles, and let stand for 20 min.

e.
Remove the prepared gel and place it in the UV
analyzer.

II.
homologous recombination

|  |  |
| --- | --- |
| **Reagent** | **Volume****(****μL****)** |
| pGEX-4T-1 | 3 |
| RV -VP7 | 1 |
| GII.4-VP1/GII.17-VP1 | 1 |
| 2x Clon Express Mix | 5 |
| Total | 10 |

Procedure:

a.
Add the above reagents to a 1.5 mL centrifuge tube. (Note: the DNA in the second row should be
added separately to the plasmid in the first row).

b.
Place in a water bath at 50 °C for 15 min.

9.
**Transformation-Heat Shock**

 Materials:

|  |  |
| --- | --- |
| **Reagent** | **Volume(μL)** |
| competent cell(DH5α/BL21/Nissle 1917) | 50 |
| Plasmid(pGEX-GII.4-VP1/pGEX-GII.17-VP1  pGEX-RV-GII.4-VP1/pGEX-RV-GII.17-VP1) | 5 |
| LB liquid medium | 950 |

Apparatus:

|  |  |
| --- | --- |
| centrifuge | 1 |
| Pipette (range: 100 μL ~ 10 00 μL ) | 2 |
| microtubes | 6 |
| Icebox | 1 |
| Water bath | 1 |
| shaker | 1 |

Procedure:

a.
Add 50 μL competent cell (DH5α/BL21/Nissle 1917) and 5
μL plasmid into the microtube.

b.
Leave the tube on ice for 30 min
.

c.
Transfer the tube to the water bath at 42 ℃ for 90
sec.

d.
Transfer the tube onto ice for 5 min.

e.
For each tube, add LB medium in it until the total volume reaches
1 mL.

f.
Place the tube in the thermostatic shaker at 37 ℃, 220 rpm for
30 min.

g.
Centrifuge the tube at 9000 x*g* for 1 min, then discard 950 μL of the liquid and mix
the content left in the tube

h.
Place Petri dishes upside down in an incubator at 37 °C
overnight.

10.
**IPTG-induced protein expression**

a.
Expanded the culture seed solution: 1% Inoculated volume is
transferred to 100 mL LB Liquid medium, and OD600 is grown at 37 °C to 0.4-0.6 (about 3 to 4
h).

b.
Performed IPTG-induced expression BL21(DE3). The final
concentration of 0.25 mM IPTG was added to induce expression.

c.
Overnight culture was performed at 16 °C, 220 rpm in
incubator.

**11.** **U****ltrasonication and purif****ication**

Material:

|  |  |
| --- | --- |
| **Reagent** | **Volume/mass** |
| Bacteria liquid medium | 90 mL |
| Lysis buffer(lysozyme) | Depend on the steps |
| BeyoGold GST-tag Purification Resin | Depend on the steps |
| cracking buffer | 0.5 mL |
| elution buffer | 9 mL |
| eluent buffer | 0.5 mL each time |
| GSH | 30 mg, 1 mL |

Apparatus:

|  |  |
| --- | --- |
| centrifuge | 1 |
| balance | 1 |
| Pipettes (100 μL-1000 μL) | 3 |
| Ice container | 1 |
| ultrasonic crusher | 1 |
| EP tubes | 8 |
| affinity column | 1 |
| refrigerator | 1 |
| centrifuge tubes | 8 |

Procedure:

a. Divide three bottles of *E. coli* BL21, 30 mL each.

b. Remove the supernatant after
centrifugation.

c. Remove the weight of the bottle and measure the wet
weight of the bacteria.

d. Configuration of the lysozyme is 100 mg/mL (23
mg + 230 μL Lysis buffer).

e. On the basis of 0.1 g sediment weight/1000 μL
of lysozyme and lysis buffer are added together, blown well, and put into a container of ice water for
ultrasonic crushing.

f. Ultrasonic cracking bacteria on ice, ultrasonic
power 40%, each ultrasonic treatment 5 sec, each interval 5 sec, a total of 3 ultrasonic
treatments.

g. The bacterial solution after three tubes of
ultrasonic crushing was added to 8 EP tubes of 1 mL respectively.

h. Centrifuge at 4 °C at 10000 x*g* for 20-30 min.

i. Take the supernatant and merge it
together.

j. Extract 1 mL BeyoGold GST-tag Purification Resin,
centrifuge 1000 x*g* at 4 °C for 10 sec, and discard the storage
solution.

k. Add 0.5 mL of cracking buffer and centrifuge at 4
°C, and repeat the steps.

l. Put in the supernatant, and place in a 4 °C
shaker for 60 min.

m. The mixture of the lysate and BcyoGold GST-tag
Purification Resin was loaded into the empty column tube of the affinity column.

n. The lid at the bottom of the purification column
was opened, and the liquid in the column was discharged under the action of gravity. About 20
mL
 of the flow fluid was collected for subsequent
analysis.

o. Wash the column twice and add 0.5 mL of lysis
buffer each time, collect, and put in the refrigerator.

p. To configure the elution buffer, first add 30 mg
GSH and 1 mL of elution buffer. Next, 9 mL of elution buffer and 1 mL of GSH were mixed at a ratio of 9:
1.

q. Wash the column twice, each time with 0.5 mL eluent
buffer, each eluent is collected into a different centrifuge tube. The eluent obtained from the collection
is the purified GST label protein sample, which is finally placed in the refrigerator.

**12. SDS-PAGE protein
electrophoresis**

Material:

|  |  |
| --- | --- |
| **Reagent** | **Volume/mass** |
| glue solution (2x) | 2.7 mL |
| lower layer of glue-grade flushing solution (2x) | 2.7 mL |
| water | -- |
| upper glue solution (2x) | 0.75 µL |
| the color upper glue buffer (2x) | 0.75 µL |
| modified coagulant | 70 µL |
| electrophoresis buffer | -- |

Apparatus:

|  |  |
| --- | --- |
| glue molds | 2 |
| glue mixing cups | 4 |
| Pipettes (100 μL-1000 μL) | 1 |
| glass plates | 4 |
| Pipette (20 μL-200 μL) | 1 |
| Pipette (2 μL-20 μL) | 1 |
| Pipette (0.1 μL-2.5 μL) | 1 |

Procedure:

I.
SDS-PAGE modified acrylamide color
gel.

1.
Assemble the glue molds and configure the lower
layer of glue 1.00 mm.

2.
Take the same volume of the lower layer of glue
solution (2
x
) and the lower layer of glue grade flushing
solution (2
x
) 2.7 mL each, and mix them in the glue mixing
cup.

3. Add 55 µL of the modified coagulant to
the mixture in Step 2 and stir gently to avoid bubbles.

4. Add the lower layer of glue solution mixed in
step 3 to the glue maker, so that the liquid level is 1.5 cm away from the upper edge of the glass plate,
and then cover the lower layer of glue solution with a layer of water to keep the gel surface flat.

5. Stand at room temperature (25 ℃) for 6-10
min, and when the boundary between the lower layer and the covering phase appears, the gelatin is said to
have solidified.

6. Configure the upper layer glue 1.00 mm.

7. Slowly pour off the covering phase, take an
equal volume of the upper glue solution (2x) and the color upper glue buffer (2x), 0.75 µL each, and
mix well in a new glue cup.

8. Add 15 microliters of modified coagulant to
the mixture in step 7 and stir gently to avoid bubbles.

9. Add the upper glue solution to the upper
layer of the lower glue until the gel solution reaches the top of the glass plate. Slowly insert the comb
into the gel to avoid bubbles.

10. Stand for 10~15 min, wait for the upper
layer of amine to solidify, carefully pull out the comb, use the gun head or syringe to absorb the
electrophoresis buffer, rinse the sample hole, and then perform SDS-PAGE electrophoresis.

II. Protein glue staining and
decolorization

Material:

Coomassie bright blue dye

Decolorizing agent :

|  |  |
| --- | --- |
| **Reagent** | **Volume****(****μL****)** |
| Ethanoic acid | 10% |
| dd water | 50% |
| Alcohol | 4 0% |

Apparatus:

|  |  |
| --- | --- |
| Plastic box | 1 |
| Horizontal shaker | 1 |

III. Dyeing and decolorization of protein glue

Procedure:

1.
Soak the protein glue in Coomassie bright blue dye for 1
hour.

2.
Decolorize the stained protein by using the decolorizing agent 3
times, each time lasting for 10 min
.

**Overview**

Both norovirus and rotavirus are pathogens that cause diarrhea in infants and
young children
1
.
Vaccination is the primary means of preventing and controlling rotavirus and
norovirus infections
2
. Rotavirus vaccines have been clinically used for many years and can
significantly alleviate symptoms caused by rotavirus infections
3
. Due to the difficulty in achieving in vitro isolation and cultivation of
norovirus, there is currently no norovirus vaccine available for use. This study is mainly based on genetic
engineering vaccines. We mainly used genetic engineering methods to construct corresponding plasmids to
develop specific vaccines against norovirus
 and
rotavirus

viruses
.

**Results of Experiments**

We will introduce the results of our project from the following
four
 aspects
:

(1)

Construction of recombinant plasmids

(2)

Protein expression and purification

(3)

Expression optimization

(4)

Verification of protein expression in
*E. coli*
Nissle 1917

**1. Construction of recombinant plasmids**

To
 construct three recombinant
plasmids
,
*i.e.*
,

pGEX-GII.4-VP1
,
pGEX-GII.17-VP1,
 and
pGEX-
RV-
GII.17-VP1,
firstly, we amplified the
GII.4-VP1 (
1620 bp
)
,
GII.17-VP1 (
1620 bp
)
 and RV
-
VP7
 (
843 bp
)
 fragments by PCR. As shown in
F
igure 1A, the three genes were amplified successfully compared to
the
15K maker.
 T
hrough the double enzyme digestion experiment, we successfully obtained
the linearized
 plasmid pGEX-4T-1
 (
Figure 1B
)
, which
was then ligated with GII.4-VP1
,
GII.17-VP1
 and RV
-
VP7 fragments
, respectively. The mappings of recombinant plasmids after ligation are shown
in Figure 1C-D.

![](https://static.igem.wiki/teams/4872/wiki/results1575.png)

Figure 1 Construction of three

plasmids

Subsequently
the plasmids pGEX-GII.4-VP1 and pGEX-GII.17-VP1 were
transformed
into
*E. coli*
BL21 (DE3) competent cells,
respectively. The plasmid pGEX-
RV-
GII.17-VP1 was
 transformed
into
*E. coli*
BL21 (DE3) and *E. coli*
Nissle 1917
, respectively. After overnight incubation, transformants were successfully
grown on LB plates (
Figure 2
A-B). We performed colony PCR identification of the transformants, and the
results are shown in the figure, about 2/3 of the transformants containing pGEX-GII.17-VP1 had the correct
bands; all of the transformants containing pGEX-GII.4-VP1 or pGEX-RV-GII.17-VP1 had the correct bands (
Figure 2
C-D). To further verify the construction of the plasmid, we sequenced the
positive transformants, and the sequencing results showed that the expression frame sequences were all
correct, indicating that the plasmids were successfully constructed (
Figure 2
E-G).

![](https://static.igem.wiki/teams/4872/wiki/results2503.png)

Figure 2 Results of plasmid transformation, identification
,
 and sequencing

**2. Protein expression and purification**

We inoculated positive transformants and induced protein expression by IPTG.
After obtaining protein lysates, we purified the target proteins using GST tags and verified the protein
expression and purification results by SDS-PAGE. The results are shown in Figure 3, we successfully
expressed GII.4-VP1 protein, but the purified protein was barely visible, which might be due to the low
expression of protein. In the future, we plan to increase the expression level of the GII.4-VP1 protein. As
shown in Figure 4, we successfully expressed and purified GII.17-VP1 protein (
60
 kDa).

            ![](https://static.igem.wiki/teams/4872/wiki/results3213.png)

Figure 3
SDS-PAGE results of pGEX-GII.4-VP1
 protein expression and purification

(P represents
" Precipitation" ,
S represents
" Supernatant" ,
T represents
" Flow through" ,
W represents
" Wash solution" and
E represents
" Eluent
"
)

           ![](https://static.igem.wiki/teams/4872/wiki/results3452.png)

Figure 4
SDS-PAGE results of pGEX-GII.
17
-VP1
 protein expression and purification

(P represents
" Precipitation" ,
S represents
" Supernatant" ,
T represents
" Flow through" ,
W represents
" Wash solution" , and
E represents
" Eluent
"
)

3.
**Expression optimization**

In addition to constructing norovirus-associated plasmids, we constructed
recombinant plasmids incorporating norovirus and rotavirus antigenic genes. Similarly, we inoculated the
transformant containing pGEX-RV-GII.17-VP1, induced its expression, and explored its optimal expression
conditions.

To find the optimal conditions for the highest protein expression, we chose
different concentrations (OD
600
=0.3/0.6/0.8/1) of the bacterial solution and different IPTG induction times
(0 h/4 h/8 h/16 h). After obtaining each protein lysate, we measured the total protein amount (A280) under
different induction conditions (
Figure
6A
). In addition, we examined the expression of the target proteins using
SDS-PAGE (
Figure
5
) and used the ImageJ software to quantify the target band
 (119 KDa) on the SDS-PAGE gel,
collected and organized the data, and plotted a line graph with OD600 as the x-axis and gray value
as the y-axis (Figure 6B).

As shown in Figure 6, the protein concentration roughly tended to increase
with increasing bacterial concentration at the same IPTG induction time. When the bacterial concentration
was 0.8 and the induction time was 8 hours, the best protein expression level was achieved, indicating that
this condition was more suitable for expressing more target proteins.

![](https://static.igem.wiki/teams/4872/wiki/results5000.png)

Figure 5
SDS-PAGE results of
RV-
GII.
17
-VP1
 protein under different expression
conditions

![](https://static.igem.wiki/teams/4872/wiki/results5091.png)

Figure 6 Effect of IPTG induction time and bacterial concentration on protein
concentration

4.
**Verification of protein expression in*****E. coli Nissle 1917***

*E. coli*
Nissle 1917 (EcN) is a Gram-negative probiotic and an ideal expression vector
for oral live vaccines
4
. So we
transformed
 plasmid
pGEX-
RV-
GII.17-VP1
 into *E. coli*
Nissle 1917. Through the SDS-PAGE gel map, it was found that we have weak
bands within the
119

kD
a
 range, which means that RV-
GII.17-VP1
 protein expression level is
weak in *E. coli*
Nissle 1917
 (Figure 7)
.
 This initially confirmed that
RV-GII.17-VP1 could be successfully expressed in Nissle 1917, but the expression conditions need to be
optimized.

![](https://static.igem.wiki/teams/4872/wiki/results5762.png)

Figure 7
SDS-PAGE results of pGEX-
RV-
GII.
17
-VP1
 protein expression in *E.coli*
Nissle 1917

(P represents
" Precipitation" ,
S represents
" Supernatant" ,
T represents
" Flow through" ,
W represents
" Wash solution" and
E represents
" Eluent
"
)

**Future plan**

At present, we have determined the optimal conditions for protein expression
in
*E**.**coli*
. Next, we will improve and optimize the protein expression method to achieve
large-scale expression of
RV-
GII.
17
-VP1 in
*E. coli*
Nissle 1917
. In addition, the next
stage
of our project will be to obtain transformed
 *E. coli*
Nissle 1917
 through tube feeding in mice,
and then measure the levels of anti-RV-GII.17-VP1 IgG in mouse serum and IgA in feces. Finally, we can
verify the immunogenicity of our oral vaccine.

**References:**

1.

Hallowell, B. D., Parashar, U. D., & Hall, A. J. (2019). Epidemiologic
challenges in norovirus vaccine development. Human vaccines & immunotherapeutics, 15(6),
1279-C1283.

2.

Esposito S, Principi N.
[Norovirus Vaccine: Priorities for Future Research and Development.](https://pubmed.ncbi.nlm.nih.gov/32733458/)
Front Immunol. 2020 Jul 7;11:1383.

3.

Offit Paul A. Challenges to Developing a Rotavirus Vaccine.[J]. Viral
immunology,2018,31(2).

4.

Jacobi, C. A., & Malfertheiner, P. (2011). Escherichia coli Nissle 1917
(Mutaflor): new insights into an old probiotic bacterium. Digestive diseases (Basel,
Switzerland), 29(6), 600-C607.