iGEM 2021 — Team Shanghai_Metropolis

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# Team:Shanghai Metropolis/Notebook

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# Notebook

# Log of July

## 2021/07/14

1. Order plasmids.
2. Order primers.
3. Order commercial kits.
4. Order reagents and apparatuses.

## 2021/07/28

1. Finalize our project and main tasks.
2. PCR.
3. LB medium preparation.

## 2021/07/29

1. OE PCR.
2. Restriction enzyme double digestion.

![](https://static.igem.org/mediawiki/2021/0/02/T--Shanghai_Metropolis--img_notebook_1.png)

## 2021/07/30

1. Restriction enzyme double digestion.
2. ClonExpress ligation reaction.
3. Plasmid transformation.
4. Construct plasmid profile(pGEX-6P-1-VP1&pGEX-6P-1-VP1-LTB)

## 2021/07/31

1. Incubation of bacteria (in shaker).
2. MRS medium preparation.

![](https://static.igem.org/mediawiki/2021/8/81/T--Shanghai_Metropolis--img_notebook_2.png)

# Log of August

## 2021/08/01

1. Plasmid extraction.
2. Restriction enzyme double digestion.
3. Plasmid transformation.

![](https://static.igem.org/mediawiki/2021/b/bb/T--Shanghai_Metropolis--img_notebook_3.png)

## 2021/08/02

1. Analysis of sequencing results.

## 2021/08/03

1. SDS-PAGE(BL21chemically competent cells)
2. Western Blot

![](https://static.igem.org/mediawiki/2021/b/bb/T--Shanghai_Metropolis--img_notebook_4.png)

## 2021/08/04

1. SDS-PAGE(ATCC 334 chemically competent cells)
2. Western Blot

![](https://static.igem.org/mediawiki/2021/d/d0/T--Shanghai_Metropolis--img_notebook_5.png)

# Attachment\_Lab Notebook

---

Email us at: zhixuansong1022@qq.com; 1956461994@qq.com
Search‘EV71terminator’ in Wechat to find more about us

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# Team:Shanghai Metropolis/Engineering

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# Engineering

# Introduction

Hand-foot-mouth disease (HFMD) is an infectious disease caused by enterovirus 71 (EV71). The virus is an
important pathogenic factor of hand, foot and mouth disease. Vp1 protein is the viral capsid protein and
promotes the infection of host cells by virus particles. Vp1 is also the main antigen gene of the EV71
virus. Generally, the vaccinated population, especially infants and young children, are more compliant
with oral vaccines, so we are trying to develop oral HFMD vaccines. Probiotics bacteria *Bifidobacteria*,
as the natural host of the intestinal tract, can adhere to intestinal epithelial cells and are ideal
oral live vaccine expression vectors, and related studies have found that their preventive effects on
gastrointestinal pathogens are more significant. Therefore, we can use the *bifidobacterium* in
lactic acid bacteria as an expression system to express EV71 vp1.

# Design

The B subunit LTB in the heat-labile enterotoxin (LT) of Escherichia coli heat-labile enterotoxin (LT)
has strong immunogenicity and adjuvant activity, and will not cause harm to the human body. LTB and a
variety of non-related proteins and their non-protein antigens can increase the mucosal IgA and humoral
immune IgG response levels of the antigen through different immunization pathways. Fusion recombinant
protein constituting EV71 vp1 and LTB is produced by bifidobacterium expression system. Vp1-LTB is
tagged by glutathione S-transferase (GST) in its N-terminal. The tag is used for recombinant protein
purification.

# Build

Plasmids pGEX-vp1 and pGEX-vp1-LTB were constructed to produce Vp1 and Vp1-LTB fusion proteins in
probiotics bacteria Bifidobacterium, respectively (Figure 1). The production is controlled by tac
promotor. The construct is confirmed by DNA sequencing.

![](https://static.igem.org/mediawiki/2021/f/fd/T--Shanghai_Metropolis--img_engineering_1.jpg)
Figure 1. Schematic map of plasmids pGEX-vp and pGEX-vp1-LTB.

# Test

Due to the relatively low rate of growth and efficiency of electroporation of *L. casei*, our team
first transformed *E. coli* BL21, which is commonly used in plasmids transformation, to verify the
expression and antigencity of VP1 and VP1-LTB proteins (Figure 2).

![](https://static.igem.org/mediawiki/2021/a/a2/T--Shanghai_Metropolis--img_engineering_2.jpg)
Figure 2. SDS-PAGE and Western Blot for expression of VP1 and VP1-LTB proteins.

# Learn

GST, GST-VP1 and GST-VP1-LTB had all been successfully expressed by E.coli BL21, and they mainly existed
in the precipitation in the form of inclusion body. The expression of VP1 and VP1-LTB was not so
effective in *E.coli* BL21. There was no obvious expression of GST-VP1 and GST-VP1-LTB in *L.
casei*.

---

Email us at: zhixuansong1022@qq.com; 1956461994@qq.com
Search‘EV71terminator’ in Wechat to find more about us

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# Team:Shanghai Metropolis/Experiments

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# Experiments

# PCR

## · Materials

| Reagent | Volume/mass |
| --- | --- |
| pUC57-VP1 | 150ng |
| pUC57-LTB | 150ng |
| PrimeSTAR Max Premix | 75μL |
| VP1-FP (10μM) | 5μL |
| VP1-RP (10μM) | 2.5μL |
| VP1-Lkr-RP (10μM) | 2.5μL |
| LTB-FP (10μM) | 2.5μL |
| LTB-RP (10μM) | 2.5μL |
| ddH2O | 30μL |

\*PrimeSTAR Max Premix includes DNA polymerase, dNTP and buffer.

| Apparatus | VAmount |
| --- | --- |
| PCR thermal cycler | 1 |
| Pipette (range: 2μL-20μL) | 1 |
| Pipette (range: 20μL-100μL) | 1 |
| Micro-centrifuge tube | 3 |

## · Procedure

1. Add 20μL pUC57-VP1 into 2 micro-centrifuge tubes with each tube 10μL. Label these tubes with
   VP1 and VP1-lkr. Then add 10μL pUC57-LTB into another micro-centrifuge tube. Label this tube
   with LTB. Use the pipette (range: 2μL-20μL) to transfer the liquid above.
2. Add VP1-FP and VP1-RP into VP1. Add VP1-FP and VP1-Lkr-RP into VP1-Lkr. Add LTB-FP and LTB-RP
   into LTB. Use the pipette (range: 2μL-20μL) to transfer the liquid above.
3. Add 25μL PrimeSTAR Max to each micro-centrifuge tubes by using a pipette (range: 20μL-100μL).
   Add 10μL ddH2O to each micro-centrifuge tubes by using a pipette (range: 2μL-20μL).
4. Shake three micro-centrifuge tubes and then insert them into the PCR Thermal Cycler. Use the
   following setting:

| Process | Temperature | Time |
| --- | --- | --- |
| Denaturation | 98℃ | 10s |
| Annealing | 65℃ | 15s |
| Extension | 72℃ | 5s |
| Back to step 1 (35X) | | |
| Final elongation | 72℃ | 5mins |
| -- | 12℃ | ∞ |

# Overlap Extension PCR (OE PCR)

## · Materials

| Reagent | Amount used | Final concentration |
| --- | --- | --- |
| PrimeSTAR Max Premix (2×) | 25μL | 1× |
| VP1-FP | 10 ~ 15pmol | 0.2 ~ 0.3μM |
| LTB-RP | 10 ~ 15pmol | 0.2 ~ 0.3μM |
| ddH2O | To 50μL | -- |
| Template (VP1-Lkr & LTB fragment) | 150ng + 150ng | -- |

\*PrimeSTAR Max Premix includes DNA polymerase, dNTP and buffer.

| Apparatus | Amount |
| --- | --- |
| Micro-centrifuge tube (1.5ml) | 3 |
| Pipette (range: 0.5 μl ~ 10 μl) | 1 |
| Pipette (range: 20 μl ~ 200 μl) | 1 |
| Pipette tips (of all three sizes) | 6 |
| Conical centrifuge tube (15 ml) | 1 |
| Micro-centrifuge tube rack | 2 |
| PCR Thermal Cycler | 1 |

## · Procedure

1. Set up a PCR system (50μL).
2. Place the micro-centrifuge tube holding the system into the PCR Thermal Cycler.
3. Set up a reaction program.

| Process | Temperature | Time |
| --- | --- | --- |
| Denaturation | 98℃ | 10s |
| Annealing | 65℃ | 15s |
| Extension | 72℃ | 5s |
| Back to step 1 (35X) | | |
| Final elongation | 72℃ | 5mins |
| -- | 12℃ | ∞ |

4. Start the PCR reaction.
5. After the reaction, take out the micro-centrifuge tubes from the PCR thermal cycler.
6. Run a DNA gel electrophoresis and extract the DNA fragments from the gel.

# Colony PCR

## · Materials

| Reagent | Amount used | Final concentration |
| --- | --- | --- |
| PrimeSTAR Max Premix (2×) | 10μL | 1× |
| Forward primer | 10 ~ 15pmol | 0.2 ~ 0.3μM |
| Reverse primer | 10 ~ 15pmol | 0.2 ~ 0.3μM |
| ddH2O | To 50μL | -- |
| Template (incubated ATCC 334 chemically competent cells with plasmids pGEX-6P-1-VP1 and pGEX-6P-1-VP1-LTB) | 1μL | -- |

\*PrimeSTAR Max Premix includes DNA polymerase, dNTP and buffer.

| Apparatus | Amount |
| --- | --- |
| Micro-centrifuge tube (1.5ml) | 3 |
| Pipette (range: 0.5 μl ~ 10 μl) | 1 |
| Pipette tips (of all three sizes) | 6 |
| Conical centrifuge tube (15 ml) | 1 |
| Micro-centrifuge tube rack | 2 |
| PCR Thermal Cycler | 1 |

## · Procedure

1. Set up a PCR system (20μL).
2. Place the micro-centrifuge tube holding the system into the PCR Thermal Cycler.
3. Set up a reaction program.

| Process | Temperature | Time |
| --- | --- | --- |
| Denaturation | 98℃ | 10s |
| Annealing | 65℃ | 15s |
| Extension | 72℃ | 5s |
| Back to step 1 (35X) | | |
| Final elongation | 72℃ | 5mins |
| -- | 12℃ | ∞ |

4. Start the PCR reaction.
5. After the reaction, take out the micro-centrifuge tubes from the PCR thermal cycler.
6. Run a DNA gel electrophoresis.

# Agarose Gel Electrophoresis

## · Materials

| Reagents | Volume/mass |
| --- | --- |
| Agarose | 1g |
| TAE Buffer | ~500mL |
| Ts-GelRed | 10μL (10000X) |
| DNA Loading Buffer | 10X in template |
| Marker | 5μL |
| Template | -- |

| Apparatus | Amount |
| --- | --- |
| Conical flask (range: 300mL, division value: 100mL) | 1 |
| Pipette (range: 2μL-20μL) | 1 |
| Pipette (range: 0.5μL-10μL) | 1 |
| Weighing paper | 1 piece |
| Electronic balance | 1 |
| Spatula | 1 |
| Microwave oven | 1 |
| Aluminum foil | 1 piece |
| Mold for creating gel | 1 |
| Electrophoresis chamber | 1 |
| UV transilluminator | 1 |
| Scalpel | 1 |

## · Procedure

### Making gel

1. Place the weighing paper on the electronic balance, weigh 1g agarose, and pour it into
   the
   conical flask.
2. Pour 100mL TAE into the conical flask and shake it.
3. Cover the conical flask with the aluminum foil and place it into the microwave oven for 3
   minutes, until the granules of agarose are well dissolved and invisible.
4. Use the pipette (range: 2μL-20μL) to transfer 10μL Ts-GelRed into the conical flask.
5. Pour the gel into the mould and insert a ‘comb’ into the gel. Wait for 10 minutes until
   the gel is solidified.

### Electrophoresis

1. Pour about 400mL TAE into the electrophoresis chamber.
2. Carefully take out the gel and replace it in the electrophoresis chamber.
3. Add the loading buffer, 1/10 of the volume of the template, into the template by using
   the pipette (range: 2μL-20μL) and shake it.
4. Use the pipette (range: 0.5μL-10μL) to add 5μL marker in the first hole of the gel, then
   add 5μL of each template into the following holes.
5. Turn on the power of the electrophoresis chamber and wait for about 40 minutes (the
   specific time should depend on the markers we use).

### Authentication and recycle

1. Take out the gel and place it on the UV tray. The UV tray will then be placed into the
   UV transilluminator.
2. Open the Image Lab and scan the gel.
3. Comparing with the marker reference, determine the target section which will be
   extracted during the recycling process.
4. Use the scalpel to cut the target section of the gel.

![](https://static.igem.org/mediawiki/2021/f/f9/T--Shanghai_Metropolis--img_experiments_1.jpg)

# DNA Gel Extraction

## · Materials

| Reagents | Volume/mass |
| --- | --- |
| Binding buffer (NTI) | 200μL |
| Agarose gel | 100mg |
| Buffer EB | 25μL |
| Wash Buffer (NT3) | 700μL |

| Apparatus | Amount |
| --- | --- |
| Micro-centrifuge tube (1.5mL) | 2 |
| Thermostat | 1 |
| Spin column | 1 |
| Centrifuge | 1 |

## · Procedure

### Gel extraction

1. Add 100mg agarose gel and 200μL NT1 into one micro-centrifuge tube.
2. Place the tube in the thermostat at 50°C for 5-10 minutes.
3. Vortex the tube after ensuring all gel dissolves.

### Binding DNA

1. Insert a spin column into a collection tube and transfer the solution into the spin
   column.
2. Centrifuge the tube at 12000rpm, 25°C for 1 minute.
3. Discard the waste liquid from the collection tube.

### Wash silica membrane

1. Add 700μL NT3 solution into the spin column.
2. Centrifuge the tube at 12000rpm, 25°C for 1 minute.
3. Discard the waste liquid from the collection tube.
4. Repeat steps 1-3 for 2 times.

### Dry silica membrane

1. Centrifuge the tube at 12000rpm, 25°C for 2 minutes
2. Discard the waste liquid from the collection tube.

### Elution

1. Insert the spin column into a new micro-centrifuge tube.
2. Add 25μL buffer EB to the center of the column membrane and incubate the column at
   room temperature for 2 minutes.
3. Centrifuge the tube at 12000rpm, 25°C for 1 minute.

# Restriction Enzyme Double Digestion

## · Materials

| Reagents | Volume |
| --- | --- |
| VP1 fragment | 10μL |
| VP1-LTB fragment | 10μL |
| ddH2O | 14μL |
| FD buffer | 4μL |
| SalI enzyme | 1μL |
| BamHI enzyme | 1μL |

| Apparatus | Amount |
| --- | --- |
| Micro-centrifuge tube | 2 |
| Pipette (range: 2μL-20μL) | 2 |
| Pipette (range: 0.5μL-10μL) | 2 |
| Thermostat | 2 |

## · Procedure

1. Add 10μL fragments (VP1/VP1-LTB), 14μL ddH2O and 2μL buffer into one micro-centrifuge tube
   with a pipette(range:2μL-20μL)
2. Add 0.5μL SalI and 0.5μLBamHI into the micro-centrifuge tube with another pipette
   (range:0.5μL-10μL)
3. Place the micro-centrifuge tube in the thermostat at 37℃ for 30 minutes.

\*The exact time and temperature should depend on the template

# ClonExpress ligation reaction

## · Materials

| Reagents | Volume |
| --- | --- |
| Ligation buffer | 10μL |
| T4 ligase | 10μL |
| VP1 fragment | 20μL |
| VP1-LTB fragment | 20μL |
| Vector (digested pGEX-6P-1) | 30μL |
| ddH2O | 10μL |

| Apparatus | Amount |
| --- | --- |
| Micro-centrifuge tube（1.5 mL） | 2 |
| Pipettes (range:5-50μL） | 1 |
| Thermostat | 1 |

## · Procedure

1. Add 20μL VP1 fragments to one micro-centrifuge tube and add 20μL VP1-LTB fragments to another
   tube.
2. Add 5μL ligation buffer, 5μL T4 ligase, 15μL digested pGEX-6P-1, and 5μL ddH2O to each
   tube.
3. Place the mixed reagents in the thermostat at 22 ℃ for 1 hour.

# *E. coli* Transformation

## · Materials

| Reagents | Volume |
| --- | --- |
| pGEX-6P-1-VP1 | 1μL |
| pGEX-6P-1-VP1-LTB | 1μL |
| DH5α/BL21 chemically competent cell | 100μL |
| Liquid LB medium (amp-) | 500μL |
| Solid LB medium (amp+) | 500μL |

| Apparatus | Amount |
| --- | --- |
| Thermostat | 1 |
| Petri dish | 2 |
| Shaker | 1 |
| Pipette (Range: 0.5-2.5μL) | 1 |
| Pipette (Range: 20-200μL) | 1 |
| Conical centrifuge tube (1.5 mL) | 2 |
| Incubator | 1 |
| Bacterial culture tube | 2 |

## · Procedure

### Transformation

1. Thaw 100μL competent cells on ice for 1-2 minutes.
2. Mix the cells with 1μL plasmids (pGEX-6P-1-VP1 or pGEX-6P-1-VP1-LTB) in one bacterial
   culture tube with one pipette (range: 0.5-2.5μL).
3. Place the tubes in ice for 30 minutes.

### Heat shock

1. Place the tubes in the thermostat at 42℃ for 60-90 seconds.
2. Place the tubes in ice bath immediately and cool them for 2-3 minutes.

### Resuscitation

1. Add 500μL LB medium (amp-) to every bacterial culture tube.
2. Mix the solution uniformly, and incubate it in the shaker at 37℃,180rpm for 45-60
   minutes.

### Incubation

1. Aspirate the transformed competent cells with one pipette (range: 20-200μL)
2. Coat them uniformly on the LB solid media (amp+)
3. Place the petri dishes at room temperature until the solution is absorbed.
4. Turn the dishes upside down and incubate the cells at 37℃ for 12-16 hours.

# *L.paracasei* Transformation

## · Materials

| Reagents | Volume |
| --- | --- |
| ddH2O | 300μL |
| 20% glycerinum solution | 400μL |
| MRS medium (amp-) | 1800μL |
| MRS solid medium (amp+) | 1800μL |
| ATCC334 Chemically Competent Cell | 200μL |
| pGEX-6P-1-VP1 | 10μL |
| pGEX-6P-1-VP1-LTB | 10μL |

| Apparatus | Amount |
| --- | --- |
| Micro-centrifuge tube (1.5ml) | 2 |
| Centrifuge | 1 |
| Electroporation cuvette | 2 |
| Electroporation machine | 1 |
| Shaker | 1 |
| Incubator | 1 |
| Petri dish | 2 |
| Pipette (Range: 0.5-2.5μL) | 1 |
| Pipette (Range: 20-200μL) | 1 |
| Bacterial culture tube | 2 |

## · Procedure

### Thaw

1. Take out the competent cells from the refrigerator at -80℃ and thaw them at room
   temperature.
2. Add 150μL ddH2O to one micro-centrifuge tube.
3. Place it at room temperature for 20 minutes.
4. Precool the centrifuge at 4℃.
5. Centrifuge the tube at 13,000rmp, 4℃ for 3 minutes.
6. Discard the supernatant.

### Resuscitation

1. Add 100μL precooled 20% glycerinum solution to the micro-centrifuge tube.
2. Centrifuge the tube at 13,000 rpm for 10 minutes.
3. Discard the supernatant.

### Resuspension

1. Add 100μL 20% glycerinum solution and 10μL pGEX-6p-1-VP1 or pGEX-6p-1-VP1-LTB to the
   centrifuge tube.
2. Transfer the resuspension into electroporation cuvette with one pipette (range:
   0.5-2.5μL).

### Electroporation

1. Set up the electroporation machine: 2000V, 25μF, 400Ω.
2. Insert the cuvette into the machine respectively, and pulse one time.

### Resuscitation

1. Aspirate the resuspension from the electroporation cuvettes with one pipette (range:
   0.5-2.5μL).
2. Turn it into 900μL MRS media in a bacterial culture tube.
3. Resuscitate the bacteria in the shaker at 37℃ for 3-4 hours.

### Incubation

1. Aspirate 100μL resuspension with another pipette (range: 20-200μL)
2. Coat the resuspension on the MRS medium (amp+)
3. Place the resuspension in the incubator at 37℃ for 36-72 hours.

# Plasmid extraction (TIANprep Mini Plasmid Kit)

## · Materials

| Reagents | Volume |
| --- | --- |
| Buffer BL | 500μL |
| Buffer P1 | 250μL |
| Buffer P2 | 250μL |
| Buffer P3 | 350μL |
| Buffer PW | 600μL |
| Buffer EB | 50-100μL |
| Bacteria | -- |

| Apparatus | Amount |
| --- | --- |
| Spin column CP3 | 1 |
| Collection tube (1.5mL) | 1 |
| Micro-centrifuge tube | 2 |
| Centrifuge | 1 |
| Vortex machine | 1 |

## · Procedure

### Column Equilibrium

1. Insert one spin column CP3 into the collection tube.
2. Add 500μL buffer BL to the tube.
3. Centrifuge the tube at 12,000rpm for 1 minute.
4. Discard the waste liquor in the collection tube.
5. Insert the spin column back to it.

### Bacteria Extraction

1. Harvest 1-5mL overnight cultured (12 - 16h) bacteria into a centrifuge tube.
2. Centrifuge the tube at 12,000rmp for 1 minute.
3. Discard the supernatant.
4. Invert the tube on the absorbent paper to dry.

### Resuspension

1. Add 250μL buffer P1 to the centrifuge tube.
2. Insert the tube into the vortex machine until the bacteria are suspended thoroughly.

### Lysis

1. Add 250μL buffer P2 to the centrifuge tube.
2. Turn it upside down for 6-8 times temperately.

### Precipitation

1. Add 350μL buffer P3 to the centrifuge tube.
2. Immediately turn it upside down for 6-8 times temperately until the solution is uniformly
   mixed.
3. Centrifuge the tube at 12,000rpm for 10min.

### Transfer

1. Aspirate the supernatant and transfer it to spin column CP3 in the collection tube.
2. Centrifuge it at 12,000rpm for 30-60 seconds.
3. Discard the waste liquor in the collection tube.

### Rinse

1. Add 600μL buffer PW to the spin column CP3.
2. Centrifuge it at 12,000rpm for 30-60 seconds.
3. Discord the waste liquor in the collection tube.
4. Insert the spin column back to the collection tube.
5. Repeat procedure 1-4.
6. Centrifuge the tube at 12,000rpm for 2 min.

### Elusion

1. Insert the spin column CP3 into a clean micro-centrifuge tube.
2. Add 50-100μL buffer EB to the center of the spin membrane.
3. Place the tube in room temperature for 2 minutes.
4. Centrifuge it at 12,000rpm for 2 minutes.
5. Turn the plasmid solution to the centrifuge tube.

# SDS-PAGE electrophoresis

## · Materials

| Reagents | Volume |
| --- | --- |
| Liquid LB medium (amp-) | 30mL |
| Ampicillin | 300μL |
| ddH2O | 1200μL |
| Loading buffer | 300μL |
| Running buffer | -- |
| Marker | 10μL |
| IPTG | 75μL |
| SurePAGE (15%BT) | 1 piece |

| Apparatus | Amount |
| --- | --- |
| Pipette (range: 0.5-2.5μL) | 1 |
| Pipette (range: 20-200μL) | 1 |
| Pipette (range: 5mL) | 1 |
| Micro-centrifuge tube(1.5mL) | 6 |
| Electrophoresis chamber | 1 |
| Bacterial culture tube | 6 |

## · Procedure

1. Transfer 400μL bacteria in each tube to six micro-centrifuge tubes with one pipette (range:
   5mL).
2. Centrifuge the six centrifuge tubes at 12,000rmp for 1 minute.
3. Discard the supernatant.
4. Add 200μL ddH2O and 50μL loading buffer to each tube.
5. Insert the prepared gel (SurePAGE 15%BT) into the electrophoresis chamber.
6. Add running buffer into the chamber until the gel is submerged.
7. Add 10μL marker to the first hole of the gel and add 10μL bacterial solution in each tube
   into the following holes with one pipette (range: 2-20μL).
8. Set up the electrophoresis chamber: 100V, 2h.
9. Start the program.

# Coomassie brilliant blue staining

## · Materials

| Reagents | Volume |
| --- | --- |
| Coomassie brilliant blue staining solution | -- |
| Gel with protein after SDS-PAGE | 1 piece |

| Apparatus | Amount |
| --- | --- |
| Electrophoresis chamber | 1 |
| Box (1dm×1dm×0.5dm) | 1 |
| Microwave oven | 1 |
| Shaker | 1 |

## · Procedure

(It is conducted after SDS-PAGE)

1. Discard the running buffer and take out the gel from the chamber.
2. Transfer the gel to a box and add Coomassie Brilliant Blue to it until the gel is
   submerged.
3. Heat up the gel in the microwave oven.
4. Place the box on the shaker for 10 minutes.
5. Take out the gel from the box and use Image Lab to scan the gel.

# Western Blot

## · Materials

| Reagents | Volume |
| --- | --- |
| Rabbit Anti-GST antibody | -- |
| Rabbit-HRP | -- |
| TBST solution | -- |
| ECL chemilumninecent liquid | -- |
| 5% defatted milk solution | -- |
| BeyoECL Plus solution | -- |
| Gel with protein after SDS-PAGE | 1 piece |

| Apparatus | Amount |
| --- | --- |
| Trans-blot transfer | 1 |
| PVDF membrane | 1 |
| Shaker | 1 |
| Box(1dm×1dm×0.5dm) | 1 |

## · Procedure

(It is conducted after SDS-PAGE)

### Transfer

1. Submerge trans-blot transfer in the ice bath.
2. Set the trans-blot transfer at 300mA.
3. Place the gel and PVDF membrane in the trans-blot transfer for 60 minutes.

### Blocking

1. Turn the gel to a box, add TBST solution into it, and rinse the gel for 1-2 minutes.
2. Discard the TBST solution.
3. Add 5% defatted milk into the box until the gel is submerged.
4. Place the box on the shaker at room temperature for 60 minutes.

### Primary antibody incubation

1. Discard the milk in the box.
2. Add Rabbit Anti-GST antibody solution to the box.
3. Place the box on the shaker at room temperature for 2 hours.
4. Recycle the rabbit Rabbit Anti-GST antibody solution.
5. Rinse the gel with TBST solution 3 times, 15 minutes for each time.

### Secondary antibody incubation

1. Discard the TBST solution in the box.
2. Add Rabbit-HRP solution to the box.
3. Place the box on the shaker at room temperature for 60 minutes.
4. Recycle the Rabbit-HRP solution.
5. Rinse the gel with TBST solution 3 times, 15 minutes for each time.

### Detection of proteins

1. Add BeyoECL Plus solution into the box.
2. Use the software *ImageLab* to scan the gel.

---

Email us at: zhixuansong1022@qq.com; 1956461994@qq.com
Search‘EV71terminator’ in Wechat to find more about us

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# Team:Shanghai Metropolis/Results

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# Results

# Overview

Our results mainly consist of two parts. The first part is about plasmid construction: amplification of
VP1, VP1-linker and LTB fragments, amplification and digestion of VP1-LTB fragments, and ligation of VP1
and VP1-LTB fragments with pGEX-6P-1 respectively. The second part is about plasmid transformation:
verification of pGEX-6P-1-VP1 and pGEX-6P-1-VP1-LTB and verification of VP1 and VP1-LTB proteins.

# Plasmid construction

In this part of the experiment, we amplified VP1 fragments from pUC57-VP1 and LTB fragments from
pUC57-LTB, then ligated and inserted them into the vectors pGEX-6P-1, and obtained pGEX-6P-1-VP1 and
pGEX-6P-1-VP1-LTB.

## ·PCR for VP1,VP1-linker and LTB fragments

Firstly, to amplify VP1 fragments and VP1-linker fragments from pUC57-VP1 and LTB fragments from
pUC57-LTB, we added VP1-FP and VP1-RP into two tubes to amplify VP1 fragments, VP1-FP and
VP1-linker-RP into another two tubes to amplify VP1-linker fragments, and LTB-FP and LTB-RP into
another two tubes to amplify LTB fragments.

### VP1-FP：

5′-CGGGATCCATGAATAAAGGAGCCATGAAACACC -3′

### VP1-RP:

5′-TTCTTCAACATCACCTGCTTGCTTTAGCAGAGAGAAGTTTGTGGCAAGAGTGGTG-3′

### VP1-linker-RP:

5′-TAGTGGTGAGAACGGTGTTGAAGAGAGACGATTTCGTTCGTTCGTCCAC-3′

### LTB-FP:

5′-TAAAGCAAGCAGGTGATGTTGAAGAAAACCCCGGGCCTGTTGACATATATATAAC-3′

### LTB-RP：

5′-GCGTCGACAAGCTTGCCCTCCAGCCTAGC-3′

To confirm whether we successfully amplified the fragments we wanted from the plasmids, we ran the
electrophoresis of the fragments in the six tubes. We then scanned the gel, compared the strong
bands with the markers, and identified VP1, VP1-linker and LTB fragments on the gel. If we got the
expected results, we can extract the three types of fragments from the gel and continue our
experiments: digestion of VP1 fragments and OE PCR of VP1-LTB fragments.

![](https://static.igem.org/mediawiki/2021/a/a9/T--Shanghai_Metropolis--img_results_1.png)
Figure 1. Gel electrophoresis of VP1, VP1-linker and LTB fragments after PCR.

**Conclusion:** Theoretically, VP1 fragment is 891bp in length; VP1-linker fragment is 948bp in
length; LTB fragment is 604bp in length. Compared with the markers, the strong bands in the six
tubes all fit in the right range, so it proved that our PCR for the three types of fragments was
successful, and we could continue our experiments.

## ·OE PCR for VP1-LTB fragments

After obtaining VP1-linker and LTB fragments from PCR, we overlapped them through OE PCR. We added
the two types of fragments, VP1-FP, and LTB-RP into one tube and waited for them to overlap. Then we
conducted double digestion on the newly ligated fragments.

To confirm whether we successfully overlapped the two fragments, we ran the electrophoresis of the
fragments in the tubes. We then scanned the gel, compared the strong bands with the markers and
identified VP1-LTB fragments on the gel. If we got the expected results, we can extract the
fragments from the gel and insert them into the vectors.

![](https://static.igem.org/mediawiki/2021/8/8f/T--Shanghai_Metropolis--img_results_2.png)
Figure 2 Gel electrophoresis of VP1-LTB fragments after OE PCR and enzyme digestion

**Conclusion:** Theoretically, digested VP1-LTB fragment is 1552bp in length. Compared with the
markers, the strong band fit in the right range, so it proved that our OE PCR for VP1-LTB was
successful, and we could continue our experiments.

## ·Clonexpress Ligation reaction for pGEX-VP1 and pGEX-VP1-LTB

We had already obtained digested VP1 fragments and VP1-LTB fragments after PCR and OE PCR. In order
to insert them into the vectors pGEX-6P-1 respectively, we first needed to use the same restriction
enzymes, SalⅠ and BamHⅠ, to digest pGEX-6P-1 and make the plasmids available for ligation. We then
run the gel electrophoresis of digested pGEX-6P-1, identified the fragments we wanted, and extracted
them from the gel. After that, we conducted ClonExpress ligation reaction to ligate VP1 and VP1-LTB
fragments with pGEX-6P-1.

![](https://static.igem.org/mediawiki/2021/e/e0/T--Shanghai_Metropolis--img_results_3.jpg)
Figure 3 gel electrophoresis of pGEX-6P-1 after double digestion

**Conclusion:** Theoretically, pGEX-6P-1 after double digestion of SalI and BamHI is 4975bp in
length. Compared with the markers, the strong band fit in the right range, so we can continue to
conduct ClonExpress ligation reaction for pGEX-6P-1-VP1 and pGEX-6P-1-VP1-LTB.

Simultaneously, we used *Snapgene* to construct the profiles of the plasmids we constructed,
pGEX-6P-1-VP1 and pGEX-6P-1-VP1-LTB. We demonstrated and understood the parts of the plasmids:
restriction gene, target gene, and multiple cloning site.

![](https://static.igem.org/mediawiki/2021/5/51/T--Shanghai_Metropolis--img_results_15.jpg)
Figure 4. plasmid profile of pGEX-6P-1-VP1 and pGEX-6P-1-VP1-LTB.

# Plasmid transformation

In this part, we transformed the plasmids we constructed into E.coli to replicate them, then extracted
and verified the plasmids, and transformed them into E.coli and L.casei again. Finally, we verified the
expression of VP1 and VP1-LTB proteins in the two types of bacteria.

## Verification of transformed plasmids

### ·Electrophoresis of pGEX-VP1 and pGEX-VP1-LTB

After transforming the plasmids into E.coli DH5α to replicate them, we picked 6 individual
colonies from each petri dish and extracted the plasmids from them. To confirm whether the
extracted plasmids were the ones we required, we ran a DNA gel electrophoresis of the
transformed plasmids; we also run gel electrophoresis of pGEX-6P-1 after single digestion on the
same gel as a negative group. We then scanned the gel, compared the brightest DNA bands with the
markers, and identifies pGEX-6P-1-VP1 and pGEX-6P-1-VP1-LTB on the gel.

![](https://static.igem.org/mediawiki/2021/e/e6/T--Shanghai_Metropolis--img_results_6.png)
Figure 5. gel electrophoresis of pGEX-6P-1-VP1 and pGEX-6P-1-VP1-LTB

**Conclusion:** Theoretically, pGEX-6P-1 is 4984 bp long in linear shape; pGEX-6P-1-VP1 is
5853 bp long in linear shape; pGEX-6P-1-VP1-LTB is 6511 bp long in linear shape. The length of
plasmids in the form of covalently closed circular DNA (cccDNA) is half of the length of
linear-shaped plasmids. Accordingly, pGEX-6P-1, which became linear-shaped after single
digestion, should be around 5000bp in length, while both pGEX-6P-1-VP1 and pGEX-6P-1-VP1-LTB,
which were in the form of cccDNA, should be around 2500bp in length. Compared with the markers,
the plasmids in the twelve groups that we extracted from E.coli DH5α all fit in the right range.
Therefore, we reached a preliminary conclusion that we succeeded in the experiments of plasmid
transformation and obtained the plasmids we wanted.

### ·Restriction enzyme double digestion of pGEX-VP1 and pGEX-VP1-LTB

We had tested the plasmids we extracted from E.coli DH5α in terms of their length. To further
confirm whether the plasmids carried the fragments we wanted, we used SalⅠ and BamHⅠ to cut off
the fragments between their cutting sites from pGEX-6P-1-VP1 and pGEX-6P-1-VP1-LTB;
simultaneously, we conducted single digestion on each group as the negative control group. Then
we run the gel electrophoresis of the digested plasmids to separate and identify the fragments
cut off from the plasmids.

![](https://static.igem.org/mediawiki/2021/7/79/T--Shanghai_Metropolis--img_results_7.png)
Figure 6. gel electrophoresis of pGEX-6P-1-VP1 and pGEX-6P-1-VP1-LTB after double digestion

**Conclusion:** Theoretically, VP1 fragment is 891bp in length; LTB fragment is 604bp in
length；pGEX-6P-1 after double digestion by SalⅠ and BamHⅠis 4975 in length. Compared with the
markers, samples 3 and 4 of pGEX-6P-1-VP1 were both correct, and sample 3 of pGEX-6P-1-VP1-LTB
was correct as well. However, in sample 4 B+S(pGEX-6P-1-VP1-LTB after double digestion by SalⅠ
and BamHⅠ), the weaker bands, which should represent the VP1-LTB fragments, did not appear on
the gel, so we recognized the plasmids transformation in sample 4 of pGEX-6P-1-VP1-LTB as a
failure. We discarded this sample and sent the samples that were proved right after enzyme
digestion to a company for sequencing.

### ·Sequencing for VP1 and VP1-LTB fragments on transformed plasmids

After enzyme digestion and gel electrophoresis of pGEX-6P-1-VP1 and pGEX-6P-1-VP1-LTB, we
tentatively confirmed that the experiment of plasmid transformation into *E.coli* DH5α was
successful, and we obtained the plasmids we wanted. To further verify whether there was any
mutation in VP1 and VP1-LTB fragments on the plasmids, we needed to obtain and compare the
upstream and downstream sequences of the two fragments.

[**-Sequencing results is placed at the end of this page**](https://2021.igem.org/Team%3AShanghai_Metropolis/Result#sequencing-results)

**Conclusion:** VP1-F-sequencing and VP1-R-sequencing, VP1-LTB-F-sequencing and
VP1-LTB-R-sequencing, were both complementary with each other according to the base pair rule.
Therefore, we confirmed that there were no mutation sites in VP1 and VP1-LTB fragments and the
plasmids we extracted from *E.coli* DH5α were correct.

## Verification of VP1 and VP1-LTB expression

After receiving the sequencing results, we had confirmed that *E.coli* DH5α successfully carried
and replicated the plasmids we constructed. The next part of our experiments is to transformed the
plasmids that were verified to be correct into *E.coli* BL21 and *L. paracasei* ATCC334
respectively and then to verify the protein expression in them.

### Verification of protein expression in *E.coli* BL21

Due to the relatively low rate of growth and efficiency of electroporation of L. casei, our team
first transformed E.coli BL21, which is commonly used in plasmids transformation, to verify the
expression and antigencity of VP1 and VP1-LTB proteins.

#### ·SDS-PAGE and Coomassie Brilliant Blue staining for whole bacteria, supernate, and precipitation

We transformed pGEX-6P-1-VP1 and pGEX-6P-1-VP1-LTB into E.coli BL21 respectively and
incubated
them. Firstly, we ran a PAGE gel of the whole bacteria, supernate, and precipitation of
E.coli
BL21 and then stained the gel through Coomassie Brilliant Blue Staining.

![](https://static.igem.org/mediawiki/2021/5/54/T--Shanghai_Metropolis--img_results_8.png)
Figure 7. PAGE gel of GST, GST-VP1 and GST-VP1-LTB after staining
 (W: whole bacteria; S: supernatant; P: precipitation)

**Conclusion:** After Coomassie Brilliant Blue Staining, we found that the extent of the
brightness of the band in the P group was comparable to that in the W group, while the band
in
the S group was nearly invisible. In other words, GST, GST-VP1 and GST-VP1-LTB had all been
successfully expressed by E.coli BL21, and they mainly existed in the precipitation in the
form
of inclusion body.

#### ·SDS-PAGE and Western Blot for further verification

We also ran another PAGE gel of L. casei and then conducted Western Blot on it. After that,
we
scanned the gel and analyzed the expression of VP1 and VP1-LTB in *E.coli* BL21
further.

![](https://static.igem.org/mediawiki/2021/d/d6/T--Shanghai_Metropolis--img_results_9.png)
Figure 8. PAGE gel of GST, GST-VP1 and GST-VP1-LTB after WB

**Conclusion:** Using primary and secondary antibodies, we successfully located GST,
GST-VP1,
and GST-VP1-LTB. However, the brightness of GST-VP1 bands and GST-VP1-LTB bands was much
lower
than that of the GST bands. This means that the expression of VP1 and VP1-LTB was not so
effective in *E.coli* BL21, so we needed to further probe into the optimum conditions
under
which *E.coli* BL21 could express GST-VP1 and GST-VP1-LTB the best.

#### ·Expression optimization

In order to find the optimum condition under which the proteins were expressed the most, we
selected bacteria solution of different concentration (OD600=0.5/0.6/0.8/1), and inducted
them
with IPTG solution of different concentration(IPTG=1mM/10mM). Then we ran a PAGE gel of them
and
then marked the proteins with Coomassie Brilliant Blue Staining Solution. To visualize and
compare the expression of proteins under different conditions, we used the software ImageJ
to
quantify specific bands on the gel, collected and arranged the data, and constructed a
broken
line graph with OD600 the x- axis and the gray value as the y-axis.

![](https://static.igem.org/mediawiki/2021/4/45/T--Shanghai_Metropolis--img_results_10.png)
Figure 9. PAGE gel of GST, GST-VP1 and GST-VP1-LTB under different expression conditions

![](https://static.igem.org/mediawiki/2021/c/c7/T--Shanghai_Metropolis--img_results_11.png)
Figure 10. Model

**Conclusion:** Via this graph of model, we determined the optimum expression conditions
of
VP1 and VP1-LTB respectively in *E.coli* BL21: VP1 was expressed the most under
OD600=0.5,
IPTG=10mM; VP1-LTB was expressed the most under OD600=0.8, IPTG=1mM. Besides, we could see
that
when OD600 was over 0.8, the expression of VP1-LTB proteins was significantly higher than
that
of VP1 proteins in *E.coli* BL21. This meant that LTB could promote the expression of
VP1
in *E.coli* BL21 during its late growth period.

### Verification of protein expression in *L. paracasei* ATCC334

#### ·Colony PCR

In order to verify whether the plasmids we constructed had been successfully transformed into
L. casei ATCC334, we conducted colony PCR on the bacteria solution and the ran a gel
electrophoresis of it.

![](https://static.igem.org/mediawiki/2021/2/2b/T--Shanghai_Metropolis--img_results_12.png)
Figure 11. gel electrophoresis of VP1 and VP1 fragments after colony PCR

**Conclusion:** As the graph of the gel showed, we successfully amplified VP1 and VP1-LTB
fragments from the plasmids carried in L. casei, which meant that L. casei successfully
carried the plasmids.

Next, we ran a PAGE gel of L. casei to further confirm whether they had expressed the
plasmids they carried. Then we stained and scanned the gel.

![](https://static.igem.org/mediawiki/2021/8/80/T--Shanghai_Metropolis--img_results_13.png)
Figure 12. PAGE gel of GST, GST-VP1 and GST-VP1-LTB after staining

**Conclusion:** After staining and scanning the gel, we did not find any obvious bands in
certain KD ranges, which meant there was no obvious expression of GST-VP1 and GST-VP1-LTB in
*L. casei.*

# Future plans

#

We combined VP1 and LTB to construct the recombinant expression system and used E.coli to express the
proteins. We detected the protein expression with IPTG solution of different concentration at different
growth stage, constructed related models, and determined the optimum expression conditions. All these
laid a solid foundation for the development of HFMD oral vaccine.

1. Currently, we just verified the viability of our concept and determined the optimized condition
   of protein expression in *E.coli*. However, because of the difficulty in protein expression
   in*L.casei*, we would need to optimize the incubation and electroporation of *L.casei*,
   or improve and reconstruct the plasmids, to realize the expression of GST-VP1-LTB in
   *L.casei*.
2. If *L.casei* did express the proteins after the improvement of our design, the next part of
   our project would be that we let the mice take the transformed *L.casei* via gavage, and
   then measured the level of anti-VP1-LTB IgG in the serum of the mice and the level of IgA in
   their feces. Finally, we could verify immunogenicity of our oral vaccine.

# Reference

1. Wang, H., & Li, Y. (2019). Recent Progress on Functional Genomics Research of Enterovirus 71.
   Virologica Sinica, 34(1), 9–21.
   <https://doi.org/10.1007/s12250-018-0071-9>
2. Li, C. Y., Wang, Y., Gu, Y. L., Gu, Y. N., Chen, H. Y., Tian, L., Chen, L. Y., & Peng, T. (2012).
   Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology,
   28(9), 956–959.
3. Wu, S. C., Liu, C. C., & Lian, W. C. (2004). Optimization of microcarrier cell culture process
   for the inactivated enterovirus type 71 vaccine development. Vaccine, 22(29-30), 3858–3864.
   <https://doi.org/10.1016/j.vaccine.2004.05.037>
4. Chen, H. L., Huang, J. Y., Chu, T. W., Tsai, T. C., Hung, C. M., Lin, C. C., Liu, F. C., Wang,
   L. C., Chen, Y. J., Lin, M. F., & Chen, C. M. (2008). Expression of VP1 protein in the milk of
   transgenic mice: a potential oral vaccine protects against enterovirus 71 infection. Vaccine,
   26(23), 2882–2889.
   <https://doi.org/10.1016/j.vaccine.2008.03.041>
5. Huda, M. N., Ahmad, S. M., Alam, M. J., Khanam, A., Kalanetra, K. M., Taft, D. H., Raqib, R.,
   Underwood, M. A., Mills, D. A., & Stephensen, C. B. (2019). Bifidobacterium Abundance in Early
   Infancy and Vaccine Response at 2 Years of Age. Pediatrics, 143(2), e20181489.
   <https://doi.org/10.1542/peds.2018-1489>
6. Yi, E. J., Shin, Y. J., Kim, J. H., Kim, T. G., & Chang, S. Y. (2017). Enterovirus 71 infection
   and vaccines. Clinical and experimental vaccine research, 6(1), 4–14.
   <https://doi.org/10.7774/cevr.2017.6.1.4>
7. 7.Yu, X. F., Pan, J. C., Meng, D. M., Wang, H. Q., Zhang, W., & Zheng, W. (2007). Zhonghua yu
   fang yi xue za zhi [Chinese journal of preventive medicine], 41(6), 461–465.

# Sequencing results

## >VP1-F-sequencing

ATCTGTTCCAGGGGCCCCTGGGATCTCCATGGGGATCCATGGGAGATAGGGTGGCGGATGTGATTGAGAGTTCTATAGGGGATAGTGTGAGCAGAGCTCTCACCCAAGCTTTACCAGCACCCACAGGCCAAAACACGCAAGTAAGCAGCCACCGGTTGGACACTGGTAAAGTCCCAGCACTCCAAGCCGCTGAAATTGGAGCATCATCAAATGCTAGTGACGAGAGTATGATTGAGACACGGTGTGTTCTCAACTCGCACAGTACAGCTGAAACTACCCTCGATAGTTTTTTTAGCAGAGCGGGGCTAGTTGGAGAGATAGACCTCCCCCTTGAAGGCACAACCAACCCGAATGGTTATGCCAACTGGGACATAGATATAACAGGCTATGCACAAATGCGTAGAAAGGTGGAGTTGTTCACCTACATGCGTTTTGACGCAGAGTTCACCTTTGTTGCATGTACACCCACCGGGGAGGTTGTCCCACAATTGCTCCAATATATGTTTGTGCCACCTGGGGCTCCCAAGCCAGACTCCAGAGAATCCCTTGCATGGCAAACTGCCACCAACCCCTCGGTTTTTGTTAAGTTGTCAGATCCCCCAGCACAGGTTTCGGTTCCATTCATGTCACCCGCAAGCGCCTATCAATGGTTCTATGACGGATACCCCACGTTCGGTGAGCACAAGCAAGAGAAAGACCTTGAATATGGGGCATGTCCAAACAATATGATGGGCACGTTCTCAGTGCGAACTGTGGGAACCTCGAAGTCCAAGTACCCTTTGGTGATTAGGATTTACATGAGGATGAAGCACGTCAGGGCGTGCATACCCCGTCCAATGCGTAACCAAAACTATCTATTCAAAGCCAACCCAAATTATGCCGGCACTCCATTAAGCCAACCGGTACCAGCCGCACGGCGATCAACACTTCTTTAGGTCGACTCGAGGATACAAGGACGATGACGATAAGTGAGCGGCCGCATCGTGACTGACTGACGATCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACCACATGCAGCTCCCGGGAGACGGTCCACAGCTTGTCTTGTAA

## >VP1-R-sequencing

GGCAGATCGTCAGTCAGTCACGATGCGGCCGCTCACTTATCGTCATCGTCCTTGTAATCCTCGAGTCGACCTAAAGAGTGGTGATCGCCGTGCGGCTGGTACCGGTTGGCTTAATGGAGTTGCCGGCATAATTTGGGTTGGCTTTGAATAGATAGTTTTGGTTACGCATTGGACGGGGTATCCACGCCCTGACGTGCTTCATCCTCATGTAAATCCTAATCACCAAAGGGTACTTGGACTTCGAGGTTCCCACAGTTCGCACTGAGAACGTGCCCATCATATTGTTTGGACATGCCCCATATTCAAGGTCTTTCTCTTGCTTGTGCTCACCGAACGTGGGGTATCCGTCATAGAACCATTGATAGGCGCTTGCGGGTGACATGAATGGAACCGAAACCTGTGCTGGGGGATCTGACAACTTAACAAAAACCGAGGGGTTGGTGGCAGTTTGCCATGCAAGGGATTCTCTGGAGTCTGGCTTGGGAGCCCCAGGTGGCACAAACATATATTGGAGCAATTGTGGGACAACCTCCCCGGTGGGTGTACATGCAACAAAGGTGAACTCTGCGTCAAAACGCATGTAGGTGAACAACTCCACCTTTCTACGCATTTGTGCATAGCCTGTTATATCTATGTCCCAGTTGGCATAACCATTCGGGTTGGTTGTGCCTTCAAGGGGGAGGTCTATCTCTCCAACTAGCCCCGCTCTGCTAAAAAAACTATCGAGGGTAGTTTCAGCTGTACTGTGCGAGTTGAGAACACACCGTGTCTCAATCATACTCTCGTCACTAGCATTTGATGATGCTCCAATTTCAGCGGCTTGTAGTGCTGGGACTTTACCAGTGTCCAACCGGTGGCTGCTTACTTGCGTGTTTTGGCCTGTGGGTGCTGGTAAAGCTTGTGTGAGAGCTCTGCTCACACTATCCCCTATAGAACTCTCAATCACATCCGCCACCCTATCTCCCATGGATCCCATGGAGATCCCAGGGGCTCCTGGAACAGACTTCCAGATCCGATTTTGGAGATGGTCGCCACCACCAAACGTGGCCTTG

## >VP1-LTB-F-sequencing

TGGAAGTTCTGTTCCAGGGGCCCCTGGGATCTCCATGGGGATCCATGGGAGATAGGGTGGCGGATGTGATTGAGAGTTCTATAGGGGATAGTGTGAGCAGAGCTCTCACCCAAGCTTTACCAGCACCCACAGGCCAAAACACGCAAGTAAGCAGCCACCGGTTGGACACTGGTAAAGTCCCAGCACTCCAAGCCGCTGAAATTGGAGCATCATCAAATGCTAGTGACGAGAGTATGATTGAGACACGGTGTGTTCTCAACTCGCACAGTACAGCTGAAACTACCCTCGATAGTTTTTTTAGCAGAGCGGGGCTAGTTGGAGAGATAGACCTCCCCCTTGAAGGCACAACCAACCCGAATGGTTATGCCAACTGGGACATAGATATAACAGGCTATGCACAAATGCGTAGAAAGGTGGAGTTGTTCACCTACATGCGTTTTGACGCAGAGTTCACCTTTGTTGCATGTACACCCACCGGGGAGGTTGTCCCACAATTGCTCCAATATATGTTTGTGCCACCTGGGGCTCCCAAGCCAGACTCCAGAGAATCCCTTGCATGGCAAACTGCCACCAACCCCTCGGTTTTTGTTAAGTTGTCAGATCCCCCAGCACAGGTTTCGGTTCCATTCATGTCACCCGCAAGCGCCTATCAATGGTTCTATGACGGATACCCCACGTTCGGTGAGCACAAGCAAGAGAAAGACCTTGAATATGGGGCATGTCCAAACAATATGATGGGCACGTTCTCAGTGCGAACTGTGGGAACCTCGAAGTCCAAGTACCCTTTGGTGATTAGGATTTACATGAGGATGAAGCACGTCAGGGCGTGTATACCCCGTCCAATGCGTAACCAAAACTATCTATTCAAAGCCAACCCAAATTATGCCGGCAACTCCATTAAGCCAACCGGTACCAGCCGCACGGCGATCACCACTCTTGCCACAACTTCTCTCTGCTAAAGCAAGCAGTGATGTTGAAGAAAACTCCGGGCCTGTTGACATATATACAGATTCCGGGATGAATTATGATAAG

## >VP1-LTB-R-sequencing

CCGCGAGGCAGATCGTCAGTCAGTCACGATGCGGCCGCTCACTTATCGTCATCGTCCTTGTAATCCTCGAGTCGACAAGCTTGCCCCTCCAGCCTAGCTTAGTTTTTTCTGTGTGCTATAACAGCTCCCTGTAGTGGAAGCTGTTATAGTGTTACAGTTTAAGGATCGGTATTGCCTCCTCTACCGCCTTTTCAAATGCAGCATAAGGCTCATTAGTATAAGTACAGTAGTTGTTATATAGGTTCCTAGCATTAGACATGCTTTTAAAGCAAACTAGTTTTTCATACTGATTGCCGCAATTGAATTGGGGGTTTTATTATTCCATACACATAATTTATCAATTTTGGTCTCGGTCAGATATGTGATTCTTAATGTGTCCTTCATCCTTTCAATGGCTTTTTTCTGGGAGTCTATATGTTGACTGCCCGGGACTTCGACCTGAAATGTTTCGCCGCTCTTAAATGTAATGATAACCATTTCTCTTTTGCCTGCCATCGATTCCGTATATGATAGTATCTTGTCATTTATCGTATATATTTGTGTGTTGCGATATTCCGAACATAGTTCTGTAATAGTCTGGGGAGCTCCGTGTGCATATAGAGAGGATAGTAACGCCGTAAATAAAACATAACATTTTACTTTATTCATAATTCATCCCGAATTCTGTTATATATGTCAACAGGCCCGGGGTTTTCTTCAACATCACCTGCTTGCTTTAGCAGAGAGAAGTTTGTGGCAAGAGTGGTGATCGCCGTGCGGCTGGTACCGGTTGGCTTAATGGAGTTGCCGGCATAATTTGGGTTGGCTTTGAATAGATAGTTTTGGTTACGCATTGGACGGGGTATCCACGCCCTGACGTGCTTCATCCTCATGTAAATCCTAATCACCAAAGGGTACTTGGACTTCGAGGTTCCCACAGTTCGCACTGAGAACGTGCCCATCATATTGTTTGGACATGCCCCATATTCAAGGTCTTTCTCTTGCTTGTGCTCACCGAACGTGGGGTATC

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