iGEM 2024 — Team subcat-union

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NoteBook
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7.26 Day1

         LB medium preparation

|  |  |  |
| --- | --- | --- |
|  | Solid LB | Liquid LB |
| Amount prepared | 1 L | 1 L |
| Actual amount prepared | 200 mL | 1 L and 500 mL |
| Tryptone | 10 g | 10 g |
| Yeast extract | 5 g | 5 g |
| Sodium chloride | 10 g | 10 g |
| water | 1 L | 1 L |
| Alga powder | 15 g | None |

\* All components shown in the table above are required for preparing 1L of
solid and liquid LB mediums.

Step 1: Weigh out each component.

Step 2: Mix the components and dissolve them in distilled water.

Step 3: Seal the container of the solution with aluminum foil

Step 4: Autoclave it for 2 hours with a maximum temperature of 120°C

         Kanamycin preparation

Antibiotics are included in the medium for two reasons. First, they
eliminate any unwanted bacteria that may have accidentally entered. Second, they help select the bacteria that
have successfully expressed the target gene. The target gene contains antibiotic resistance, meaning the antibiotics
will not affect those bacteria, while the others will be eliminated(Water + kanamycin powder = kanamycin solution with a storage concentration
of 50 mg/ml).This solution is then diluted by LB medium into a concentration of 50
ug/mL.

         Culturing the bacteria

1. Add 5 mL of LB medium
2. Add 5 μL of antibiotic solution in this case kanamycin solution
3. Add 200 μL of bacteria sample to the solution
4. Shake it for 12 h under 37°C

7.27 Day2

         Plasmid extraction

Procedures

1.  Place the
absorption column into the collection tube, add 500 μL of buffer S, and
then centrifuge with 12000 spins per minute for 1 minute. Remove the waste liquid from the collection tube and
place the absorption column back in it.

2.  Centrifuge 1.5 mL
of bacterial solution for 2 minutes with 8000 spin per minute. Collect the bacteria in solid form and remove the
medium.

3.  Add 250
μL of buffer
SP1 and blow it with the pipette until the bacteria is completely suspended.

4.  Add 250
μL of buffer
SP2, immediately mix it gently, and let it sit at room temperature for 2-4 minutes.

5.  Add 350
μL of buffer
SP3 and immediately mix it gently.

6.  Centrifuge the
solution for 5 minutes at 12000 spins per minute. Place the supernatant in the absorption column and centrifuge
at 8000 spins per minute for 30 seconds. Remove the liquid from the collection tube.

7.   Add 500
μL of buffer
DW1, centrifuge it for 30 seconds at 9000 spins per minute, and then remove the liquid from the collection
tube.

8. Repeat step 7.

9. Centrifuge the empty absorption column for 1 minute with 9000 spins per
minute.

10. Place the absorption column in a clean 1.5 mL centrifuge tube, add 50-100
μL Elution
Buffer (the more buffer added, the lower the concentration), leave it at room temperature for 1
minute, and then centrifuge it for 1 minute. Preserve the DNA solution in the tube.

         Different buffers

1. Buffer SP1

Resuspension buffer to provide an optimum pH for further lysis.

It contains lysosome, glucose, EDTA, and Tris.HCl.

Lysosome is responsible for breaking the cell wall of the bacterial

Glucose is for keeping the solution isotonic

EDTA inactivates many enzymes that may harm the plasmid

Tris.HCl acts as a
buffer that maintains the solution at a pH of 8

2. Buffer SP2

This is the lysis buffer.

Contains NaOH and dodecyl sulfate (SDS)

SDS solubilizes the
phospholipid and denature the protein.

NaOH provides a highly alkaline
condition that denatures the plasmid.

3. Buffer SP3

potassium acetate solution, which has a pH of 4.8. it brings the pH back to neutral and allows the participation of
proteins.

         Polymerase chain reaction

Vector PET-Dual

primer A-1F from 5’ and primer A-1R from 3’ for RH3 (failed to obtain) and
PPG.

primer A-2F from 5’ and primer A-2R from 3’ for P4H.

Vector PET

Primer C-1F from 5’ and C-1R from 3’ for PKG, DKG and EPG.

Add 25 μL of Phanta Mix 2 μL of primer F and
primer R,
and 19 μL
of water.

Put the mixture in the PCR machine, and it will use 3
different temperatures to unwind and replicate the DNA segment.

         Agar gel electrophoresis

Make use of the fact
that DNA is negatively charged at its phospho-group, which
means it will move toward the positive charge at different speeds due to the difference in size.

Preparation of 1% agarose gel: 100 mL of TAE, 1 g of agarose and 5
μL of DNA
dye.

While adding the
DNA sample, the sample should be first mixed with the loading
buffer.

A ladder that measures the length of the target DNA will be
used.

7.28 Day3

         Agar gel electrophoresis of the product from 7.27 (same procedures)

         Gel extraction

To obtain target DNA segments from the alga gel once used in the
electrophoresis.

1.          Cut the gel
containing the target DNA fragment and weigh it out.

2.          Add Buffer B2 that is 3-6 times heavier than the piece of gel. Water bath it at 50°C for 5-10 minutes to melt the gel.

3.          Place the solution in an absorption column in the collection tube and centrifuge it for 30 seconds at 8000 spins per minute. Remove the waste liquid from the collection
tube.

4.          Add 500uL of Wash Solution, centrifuge it for 30 seconds with 9000 spins
per minute, and remove the waste liquid in the collection tube.

5.          Repeat step 4.

6.          Centrifuge the absorption column for 1 minute with 9000 spins per
minute.

7.          Place the absorption column in a clean 1.5mL centrifuge tube and add
15-40uL of elution buffer at the center of the column (the
more elution buffer added, the lower the concentration). Let
it sit at room temperature for 1 minute, and then centrifuge
it for 1 minute. Preserve the DNA solution in the tube.

         Homologous recombination

Homologous recombination of PET24a is for putting the target DNA segment
into the vector by enzyme.

Use 2\*Clon express reaction system (10uL)

|  |  |
| --- | --- |
| 4uL | PET24a |
| 1uL | Fragment (3,4,5) |
| 5uL | 2\*Clon |

Water bath it at 50°C

         Transformation

To put the plasmid that contains the target gene
fragment into a cell/bacteria in order to culture more

1.          Add 10mL of homologous recombination product into the DH5alpha cell

2.          Put it in the ice for 30 minutes

3.          Put the mixture into the water bath for 45 seconds at 42°C

4.          Put it back to the ice for 3 minutes

5.          Add 900mL of LB medium and put it into the shake incubator for 30 minutes
at 37°C

6.          Centrifuge the tubes for 10 minutes with 8000 spins per minute

7.          Remove the supernatant, leave 100 uL, and mix them by blowing them with the pipette

8.          Add into solid LB medium in a petri dish

9.          Apply them evenly with a coating rod and cultivate it overnight

7.29 Day4

The following letters
represent one specific plasmid or E. coli
transformed using the plasmid.

A: pET-Dual-HisRh3CP15VP4-P4H

B: pET-Dual-HisPPGP15VP4-P4H

C: pET-24a-DKG50-P15VP4

D: pET-24a-PKG50-P4VP4

E: pET-24a-EPG50-P8VP4

A B was successfully cultured; therefore, it will be
PCR and be examined by electrophoresis.

C D E wasn’t successfully cultured; therefore, have to be
repeated.

Extraction of the
plasmid of pET Dual RH3C-P4H failed the first time.

         Solid LB medium preparation

|  |  |  |
| --- | --- | --- |
|  | Solid LB | Liquid LB |
| Amount prepared | 1 L | 1 L |
| Actual amount prepared | 200 mL | 1 L and 500 mL |
| Tryptone | 10 g | 10 g |
| Yeast extract | 5 g | 5 g |
| Sodium chloride | 10 g | 10 g |
| water | 1 L | 1 L |
| Alga powder | 15 g | None |

\*All amounts of components shown in the table above is the amount required
for preparing 1L of both LB mediums.

Step 1: weigh out each component.

Step 2: mix the components together and dissolve them in distilled
water.

Step 3: seal the container of the solution with aluminum foil

Step 4: autoclave it for 2 hours with a maximum temperature of
120°C

         Plasmid extraction

1. Place the absorption column into the collection tube, add 500 μL of buffer S and
   then centrifuge with 12000 spins per minute for 1 minute. Remove the waste liquid in the collection tube and
   place the absorption column back in the collection tube.
2. Centrifuge 1.5 mL of bacterial solution for 2 minutes with 8000 spin per minute. Collect the bacteria in
   solid form and remove the medium.
3. Add 250 μL of buffer SP1, blow it with the pipette until the bacteria is
   completely suspended.
4. Add 250 μL of buffer SP2, immediately mix it genteelly and place it in room
   temperature for 2-4 minutes.
5. Add 350 μL of buffer SP3, immediately mix it genteelly.
6. Centrifuge the solution for 5 minutes with 12000 spin per minute. Place the supernatant in the absorption
   column and centrifuge with 8000 spin per minute for 30 seconds. Remove the liquid in the collection
   tube.
7. Add 500 μL of buffer DW1, centrifuge it for 30 second with 9000 spin per minute
   and then remove the liquid in the collection tube.
8. Centrifuge the empty absorption column for 1 minute with 9000 spin per minute.
9. Repeat step 8
10. Place the absorption column in a clean 1.5 mL centrifuge tube, add 50-100 μL
    Elution Buffer (the more buffer added, the lower the concentration), place it in
    room temperature of 1 minute and then centrifuge it for 1 minute. Preserve the DNA solution in the
    tube,

         Culture Bacteria (C D E)

1. Add 5 μL of the cells into the petri dish that is covered by solid LB
   medium.
2. Add 10 μL of liquid LB medium at the point where the cell is added
3. Spread them evenly on the plate with the coating rod.
4. Culture them overnight

         PCR

PET dual

1. MCS1 multiple cloning site 1 RH3CVP4

2. MCS2 multiple cloning site 2 P4H~750bp

A. Make a new medium and draw a grid.

Only take a separate colony.

Restripe the bacteria that were cultivated yesterday into the newly
prepared medium.​

Repeat this step four times.

keep the environment 37℃.

B. colony verification (PCR + agarose gel electrophoresis, AGE）

The reaction system has a total of 100 μL

5’ Primer→4 μL

3’Primer→4 μL

2x PCR Mix →50 μL

add water→to 50
μL

Pour 100 μL of colonies into the test tube on average 5 times.

3. PCR (2x PCR Mix)

Denaturation 94℃ 5 min

Degeneration 94℃ 30 s

Annealing 50-60℃ 30 s

Stretch 72℃ 30-60 sec/kb

Final Stretch 72℃ 10 min

Degeneration → Stretch（30-35 cycle）

4. Make agarose gel, wait for 20 minutes until it solidifies, and put the
PCR product into

the well. Wait 30 minutes.

         Electrophoresis of A B that are previously PCR

Agarose gel preparation

|  |  |
| --- | --- |
| Component | Volume |
| TAE | 100mL |
| Agarose | 1g |
| DNA dye | 5uL |

1. Mix 100mL of TAE with 1g of agarose gel.
2. Seal the container with aluminium foil.
3. Heat it in a microwave for 3 minutes until it resolves.
4. Add 5uL of DNA dye and mix.
5. Pour the solution into the model and wait until it cools down and forms
   solid.

While adding the
DNA sample, the sample should be first mixed with the DNA
loading buffer.

A ladder that measures the length of DNA in BP will be used.

Electrophoresis for 30 minutes with 120 V.

7.30 Day5

         PCR of B\C\D\E that was cultured (PPG DKG PKG EPG)

Denaturation 94℃ 5min

Degeneration 94℃ 30s

Annealing 50-60℃ 30s

Stretch 72℃ 30-60sec/kb

Final Stretch 72℃ 10min

Degeneration → Stretch （30-35 cycles）

100 μL PCR reaction system

|  |  |
| --- | --- |
| 5’ primer | 4 μL |
| 3’ primer | 4 μL |
| 2\*PCR mix | 50 μL |
| DD water | 42 μL |

         Culturing bacteria

pick the bacteria from the petri dish

put them into the tube that contains LB medium

culture it overnight

7.31 Day6

         Electrophoresis of plasmid B (HisPPGP15VP4-P4H) and plasmid E
(EPG50-P8VP4)

         Culture plasmid A-E in BL21

         SDS-Page electrophoresis gel preparation

         PCR verification of the A-E in BL21

         0.2 mM IPTG (Isopropyl-beta-D-thiogalactopyranoside)
inducing protein expression in BL 21.

         Electrophoresis

Gel preparation

|  |  |
| --- | --- |
| TAE | 100mL |
| Agarose | 1g |
| DNA dye | 5uL |

1. Mix 100 TAE
   with 1g of agarose.
2. Seal the
   container with aluminium foil.
3. Heat it in
   the microwave for 3 minutes until it boils.
4. Add 5uL of
   nuclei acid dye and mix.
5. Pour the
   solution into the electrophoresis container and wait until it cools down and forms a
   solid.
6. Load the 2K
   ladder so the DNA can be measured in BPs.
7. Mix the DNA
   sample with the loader and inject it into the gel.
8. Electrophoresis it for 30 minutes with 120 V.

         Culture plasmid A-E in BL21

Select the bacteria colony from the petri dish, Add it to the tube containing LB medium, Shake it for
7h

         SDS-Page electrophoresis gel preparation

|  |  |
| --- | --- |
| Upper-layer gel solution | 5mL |
| Upper-layer gel buffer | 5mL |
| Lower-layer gel solution | 5mL |
| Lower layer gel buffer | 5mL |
| Double distilled water | Enough |
| coagulant TEMED | 20uL |

1.      Prepare the lower-layer
gel by adding the same amount of lower-layer gel solution and
buffer.

2.      Inject them into the container carefully to form a flat surface.

3.      Add double distilled water to the surface to keep it flat.

4.      When the lower layer of
gel is solidified, pour away the water.

5.      Mix the lower layer gel buffer and solution.

6.      Inject it carefully into the container.

7.      Push the comb inside the upper layer.

8.      Leave it at
room temperature for 20 minutes until it solidifies.

9.      Keep it moist and store in 4°C.

         PCR verification of the A-E in BL21

Denaturation 94℃ 5min

Degeneration 94℃ 30s

Annealing 50-60℃ 30s

Stretch 72℃ 30-60sec/kb

Final Stretch 72℃ 10min

Degeneration → Stretch （30-35 cycles）

100uL PCR reaction system

primer A-1F from 5’ and primer A-1R from 3’ for RH3

primer A-2F from 5’ and primer A-2R from 3’ for P4H.

|  |  |
| --- | --- |
| 5’ primer | 4uL |
| 3’ primer | 4uL |
| 2\*PCR mix | 50uL |
| ddH2O | 42uL |

Add this reaction system to the plasmid
extraction.

         IPTG promote protein expression

IPTG diluted to 0.2 mM

8.1 Day7

         Protein purification

         Electrophoresis of the purified protein using the SDS-PAGE gel prepared on
7.31.

         Purification of the protein

1. Collect the bacteria solution promoted overnight.
2. Centrifuge it, remove the supernatant (LB medium) and weigh the remaining bacteria.
3. Add lysis buffer; 1 g of bacteria should be mixed with 4 mL of lysis
   buffer.
4. Mix it evenly and use ultrasonication to break down the bacterial wall.
5. Centrifuge the solution and collect the supernatant, place it on the ice surface.
6. Add the lysis buffer and His-tag purification resin into the empty affinity chromatography column.
7. Add the supernatant in the column, collect the run-through fluid and repeat 3-5
   times.
8. Wash the column with a wash buffer 5 times.
9. Elute the bound proteins with a wash buffer containing imidazole 3 times.

         Electrophoresis of the purified protein

1.      Inject the sample of protein into the gel

2.      Electrophoresis it with 100 V till it passes the upper layer, then use 200 V.

3.      Take the gel and submerge it in the Coomassie Blue Staining
solution.

4.      Wash it in the destaining solution overnight.

8.2 Day8

         Use native PAGE electrophoresis (10% of separation gel) to examine the
protein.

Total 15mL of separation gel.

1.      Add 6.1mL of distilled water.

2.      Add 5 mL of 30% Arc-Bis.

3.      Add 3.75 mL of gel buffer A.

4.      Add 10 % APS for 0.15 mL.

5.      Add 0.006 mL of TEMED and pour the mixture into the gel container.

6.      Wait for the separation gel in the container to solidify.

7.      Prepare the concentration gel.

8.      Add 1.33 mL of distilled water.

9.      Add 0.67 mL of 30% Arc-Bis.

10.  Add 2 mL of gel buffer B.

11.  Add 0.04 mL of 10% APS.

12.  Add 0.004 mL of TEMED.

13.  Pour it in the container after the separation gel is solidified and then
push the comb in the gel.

14.  After the concentration gel solidifies, remove the comb and load the
sample.

15.  Electrophoresis for 20 minutes with 80 V and then use 100 V till the
end.

8.3-8.14 wiki writing

🔝

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![](https://static.igem.wiki/teams/5531/s8/2.png)
Engineering
![](https://static.igem.wiki/teams/5531/s8/1.png)

Overview

Rotaviruses (RVs) in the family Reoviridae are the most common
causative agents of severe gastroenteritis in children under five years of age, leading to significant morbidity
and mortality worldwide, particularly in developing nations (Cohen et al., 2022). The RV virion is composed of
an approximately 80 nm in diameter triple-layered protein capsid that encapsulates 11 segments
of double-stranded RNA genome[1]. When observed under an electron microscope, rotavirus appears as a
particle with short protrusions and a smooth outer edge, resembling a wheel shape (Figure 1).

![](https://static.igem.wiki/teams/5531/eng/1.png)

Figure 1. The schematic
diagram of rotavirus structure.

Before introducing vaccines for RV, most young
children will likely be infected by RV at least once before age 5. After the vaccine was introduced, the
efficacy in developed countries reached 80%-90%, yet the effectiveness significantly decreased in low- and
middle-income countries by 40%. Though the cause of this drastic decline remains unclear, studies point out
potential intestine-related issues, including coadministration of the oral poliovirus vaccine, malnutrition,
and changes in microbiome composition. There are also safety
issues related to intussusception, which is caused by the replication of bacteria in the intestine. To address
these problems, nonreplicating subunit RV vaccine is urgently needed, as it can bypass the intestine
system[2].Current subunit
vaccines use trimers like Folden, which are non-human protein sources. Fusing them with the protein antigen
can form a stable trimer, yet this will decrease the immune response.

In this study, we
experimented with humanized type III collagen (partial sequence GERGAPGFRGPAGPNGIPGEKGPAGERGAPn of Rh3, a type III collagen, a homotrimer) or model collagen sequences
(POGn,
where O represents hydroxyproline) were used as novel trimeric motifs. The Vp4 antigenic
trimer was formed using synthetic biology methods by fusion expression with the rotavirus Vp4 protein. Since
prokaryotes lack proline hydroxylation-modifying enzymes (prolyl 4-hydroxylases (P4H)),
and hydroxylation is essential for collagen stability, we co-expressed P4H derived from mega viruses with the Vp4 proteins described above to achieve
stability of Vp4 oligomers.

![](https://static.igem.wiki/teams/5531/eng/2.png)

Figure 2. Schematic
representation of the Rh3/POG-conjugated VP4 design as a trimeric vaccine antigen.

Furthermore, we utilized to reported artificially designed
proline-free heterotrimeric collagen-like protein motifs ((PKG)n, (DKG)n, (EPG)n), where three distinct serological VP4 proteins (P4, P8, P15) were fused to one of the heterotrimeric collagen-like
motifs. The fusion-expressed proteins were purified and mixed in vitro to form oligomers. In this way, a
multivalent-oligomeric rotavirus vaccine in E. coli is possible without P4H modification.

![](https://static.igem.wiki/teams/5531/eng/3.jpg)

Figure 3. Schematic
representation of PKG/DKG/EPG-conjugated VP4 design as a heterotrimeric vaccine antigen.

Part 1: BBa\_K5531010 (pET-Dual-HisRh3C-P15VP4-P4H)

Design 1:

This targeted gene, Rh3C-P15VP4 (BBa\_K5531000), synthesized by a
biotech company, contains humanized type III collagen fused with the VP4 protein from the rotavirus with
serotype P15. Meanwhile, P4H (BBa\_K5531012) is also co-expressed
in this plasmid due to the lack of proline hydroxylation-modifying enzymes (prolyl 4-hydroxylases (P4H)) in
procaryotes, which is essential for collagen stability. The pET-Dual-N-His-TEV
(BBa\_K5531006) serves as the vector.
The homologous recombination method was employed to construct pET-Dual-HisRh3C-P15VP4-P4H (BBa\_K5531010).

![](https://static.igem.wiki/teams/5531/eng/44.jpg)

Figure 4. The plasmid
map of pET-Dual-HisRh3C-P15VP4-P4H

Build 1:

We isolated pET-Dual-N-His-TEV vectors
from bacterial solutions primarily through centrifugation. The vectors were then recovered from the
remains in the absorption column. Subsequently, we linearized the vectors using restriction enzymes and
proceeded to analyze the products through electrophoresis.
The electrophoresis result displayed
correctness toward the expected outcome (HisRh3C-P15VP4 is 2000 bp, and P4H is 755 bp). We selected several individual colonies for sequencing. Figure 5C shows the success in pET-Dual-HisRh3C-P15VP4-P4H
construction.

![](https://static.igem.wiki/teams/5531/eng/55.jpg)

Figure 5. The results of pET-Dual-HisRh3C-P15VP4-P4H

Test 1:

Later, the plasmid mounted with the gene of Rh3C-P15VP4 was transferred
to E.coli DH5α to replicate. The extracted plasmid was transferred into E.coli
BL21, which can help express His-Rh3C-P15VP4. After the colony PCR of E.coli
BL21 was finished and verified, the bacteria were cultured and treated with 0.2 mM IPTG, which can promote
protein expression.

After promoting the protein expression overnight, the protein was
purified via His-tag Purification Resin and went through SDS-PAGE electrophoresis and Native-PAGE
electrophoresis. The target protein Rh3C-P15VP4 has a size of 85 kDa (Figure 6).

![](https://static.igem.wiki/teams/5531/eng/66.jpg)

Figure 6. The expression of His-Rh3C-P15VP4 using
E. coli BL21. SDS-PAGE gels showing the purification results. The loading
sequence is protein marker, whole cell lysate, precipitate, supernatant, flow-through, unwanted proteins, and
target protein.

Collagen-VP4 A was diluted to a concentration
of 0.5 mg/mL and incubated at 37°C for 1 hour before undergoing native-PAGE and SEC analysis. As illustrated
in Figure 7, the samples exhibited two distinct
clusters: high-molecular-weight and low-molecular-weight
states,
each displaying multiple bands indicating varying levels of
proline hydroxylation.. Based on the
construct design, we hypothesized that the high-molecular-weight clusters corresponded to
trimer assemblies, while the low-molecular-weight clusters corresponded to the monomers. This hypothesis was
confirmed by the SEC analysis, as depicted in Figure 7. The SEC peaks for
collagen-VP4 A ranged from 0.5 to 0.8 CV, with the prominent peaks at 0.56 CV and 0.66
CV, respectively, suggesting the presence of trimeric and monomer
macromolecules.

![](https://static.igem.wiki/teams/5531/eng/77.jpg)

Figure 7. Native-PAGE analysis (left)of oligomeric
states of collagen A; Size exclusion chromatographic (SEC) analysis(right) of oligomeric states of collagen
A.

Learn 1:

The first is that the activity of bacteria should be considered, as
some of them failed to be cultured under the correct environment. The selection of primers and PCR temperature for different stages should
be reevaluated, as the formation of primer dimers during PCR amplification may lead to inaccurate PCR
results.

###### Part 2: BBa\_K5531011 (pET-Dual-HisPPG-P15VP4-P4H)

Design 2:

The collagen sequence (POGn, O is
hydroxyproline) was synthesized with the VP4 gene from the rotavirus with serotype P15. Meanwhile, P4H (BBa\_K5531012) is
also co-expressed in this plasmid due to the lack of proline hydroxylation-modifying enzymes (prolyl
4-hydroxylases (P4H)) in procaryotes, which is essential for collagen
stability. The pET-Dual-N-His-TEV (BBa\_K5531006) serves as the vector. The homologous recombination method was employed to
construct pET-Dual-HisPPG-P15VP4-P4H(BBa\_K5531011).

![](https://static.igem.wiki/teams/5531/eng/88.jpg)

Figure 8. The plasmid
map of pET-Dual-HisPPG-P15VP4-P4H

Build 2:

We isolated pET-Dual-N-His-TEV
vectors from bacterial solutions primarily by centrifugation. The vectors were then obtained from the remains
in the absorption column. Subsequently, we linearized the vectors using restriction enzymes and conducted
electrophoresis to analyze the products. The electrophoresis result displayed
consistency toward the expected outcome (HisPPG-P15VP4 is
2000 bp
and P4H is 630 bp), indicating the success in pET-Dual-HisPPG-P15VP4-P4H
construction. We selected several individual colonies for
sequencing. Figure 9C shows the success
ofpET-Dual-HisPPG-P15VP4-P4H
construction.

![](https://static.igem.wiki/teams/5531/eng/99.jpg)

Fig 9. The results of pET-Dual-HisPPG-P15VP4-P4H

Test 2:

Later, the plasmid mounted with the gene of PPG-P15VP4 was transferred
to E.coli
DH5α to replicate. The extracted plasmid was transferred into E.coli
BL21, which can help express His-PPG-P15VP4. After the
colony PCR of E.coli
BL21 was finished and verified, the bacteria were cultured and treated with 0.2 mM IPTG, which can promote
protein expression.

The protein expressed via E.coli
BL21 was purified via His-tag Purification Resin and went through SDS-PAGE electrophoresis. The target protein
PPG-P15VP4
has a size of 59 kDa (Figure 10).

![](https://static.igem.wiki/teams/5531/eng/1010.jpg)

Figure 10. His-PPGP15VP4 using E. coli BL21. SDS-PAGE gels showing the purification results. The loading
sequence is protein marker, whole cell lysate, precipitate, supernatant, flow-through, unwanted proteins, and
target protein.

Collagen-VP4 A was diluted to a concentration of 0.5 mg/mL and
incubated at 37°C for 1 hour before undergoing native-PAGE and SEC analysis. As illustrated in Figure
11, the
samples were divided into two distinct clusters: high-molecular-weight and low-molecular-weight states. Each
cluster contained multiple bands, indicative of varying degrees of proline hydroxylation. Based on the construct
design, we hypothesized that the high-molecular-weight clusters represented the trimer assemblies, while the
low-molecular-weight clusters corresponded to the monomers. This hypothesis was confirmed by the SEC analysis,
as depicted in Figure 11. The peaks for collagen-VP4 B ranged from 0.5 to
0.8 CV, with the prominent peaks at 0.56 CV and 0.66 CV, respectively, suggesting the presence of trimer and
monomer macromolecules.

![](https://static.igem.wiki/teams/5531/eng/1111.jpg)

Figure 11. Native-PAGE analysis
(left)of oligomeric
states of collagen B; Size exclusion chromatographic (SEC) analysis(right) of oligomeric states of collagen
B

Learn 2:

It is crucial to consider the activity of bacterial strains when
conducting experiments, as the initial activity of the strains plays a vital role in the entire experimental
process. Proper preservation of the bacterial strains is essential to ensure the orderly progression of
subsequent experiments.

###### Part 3: BBa\_K5531007 (pET24a-DKG50-P15VP4)

Design 3:

This targeted gene, DKG50-P15VP4(BBa\_K5531002), synthesized by a
biotech company, contains proline-free heterotrimeric collagen-like protein motif DKG50 fused with the VP4
protein from the rotavirus with serotype P15. The pET24a(BBa\_K5531005) serves as the vector.
The homologous recombination method was employed for the construction of pET24a-DKG50-P15VP4(BBa\_K5531007).

![](https://static.igem.wiki/teams/5531/eng/12.jpg)

Figure 12. Plasmid map
of pET24a-DKG50-P15VP4

Build 3:

We isolated pET24a vector from bacterial solution primarily by
centrifugation. The vectors were then obtained from the remains in the absorption column. Subsequently, we
linearized the vectors using restriction enzymes and conducted electrophoresis to analyze the products. The
electrophoresis’s result consistently predicted the expected outcome (DKG50-P15VP4 is 2000 bp). We selected several individual colonies for sequencing. Figure 13 shows the success of pET24a-DKG50-P15VP4 construction.

![](https://static.igem.wiki/teams/5531/eng/13.png)

Figure 13. The results of pET24a-DKG50-P15VP4

Test 3:

Later, the plasmid mounted with the gene of DKG50-P15VP4 was
transferred to E.coli
DH5α to replicate. The extracted plasmid was transferred into E.coli
BL21, which can help express His-DKG50-P15VP4. After the colony PCR of E.coli
BL21 was finished and verified, the bacteria were cultured and treated with 0.2 mM IPTG, which can promote
protein expression.

After promoting the protein expression overnight, the protein was
purified via His-tag Purification Resin and went through SDS-PAGE electrophoresis. The target protein
DKG50-P15VP4 has a size of 68.1 kDa.

![](https://static.igem.wiki/teams/5531/eng/1414.jpg)

Figure 14. The
expression of DKG50-P15VP4 using E.coli BL21

Learn 3:

We should continue optimizing the recombinant protein technology
pathway for the vaccine, exploring various forms of the VP4 protein to improve its protective efficacy and
identify the vaccine antigen. Additionally, we will develop adjuvants and formulations, focusing on designing
adjuvants to boost immune responses and creating suitable vaccine formulations.

###### Part 4: BBa\_K5531009 (pET-24a-EPG50-P8VP4)

Design 4:

Using the backbone of pET-24a(BBa\_K5531005), proline-free
heterotrimeric collagen-like protein motif EPG50 is fused with serological P8-VP4 via chemical
synthesis. The homologous recombination method was employed for the construction of
pET-24a-EPG50-P8VP4(BBa\_K5531009).

![](https://static.igem.wiki/teams/5531/eng/15.jpg)

Figure 15. Plasmid map of pET-24a-EPG50-P8VP4

Build 4:

We isolated pET-24a vectors from bacterial solutions primarily by
centrifugation. The vectors were then obtained from the remains in the absorption column. Subsequently, we
linearized the vectors using restriction enzymes and conducted electrophoresis to analyze the products. The
electrophoresis result displayed correctness toward the expected outcome (EPG50-P8VP4 is
2000 bp).
We selected several individual colonies for
sequencing. Figure 16 shows the success of
pET24a-EPG50-P8VP4 construction.

![](https://static.igem.wiki/teams/5531/eng/16.png)

Figure 16. The results of pET24a-EPG50-P8VP4

Test 4:

Later, the plasmid mounted with the gene of EPG50-P8VP4 will then be
transferred to E.coli
DH5α to replicate. The extracted plasmid was transferred into E.coli
BL21, which can help express His-EPG50-P8VP4. After the colony PCR of E.coli
BL21 was finished and verified, the bacteria were cultured and treated with 0.2 mM IPTG, which can promote
protein expression.

After promoting the protein expression overnight, the protein was
purified via His-tag Purification Resin and went through SDS-PAGE electrophoresis and Native-PAGE
electrophoresis. The target protein EPG50-P8VP4 has a size of 67.5 kDa.

![](https://static.igem.wiki/teams/5531/eng/1717.jpg)

Figure 17. The
expression of EPG50-P8VP4 using E.coli BL21.

Learn 4:

After protein cell lysis and purification, it is important to ensure
the timely use and proper storage of the protein. Store the purified protein in suitable conditions to maintain
its stability and activity for future experiments. Additionally, avoid repeated freeze-thaw cycles and consider
aliquoting the protein for multiple usages to minimize degradation and maintain its integrity.

###### Part 5: BBa\_K5531008 (pET24a-PKG50-P4VP4)

Design 5:

This targeted gene, PKG50-P4VP4(BBa\_K5531003), synthesized by a
biotech company and constructed by a biotech company, contains proline-free heterotrimeric collagen-like protein motifs PKG fused P15VP4 protein. The pET24a(BBa\_K5531005) serves as the vector.
The homologous recombination method was employed for the construction of pET24a-PKG50-P4VP4(BBa\_K5531008).

![](https://static.igem.wiki/teams/5531/eng/18.png)

Figure 18. Plasmid map of pET-24a-PKG50-P4VP4

Build 5:

We isolated pET-24a vectors from bacterial solutions primarily by
centrifugation. The vectors were then obtained from the remains in the absorption column. Subsequently, we
linearized the vectors using restriction enzymes and conducted electrophoresis to analyze the products. The
electrophoresis’s result displayed consistency toward the expected outcome (PKG50-P4VP4 is 2000 bp); We selected several individual colonies for sequencing. Figure 19 shows the success of pET24a-PKG50-P4VP4 construction.

![](https://static.igem.wiki/teams/5531/eng/19.png)

Figure 19. The results of pET24a-PKG50-P4VP4

Test 5:

Later, the plasmid pET-24a-PKG50-P4VP4 will then be transferred to
E.coli
DH5α to replicate. The extracted plasmid was transferred into E.coli
BL21, which can help express His-PKG50-P4VP4. After the colony PCR of E.coli
BL21 was finished and verified, the bacteria were cultured and treated with 0.2 mM IPTG, which can promote
protein expression.

After promoting the protein expression overnight, the protein was
purified via His-tag Purification Resin and went through SDS-PAGE electrophoresis and Native-PAGE
electrophoresis. The target protein PKG50-P4VP4 has a size of 68.1 kDa.

![](https://static.igem.wiki/teams/5531/eng/20.jpg)

Figure 20. The
expression of PKG50-P4VP4 using E.coli BL21

Learn 5:

During protein purification, it is important to follow standard operating
procedures for purification columns. Careful loading of the protein sample onto the column is essential to
prevent the formation of air bubbles and to ensure uniform distribution within the resin.

Other Test:

The collagen of the C-D-E-VP4 complex (in a 1:1:1 ratio) was diluted
to a concentration of 0.5 mg/mL and incubated at 37°C for 1 hour prior to native-PAGE and SEC analysis. As
illustrated in Figure 21, the samples were divided into two distinct clusters: high-molecular-weight and
low-molecular-weight states. Each cluster contained multiple bands, indicative of varying degrees of proline hydroxylation. Based on
the construct design, we hypothesized that the high-molecular-weight clusters represented the trimer assemblies,
while the low-molecular-weight clusters corresponded to the monomers. This hypothesis was confirmed by the SEC
analysis, as depicted in Figure 21. The peaks for the C-D-E-VP4 complex also ranged from 0.5 to 0.8 CV, with
the prominent peaks at 0.57 CV, 0.68 CV, 0.72 CV, and 0.77 CV, indicating a trimer at
0.57 CV and the other peaks corresponding to the monomers of C, D, and E. It confirms the reliability of our
experimental design.

![](https://static.igem.wiki/teams/5531/eng/21.jpg)

Figure 21. Native-PAGE
analysis of oligomeric states of collagen c-d-e complex(left); Size exclusion
chromatographic (SEC) analysis of oligomeric states of collagen c-d-e complex(right)

References

[1] Rotavirus Vaccine - Gastrointestinal ADRs | Pharmacovigilance. https://www.pharmacy180.com/article/rotavirus-vaccine-3274/

[2] World Health Organization (WHO). (2020). Rotavirus vaccines: WHO position paper, January 2018. Weekly Epidemiological Record, 93(6), 53-72.

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Experiments
![](https://static.igem.wiki/teams/5531/s8/1.png)

Bacterial colony culture

Materials

√         LB solution

√         Antibiotics

√         Target gene

√         15 mL bacterial culture tube

√         Flask

Procedure

√         Disinfection

√         Prepare tubes

√         Preparing sterile LB 5 ml in the flask

√         Put in 5 μL Ampicilin

√         Put in 20 μL ‘pET-Dual-HisRh3CP15VP4-P4H’

√         Put the bacterial culture in the shaker

Plasmid extraction

Materials

√         Buffer SP1 with RNase A: 250 μL

√         Buffer SP2: 250 μL

√         Buffer SP3: 350 μL

√         Buffer S: 500 μL

√         Buffer DW1: 500 μL

√         Wash Solution: 500 μL

√         Absorption column

√         Centrifuge

√         Collection tube

Procedure

1. preparation

Check whether RNase A has already been added into buffer SP1

Check whether ethanol has already been added to the wash solution

2. Adsorption column equilibrium

Add 500 μL Buffer S into the
adsorption column, and centrifuge for 1min with 12,000 g.

Drain the waste liquid from the collection pipe.

Put the adsorption column back in the collection tube.

3. Collect bacteria for minutes and discard the medium. Take 1.5-5 mL of
overnight cultured bacterial solution and centrifuge it by 8,000 g

4. Add 250 μL Buffer SP1 to the sediments and entirely suspend the bacteria.

Add 250 μL Buffer SP2 and gently
reverse the centrifuge tube immediately

5. Centrifuge 5-10 times and mix well. Let the mixture stand at room
temperature for 2-4 minutes.

6. Add 350 μL Buffer SP3 and gently reverse the centrifuge tube immediately

Mix 5-10 times.

7.12,000 g centrifuge for 5-10 min. Transfer the supernatant into the
adsorption column,

Centrifuge the supernatant at 8,000 g for 30 seconds and drain the liquid
in the collection tube.

8.(Optional) Add 500 μL Buffer DW1 and centrifuge 9,000 g, 30 s. Drain the collection
tube.

9. Add 500 μL Wash Solution and centrifuge 30 s by 9,000 g

10. Repeat Step 9.

11. Centrifuge the empty adsorption column at 9,000 g for 1 min. Drain the
ethanol from the adsorption column.

12. Place the adsorption column into a clean 1.5 mL, add 50-100
μL Elution Buffer, place
it at room temperature for 1 minute, and then centrifuge it for 1 minute.

PCR

Materials

√         5’ primer: 4 μL

√         3’ primer: 4 μL

√         PCR mix: 50 μL

√         ddH2O: 42 μL

√         PCR tube

Procedure

1. Put all reagents on ice

2. Mixing 50 μL primers (3’primer 4 μL, 5’primer 4 μL, plasmid 2 μL, water 42 μL) into a homogenous state and transfer the solution into the PCR
tubes

3. Placing tubes in the PCR machine

Prepare negative control without template DNA

Prepare positive control with template DNA of known size and appropriate
primers

conditions: at 95℃double helical DNA
unwinding, at 65℃ primer matching with DNA template, at 72℃ DNA
replication

Agarose Gel Electrophoresis

Materials

√         TAE:120ml

√         Agarose:1.20 g

√         Nucleic acid dye:5μL

√         Machine

√         Mode

Procedure

√         Preparing TAE (TAE powder and distilled water)

√         After adding 120 ml TAE and 1.2g Agarose into the flask, heat the solution
in the microwave.

√         Waiting for 3 to 5 minutes to cool down the solution naturally.

√         Adding 5 μL nucleic acid dye in
the solution.

√         Adding the solution into the mode.

DNA extraction from agarose gel

Materials

√         Wash Solution added with ethanol

√         Buffer B2

√         Water bath

         Wash solution: 500 μL

         Elution Buffer:15-40 μL

√         1.5 mL centrifuge tubes

Procedure

1. Turn the water bath to 50℃.

2. Cut the agarose gel containing the target DNA fragment and weigh it
out.

3. Add Buffer B2 3-6 times the weight of the gel, 50°C water bath for 5-10
minutes.

4.(Optional) For <500 bp segments, add isopropyl alcohol that is 1/3
Volume of Buffer B2.

5. Transfer the solution into the adsorption column and centrifuge for 30
s at 8,000 g.

6. Add 500 μL Wash Solution and centrifuge 9,000 g for 30 seconds.

7. Repeat Step 6.

8. Centrifuge the empty adsorption column at 9,000 g for 1 min.

9. Put the adsorption column into a clean 1.5 mL centrifuge tube and drain
the ethanol from the collection column.

10. Add 15-40 μL Elution Buffer to the center of the collection column, let it stand at
room temperature for 1 minute, and centrifuge for 1 minute. Then, preserve the DNA solution in the tube.

Homologous recombination

Materials

√         pET 24a: 4 μL

√         DNA fragment: 1 μL (according to the length)

√         2\* Clon Express reaction system: 5 μL

Procedure

√         Adding all materials into tubes

√         Water bath at 50
℃ for 15
minutes

Transformation

Materials

√         homologous
recombination system products

√         receptive cells: 100 μL

Procedure

1. The 10 μL homologous recombination system was extracted and added to
100μL
receptive cells.

2. First ice bath for 30 minutes

3. Heat shock 42℃.

4. Then ice bath for 2 minutes, add 900 μL liquid LB.

5. Culture in Petri dishes.

Expression and purification

The construction of an expression vector for collagens a, b, c, d, and
e was
undertaken to facilitate the production of fusion proteins in E. coli BL21 (DE3). The plasmid DNA was successfully transformed into
E. coli BL21 (DE3), enabling the expression of the collagens above using an auto-induction technique. The induction of
collagens a, b, c, d, and e was carried out using the auto-induction method in E. coli BL21 (DE3) at a temperature of 18 °C. Following a 36-hour induction
period, the cells were harvested via centrifugation. The resulting cell pellet was resuspended in a lysis buffer
comprising pH 8.0, 25 mM Tris-HCl, 300 mM NaCl, 0.5 mM TCEP, 1 mM PMSF, 0.1 mg/mL lysozyme, and 25 U/mL
SuperNuclease, and incubated for 30 minutes at room temperature. Subsequently, the
cells were lysed by sonication on ice, and the cell debris was removed by centrifugation at 18,000 rpm at 4 °C
for 30 minutes. The supernatant was then collected and subjected to purification using Ni-TED agarose. Further
collagens a, b, c, d, and e purification was achieved on a Superdex 75 10/300 GL
column with a protein storage buffer consisting of pH 7.4, 25 mM Tris-HCl, 300 mM NaCl, and 0.5 mM TCEP.

SDS polyacrylamide gel electrophoresis (SDS-PAGE)

Materials

√         SDS-PAGE gel mold set

√         SDS-PAGE color preparation kit

√         Coagulant TEMED

√         Electrophoresis buffer

Procedure

1. Assemble the glue mold and configure the lower layer of glue first
(take a piece of 0.75/1.00/1.50mm thick mini glue as an example).

2. Take 2.0/2.7/4 mL of the lower-layer glue solution (2X) and the
lower-layer glue buffer (2X) in the same volume and mix it well.

3. Add the improved coagulant 40/55/80 μL to the mixed solution in Step 2 and stir gently to avoid bubbles.

4. Add the lower layer of glue solution mixed in step 3 to the glue-making
mold so that the liquid level is 1.5 cm away from the upper side of the glass plate, and then cover the lower
layer of glue solution with a layer of water or alcohol to keep the gel surface flat and do not produce
bubbles.

5. Stand at room temperature (25°C) for 6 to 10 minutes, 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.

7. Slowly pour off the covering phase, take the same volume of the upper
adhesive solution (2X) and the color upper adhesive buffer (2X), 0.5/0.75/1.0 mL each, and mix them in a new
dispensing cup.

8. Add the modified coagulant 10/15/20 μL to the mixed solution 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 minutes, wait for the upper layer of glue to solidify,
carefully pull out the comb, use the gun head or syringe to absorb the electrophoresis buffer, and wash the
sample hole clean then the SDS-PAGE electrophoresis operation can be performed.

Coomassie Bright Blue R250 solution

Take 250 mL of methanol solution, add 0.5 g of Coomassie bright blue R250
powder, stir the solution thoroughly, add 50 mL of acetic acid, mix again, and finally add 500 mL of distilled
water. Store the dye solution at room temperature.

SDS-PAGE decolorising solution

70 mL of glacial acetic acid, 50 mL of anhydrous ethanol, and distilled
water to 1 L.

Store at room temperature.

Native polyacrylamide gel electrophoresis (native-PAGE)

A 10% native-PAGE gel for electrophoresis was prepared by combining 2.5 mL
of 40% acrylamide solution, 2.5 mL of 1.5 M Tris-HCl buffer at pH 8.8, and 5 mL of deionised water. The
polymerisation was initiated by adding 50 μL of 10% ammonium persulfate and 10 μL of TEMED. For sample preparation, 20 μL of a 0.5 mg/mL collagens a, b, and c-d-e (1:1:1) solution were mixed with
5 μL of sample buffer
containing 250 mM Tris-HCl at pH 6.8, 50% glycerol, 0.5% bromophenol blue, and 0.05% Triton X-100. The samples
were loaded onto the gel and electrophoresed in a Tris-glycine running buffer of 25 mM Tris-HCl, 250 mM glycine,
and 0.01% Triton X-100. After electrophoresis, the gel was stained with Coomassie Brilliant Blue and destained
using a solution with 10% methanol and 7% acetic acid for enhanced clarity.

Size exclusion chromatographic (SEC) analysis

SEC was performed with a SuperdexTM 75 Increase
10/300 GL analytical column (GE Healthcare) at 4 ℃. The column was
pre-equilibrated and eluted with buffer (25 mM Tris-HCl, 300 mM NaCl, 0.5 mM TCEP, and 0.01% Triton X-100, pH
7.4) at a 0.3 mL/min flow rate. Approximate 0.5 mg/mL collagens a, b, and c-d-e (1:1:1) were pre-incubated
separately in 0.5 mL volume for 63 min at 37 ℃ before loading onto the column at a 0.3 mL/min flow rate.

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![](https://static.igem.wiki/teams/5531/s8/2.png)
Results
![](https://static.igem.wiki/teams/5531/s8/1.png)

Overview

Currently, trimeric motifs such as Foldon from non-human protein sources expressed in fusion with antigenic
proteins can spontaneously form stabilizing trimers from antigens. However, the potent immunogenicity of
these motifs can weaken the immune response of the target proteins, limiting their use in vaccine antigen
design.
In this study, we explored the use of humanized type III collagen (specifically, a partial sequence
GERGAPGFRGPAGPNGIPGEKGPAGERGAPn of Rh3, a type III collagen, forming a homotrimer) and model collagen
sequences (such as POGn, where O represents hydroxyproline) as innovative trimeric motifs, synthesized
through synthetic biology approaches.

The Vp4 antigenic trimer was engineered via synthetic biology techniques by fusing it with the rotavirus
Vp4 protein. Since prokaryotes lack proline hydroxylation-modifying enzymes (prolyl 4-hydroxylases (P4H)),
and hydroxylation is essential for collagen stability, we co-expressed P4H derived from mega viruses with
the Vp4 proteins described above to achieve stability of Vp4 oligomers.

Furthermore, we utilized to reported artificially designed proline-free heterotrimeric collagen-like
protein motifs ((PKG)n, (DKG)n, (EPG)n), three different serological Vp4 proteins (P4, P8, P15) were fused
to one of the heterotrimeric collagen-like motifs. The fusion-expressed proteins were purified and mixed in
vitro to form oligomers. In this way, a multivalent-oligomeric rotavirus vaccine in E. coli is possible
without P4H modification.

Plasmid construction

Using PET-Dual and PET-24a as vector plasmids, we put PPG, P4H, and Rh3C in PET-Dual, and EPG50 with p8,
PKG50 with p4, and DKG50 with p15 were inserted into PET-24a. Based on the homologous recombination
technique, we added restriction sites to primers and applied the same restriction enzymes to the plasmids;
then, the PCR fragment and linearized vector can be linked via homologous recombinase.

We first constructed the pET-Dual-HisRh3CP15VP4-P4H plasmid, amplified the Rh3C-P15-26-476 and P4H
fragments by PCR, and obtained the linear plasmid backbone pET-Dual-N-His-TEV (Dual protein prokaryotic
expression plasmid) by double digestion, and then, by homologous recombination, the Subsequently, by
homologous recombination, the HisRh3CP15VP4 fragment was inserted between Xba1 and Sal1, and the P4H
fragment was inserted between Nde1 and Xho1, and the recombinant plasmid pET-Dual-HisRh3CP15VP4-P4H was
finally obtained.

Then, we continued to construct the pET-Dual-HisPPGP15VP4-P4H plasmid, amplified the PPG-P15-26-476 and P4H
fragments by PCR, and obtained the linear plasmid backbone pET-Dual-N-His-TEV (Dual-Protein Prokaryotic
Expression Plasmid) by double-enzymatic cleavage, and then, using homologous recombination, we inserted the
HisRh3CP15VP4 fragment between Xba1 and Sal1, and the P4H fragment between Nde1 and Xho1. Subsequently, by
homologous recombination, the HisPPGP15VP4 fragment was inserted between Xba1 and Sal1, and the P4H fragment
was inserted between Nde1 and Xho1, and the recombinant plasmid pET-Dual-HisPPGP15VP4-P4H was finally
obtained

Moreover, we continued to construct pET-24a-DKG50-P15VP4 plasmid, amplified DKG50-P15-26-476-FLAG fragments
by PCR and obtained linear plasmid backbone pET-24a by double digestion, and then, using homologous
recombination, we inserted the DKG50-P15VP4 fragments between Xba1 and Xho1 and finally obtained the
recombinant plasmid pET-24a-DKG50-P15VP4. Xho1 to obtain the recombinant plasmid pET-24a-DKG50-P15VP4.

Next, we continued to construct pET-24a-PKG50-P4VP4 plasmid, amplified PKG50-P4-26-476-HA fragments by PCR
and obtained linear plasmid backbone pET-24a by double digestion, and then, using homologous recombination,
we inserted the PKG50-P4VP4 fragments between Xba1 and Xho1 and finally obtained the recombinant plasmid
pET-24a-PKG50-P4VP4.

Finally, we constructed pET-24a-EPG50-P8VP4 plasmid, amplified EPG50-P8-26-476-c-Myc fragments by PCR, and
obtained linear plasmid backbone pET-24a by double digestion. Then, using homologous recombination, we
inserted the EPG50-P8VP4 fragments between Xba1 and Xho1 and finally obtained the recombinant plasmid
pET-24a- EPG50-P8VP4.

![](https://static.igem.wiki/teams/5531/res/11.jpg)

Figure 1. Electrophoresis for the target genes PKG50, DKG50, PPG, EPG50, and P4H.

Figure 1 shows that the fragment lengths are consistent with the results. It indicates that we have
successfully amplified the target genes. After electrophoresis, the bands that contain the target DNA
produced the required DNA fragment by gel extraction. The DNA extracted from the gel was inserted into the
plasmid pET24a or pET-Dual via homologous recombination.

Subsequently, the concatenated plasmids A-E were introduced into E. coli DH5α through transformation. In Figure 2A, successful growth of isolated colonies is depicted, with selected colonies undergoing verification. Figure 2B shows that the length of Rh3CP15VP4 is about 2000 bp, and P4H is about 750 bp. After the extraction of the plasmid from the positive colony, the sequencing results in Figure 2C showed that Rh3CP15VP4 and P4H do not contain any gene mutation. It proves that the plasmid pET-Dual-HisRh3CP15VP4-P4H was successfully constructed.

![](https://static.igem.wiki/teams/5531/res/2.jpg)

Figure 2. The culture plate for E. coli DH5α(A), verification of colonies(B), and the sequencing results of pET-Dual-HisRh3CP15VP4-P4H(C).

The plasmid pET-Dual-HisPPGP15VP4-P4H was successfully constructed the same way as pET-Dual-HisRh3CP15VP4-P4H via homologous recombination, as shown in Figure 3.

![](https://static.igem.wiki/teams/5531/res/3.jpg)

Figure 3. The culture plate for E. coli DH5α (A), verification of colonies (B), and the sequencing results of pET-Dual-HisPPGP15VP4-P4H (C).

The plasmid pET-24a-DKG50-P15VP4, pET-24a-PKG50-P4VP4, and pET-24a-EPG50-P8VP4 were successfully constructed using homologous recombination, as shown in Figure 4.

![](https://static.igem.wiki/teams/5531/res/4.jpg)

Figure 4. The culture plates for E. coli DH5α (A), verification of colonies (B), and the sequencing results of pET-24a-DKG50-P15VP4, pET-24a-PKG50-P4VP4, pET-24a-EPG50-P8VP4 (C).

Protein Expression and Purification

We first transformed the five plasmids into E.coli BL21 (DE3) to test the protein's expression. Figure 5A shows that the isolated colonies were successfully grown and selected for colony verification. After the E.coli BL21 had been cultured for 12-16h, single colones were selected and then examined via PCR. Figure 5B shows the electrophoresis results after PCR.

![](https://static.igem.wiki/teams/5531/res/5.jpg)

Figure 5. The culture plates for E. coli BL21 (A), verification of colonies (B) results of pET-Dual-HisRh3CP15VP4-P4H, pET-Dual-HisPPGP15VP4-P4H, pET-24a-DKG50-P15VP4, pET-24a-PKG50-P4VP4, pET-24a-EPG50-P8VP4.

After the colonies (BL21) were verified, they were correctly inoculated into liquid LB medium and cultured until reaching an OD600 of 0.6. Inducer IPTG was then added to induce protein expression. Following overnight cultivation at 16°C, the cells were lysed, and the protein was purified for subsequent analysis via SDS-PAGE and Native-PAGE electrophoresis.

SDS-PAGE gels showing the purification results. Protein purification of the five proteins A-E was examined using nickel column affinity chromatography. As can be seen from the figure, there is a single band at the size of the target proteins. This further indicates that the proteins can be correctly folded and expressed after induction and are solubilized for expression.

![](https://static.igem.wiki/teams/5531/res/6.jpg)

Figure 6. The expression of His-Rh3CP15VP4 (A), His-PPGP15VP4 (B), DKG50-P15VP4 (C), PKG50-P4VP4 (D), EPG50-P8VP4 (E) using E. coli BL21. Tips: The sample order from left to right is protein marker, whole cell lysate, precipitate, supernatant, flow-through, unwanted proteins, and target protein.

Functional Analysis

Collagen-VP4 A, B, C, D, and E were fermented separately in 400 mL of inductive culture medium. The proteins were then purified using a one-step nickel affinity chromatography method, as shown in Figure 6. The yield rates for each collagen were 3.379, 2.557, 2.256, 3.723, and 1.085 mg/L, respectively. Collagen-VP4 A, B, and the C-D-E-VP4 complex (in a 1:1:1 ratio) were diluted to 0.5 mg/mL and incubated at 37°C for 1 hour before undergoing native-PAGE and SEC analysis. As illustrated in Figure 7, the samples were divided into two distinct clusters: high-molecular-weight and low-molecular-weight states. Each cluster contained multiple bands, indicative of varying degrees of proline hydroxylation.

![](https://static.igem.wiki/teams/5531/res/7.jpg)

Figure 7. Native-PAGE electrophoresis showing the protein trimer formed by Rh3CP15VP4(A) and PPGP15VP4(B). The heterotrimeric collagen is composed of DKG50-P15VP4(C), PKG50-P4VP4(D), and EPG50-P8VP4(E).

Based on the construct design, we hypothesized that the high-molecular-weight clusters represented the trimer assemblies, while the low-molecular-weight clusters corresponded to the monomers. This hypothesis was confirmed by the SEC analysis, as depicted in Figure 8. The peaks for collagen-VP4 A and B ranged from 0.5 to 0.8 CV, with the main peaks at 0.56 CV and 0.66 CV, respectively, suggesting the presence of trimer and monomer macromolecules. The peaks for the C-D-E-VP4 complex also ranged from 0.5 to 0.8 CV, with the main peaks at 0.57 CV, 0.68 CV, 0.72 CV, and 0.77 CV, indicating a trimer at 0.57 CV and the other peaks corresponding to the monomers of C, D, and E. Additionally, we calculated the peak area ratio of trimer to monomer for the main peaks to assess the assembly efficiency of the collagens. The ratios were 1.27 for collagen-VP4 A, 1.24 for collagen-VP4 B, and 0.60 for the C-D-E VP4 complex/monomers.

![](https://static.igem.wiki/teams/5531/res/8.jpg)

Figure 8. Size exclusion chromatographic (SEC) analysis of homotrimeric collgen-VP4, Rh3CP15VP4(A), and PPGP15VP4(B), heterotrimeric C-D-E-VP4 complex.Tips: DKG50-P15VP4(C), PKG50-P4VP4(D), and EPG50-P8VP4(E).

Future plan

Further research is essential for the advancement of a recombinant human rotavirus vaccine. Evaluation of immunogenicity in larger animal models such as pigs and monkeys is imperative, as certain rotavirus antigens that elicit robust protective immunity in small animals may not confer the same effectiveness in larger species. Collaboration with biotechnology firms to conduct animal trials for dose determination is recommended. Additionally, the timeframe for observing vaccine efficacy and the likelihood of adverse reactions post-vaccination are critical considerations. These aspects warrant careful investigation to determine the duration of vaccine protection and the associated risk of vaccine-related illness in the vaccinated population.

Our vaccine now consists of three different serological Vp4 proteins (P4, P8, P15); we can try more different proteins in the future. In the future, we can work with various hospitals to get their data and further improve our vaccines.

We should continue optimizing the vaccine's recombinant protein technology route, exploring more forms of VP4 protein to enhance its protection and determine the vaccine's antigen. Adjuvant and formulation development involves designing adjuvants to enhance immune responses and appropriate vaccine formulations. Animal experiments: The vaccine's safety, immunogenicity, and preliminary efficacy were tested in animal models. This stage aids in pinpointing potential side effects and dose responses.

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