iGEM 2023 — Team bjhs

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each
day for your project.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/4861/wiki/notebook.png)

# **Wet Lab** **Notebook**

## **2023.6.20**

Order Experiment Materials

## **2023.7.1**

## Online Meetup for determining lab schedules

## **2023.7.6**

1.

Project Design Review

2.

Lab Safety Training

## **2023.7.7**

1.Configure the culture medium
2.Extract plasmids
3.digestion
4.Switch

## **2023.7.8**

1.Agarose gel electrophoresis
2.Rubber recycling
3.T4 DNA Enzyme connection
4.Conversion (DH5α) Conversion BL21

## **2023.7.9**

1.Pick single clone, shake bacteria
2.Make protein glue
3.plasmid extract
4.digestion

## **2023.7.10**

Group A: plasmid extract, enzyme cutting,Agarose gel electrophoresis
Group B: Sample pretreatment

## **2023.7.11**

Send for sequencing

## **2023.7.1****2**

1.Expanded training (need to start from 9 o’clock)
2.Sort out the SDS-PAGE results to determine the best expression conditions
3.Add IPTG
4.Collect bacteria

## **2023.7.1****3**

1.Collect protein
2.Pretreatment of dialysis bag (estimated to take 30min)
3.Contains body complex (dialysis)

## **2023.7.1****4**

1.Nickel column purification
2.BCA Quantitative
3.SDS-PAGE Glue to detect protein purity

## **2023.7.1****5**

Utilize ELISA examine SARS-CoV-2 RBD with people ACE2 the binding ability of protein

## **2023.7.1****6**

Data curating and analysis

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/4861/wiki/engineering-success/engineering.png)

**Overview**

The aim of the experiment is to produce a Recombinant Protein Vaccine with Spike-Protein as an
antigen in E. coli and assesse its functionality. To achieve this idea, we design the following methods:

![](https://static.igem.wiki/teams/4861/wiki/engineering-success/enginnering01.png)

Each of these components came with its own set of challenges and obstacles, both in the design
process and during practical implementation.

**Engineering** **Cycle**

1.
**Design**

Spike protein is the key protein for Covid-19 to infect the human body. It mediates the entry of
the virus into host cells by binding to the ACE2 receptor on the surface of host cells.*(R. Lu et al, 2020)* Therefore, spike protein is the preferred choice for designing recombinant protein vaccine
against Covid-19. The spike protein is divided into two sub-units *(P. Zhou et al., 2020)*, S1 and S2, in which the S1 sub-unit contains a receptor binding domain (RBD), which is the key
structure for the virus to enter the host cell.*(A. C. Walls et al., 2020)* Therefore, it is appropriate to select RBD as the target antigen for designing recombinant
protein vaccines.

The Recombinant Protein Vaccine
was
planned to combine the Spike protein in E. coli with the four RBD fragments of the SARS-CoV-2
virus(Wuhan, Delta, BQ1.1, XBB1.5). Using RBD as an immunogen produces Anti-RBD antibodies after
vaccination and finally inhibits virus invasion and infection(Figure 1.1).

![](https://static.igem.wiki/teams/4861/wiki/engineering-success/1-1.png)

***Figure 1.1****Diagram of Spike Protein combines hACE2 through RBD and produce
anti-body*

The experiment will
be
separate
d
 into three parts.

In the first part, we will build sample plasmids and prove
they are
 successfully built. We
used
 the pET-28a(+) vector as the viral vector and transferred the RBD fragments in the four
virus samples into the pET-28a(+) vector by enzyme digestion and enzyme chaining. RBD samples will go
through Gel electrophoresis of nucleic acids to assess their success.

In the second part, we will transfer the plasmid samples to BL21(DE3) Competent E. coli. We will
induce plasmid with the Isopropyl β-D-1-thiogalactopyranoside induction in different concentrations and
temperatures. To test the optimal concentration for the plasmid, we will use SDS-PAGE discontinuous
electrophoretic system and record the optimal concentration. We will purify the sample with His-Tag.

In the third part, we use the ELISA Test to assess the success of Anti-RBD
antibodies.

2.
**Build**

**Part I****-Plasmid Construction**

In building experiment, the pET28a(+) vector was employed as the expression vector throughout the
experimental procedures (Figure 2.1). Initially, four viral cultures were cultivated for 12-16 hours in
LB medium, and subsequently, the Vazyme DC201 Protocol was employed to extract the respective plasmids.
Evaluation of the plasmid concentrations facilitated the selection of the most suitable plasmid for
subsequent double digestion, targeting a specific region within the pET28a(+) vector (Figure 2.2). The
insertion event was targeted at approximately 6000 base pairs of the pET28a(+) vector.

![](https://static.igem.wiki/teams/4861/wiki/engineering-success/2-1.png)

***Figure 2.1****Diagram of pET28a(+) vector*

![](https://static.igem.wiki/teams/4861/wiki/engineering-success/2-2.png)

***Figure 2.2****Diagram of the process of double digestion*

**Part II****-Cells Transformation**

Two distinct strains of Escherichia coli were utilized in this study: DH5α and BL21. DH5α was
specifically chosen for cloning purposes. Initially, the plasmid was transferred into DH5α cells.
Following
that
 the plasmid was subsequently transferred into BL21 cells to facilitate protein expression
(Figure 2.3).

![](https://static.igem.wiki/teams/4861/wiki/engineering-success/2-3.png)

***Figure 2.3****Diagram of the process of pET28a(+) plasmid transform in
BL21(DE3)*

3.
**Test**

In order to prove that every step of the experiment is successful, we need to prove the success
of each step through different verification methods.

**Test I-****Plasmid Construction Validation**

In order to prove that four recombinant plasmids (
Wuhan-pET28a
, Delta-pET28a, BQ1.1-pET28a, XBB1.5-pET28a) were digested successfully.
According to the
electrophoretogram(figure 3.1)
, t
he criterion for judging success is two parallel lines at 760bp and 6000bp.

![](https://static.igem.wiki/teams/4861/wiki/engineering-success/3-1.png)

***Figure 3.1****E**lectrophoretogram consist of highlight marker(M),* *Wuhan-pET28a**plasmid, Delta-pET28a plasmid, BQ1.1-pET28a plasmid, XBB1.5-pET28a
plasmid*

Subsequently, for further confirmation, we sent the recombinant plasmid to a biological company
(Azenta) for sequencing, and the sequencing results are shown in the figure
3
.2. This visual confirmation signifies the achievement of our plasmid construction endeavor and sets
the stage for the subsequent protein expression phase.

![](https://static.igem.wiki/teams/4861/wiki/engineering-success/3-2.png)

***Figure*** ***3******.******2******.****Sequencing results*

**Test II-****Protein Expression and** **O****ptimal** **E****xpression** **C****oncentration** **of Determination**

Protein expression was induced with the use of IPTG reagent, and an
analysis of IPTG data allowed for the determination of the optimal concentration for protein
expression. To this end, seven different concentrations of IPTG (0.002 mol/L, 0.001 mol/L, 0.00075
mol/L, 0.0005 mol/L, 0.00025 mol/L, 0.0001 mol/L) were introduced into 100 ml culture medium, with
overnight cultures performed at 16
°
C and 3-hour cultures at 37
°
C (Figure
3.3
). Subsequently, based on the conditions yielding calculates the optimal
IPTG concentrations.

![](https://static.igem.wiki/teams/4861/wiki/engineering-success/3-3.png)

***Figure*** ***3.3****Diagram of the process cell expression*

Based on the
SDS-PAGE
results
(figure 3.
4
) through E-Gel Imager and
the relevant A280
data(figure 3.
5 & 3.6
)
, we could conclude that the optimal concentration of IPTG to induce
protein expression in this experiement is 0.5 mM.

![](https://static.igem.wiki/teams/4861/wiki/engineering-success/3-4.png)

***Figure 3.******4****E**lectrophoretogram of IPTG induction in 16°C and 37°C
condition*

![](https://static.igem.wiki/teams/4861/wiki/engineering-success/3-5.png)

***Figure 3.******5****Chart of protein concentration in 16°C condition*

![](https://static.igem.wiki/teams/4861/wiki/engineering-success/3-6.png)

***Figure 3.******6****Chart of protein concentration in 37°C condition*

**Test III-ELISA** **Essay**

The Enzyme-Linked Immunosorbent Assay (ELISA) is employed for testing and assessing the binding
affinity between the SARS-CoV-2 RBD and the human ACE2 protein.

We immobilize the RBD onto an ELISA high binding plate, resulting in the generation of S2 Biotin.
Subsequently, S2 Biotin binds to Streptavidin-labeled HRP (horseradish peroxidase) conjugate. Upon the
addition of Tetramethylbenzidine (TMB) substrate solution, a colorimetric reaction occurs. We then
measure the Optical Density (OD) and record the data (Figure 3.
7
). This process allows us to quantify the interaction between these proteins with precision and
biological relevance.

![](https://static.igem.wiki/teams/4861/wiki/engineering-success/3-7.png)

***Figure 3.******7****The graph records the product reaction in 450nm OD*

4.
**Learn**

**Improvement I**

Plasmid
c
onstruction and
e
xpression
p
rocess
p
roceeded
s
moothly, but
f
urther
c
odon
o
ptimization or
t
ranscription-
t
ranslation
o
ptimization of the
p
lasmid could be studied and
i
mplemented to
e
nhance
p
rotein
e
xpression from the
g
ene
s
equence.

**Improvement II**

While
r
elative
o
ptimal
e
xpression
c
onditions were
i
dentified under the
g
iven
s
ettings, the
u
ltimate
c
onditions for
p
rotein
e
xpression may
r
equire
f
urther
e
xploration to specify more details,
e
specially
c
onsidering the
u
pcoming
l
arge-
s
cale
i
ndustrial
p
roduction, where
c
ost-
e
fficiency is
c
rucial. Further
r
esearch is
n
eeded to
a
chieve
s
ufficient
p
rotein
y
ield under
c
ost-
e
ffective
c
onditions.

**Improvement III**

According to the ELISA curves, we could see the trend that clarifies the
certain impact of the interaction between these 4 groups of  SARS-CoV-2 RBD and the human ACE2
protein.  However,
the
e
fficacy of SARS-CoV-2 RBD
p
roduced in Prokaryotic Cells to
i
nduce
n
eutralizing
a
ntibodies
r
equires
f
urther
v
alidation and
e
xperimentation. Further
e
xperiments are
r
equired to
d
etermine the
a
bility of the
p
roduced RBD to
e
ffectively
i
nduce
n
eutralizing
a
ntibodies.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/4861/wiki/experiments.png)

**Part I   Construction of plasmids**

**1.** **Escherichia coli culture**

|  |  |
| --- | --- |
| LB culture medium/1L | |
| Tryptone | 10g |
| Yeast extract | 5g |
| NaCl | 10g |

|  |  |
| --- | --- |
| Agar | 15g (1.5%) |

2.
**Plasmid Extraction**

Apparatus: Centrifuge

Using
Vazyme
DC201 plasmid test kit:

1. Take 1-5 ml of the bacterial
solution cultured overnight (12-16 h), add it into the centrifuge tube, and centrifuge at 10,000
rpm(11,500 × g) 1 minute. Discard the culture medium, invert on absorbent paper to absorb the residual
liquid.

2. Add 250 μl Buffer P1
into the centrifuge tube with bacterial precipitation and mixed with
pipette
s
 or vortex oscillations.

3. Add 250 μl Buffer P2 to
step 2 and gently mix it upside down for 8-10 times to fully crack the bacteria.

4. Add 350 μl Buffer P3 to
step 3 and immediately gently turn the solution upside down 8-10 times to completely neutralize the
BufferP2. A white flocculent precipitate should appear. Centrifuge at 12,000 rpm(13,400 × g) for 10 min.

5. The FastPure DNA Mini Columns
adsorption column was placed in the Collection Tube 2ml. Put Step 4
supernatant
carefully to the adsorption
column with a pipette, taking care not to absorb precipitate, and centrifuge 30-60 sec at 12,000 rpm(13,400 × g)
. Drain the waste liquid from the collection tube and put the adsorption column back into the collection
tube.

6. Add 500
μl
 Buffer PW1 and centrifuge 30-60 sec at 12,000 rpm(13,400 ×
g)
.

7. Add 600 μl Buffer
PW2(check whether it has been diluted with anhydrous ethanol) to the adsorption column. 12,000
rpm(13,400 x g) centrifuge 30-60 sec. Discard the waste liquid and put the adsorption column back into
the collection tube.

8. Repeat Step 7.

9. Place the adsorption column
back into the collection tube. Centrifuge at 12,000 rpm(13,400 × g) for 1 min to dry the adsorption
column, the purpose is to adsorb
t
he residual bleach liquid in the
column is completely removed.

10. Place the adsorption column
in a new sterilized 1.5 ml centrifuge tube. Add a 30-100 μl ddH2O to the center of the membrane of
the column adsorption column. Let stand at room temperature for 2 min, centrifuge at 12,000 rpm(13,400 ×
g) for 1 min and wash
r
emove the DNA.

11. Measure the
concentration.

**3. Digestion**

Gene synthetic plasmid
treatment: centrifuge at 5000rpm for 1min, and then dissolve in 20ul water with a concentration of
200ng/μL.

There are 5 samples in
total (wuhan, Delta, BQ1.1, XBB1.5 and pET-28a)

Reaction condition:
37
℃
/30min

Apparatus:
PCR
 amplifier

Reaction system:

1.
20ul system for DNA fragments

|  |  |
| --- | --- |
| Nco I | 1 ul |
| XhoI | 1ul |
| 10X buffer | 2ul |
| DNA fragment | 5ul |
| ddH2O | 11ul |

2.50ul system for pET-28a

|  |  |
| --- | --- |
| NcoI | 1ul |
| XhoI | 1ul |
| 10X buffer | 5ul |
| pET-28a | 5ul |
| ddH2O | 38ul |

4.
**T4 DNA Enzyme ligation**

A
pparatus:
PCR
 amplifier

1.
Set up the following reaction in
a microcentrifuge tube on ice.

|  |  |
| --- | --- |
| COMPONENT | 20ul REACTION |
| T4 DNA Ligase Buffer (10X) | 2 ul |
| V ector DNA (4 kb) | 50 ng (0.020 pmol) |
| I nsert DNA (1 kb) | 37.5 ng (0.060 pmol) |
| Nuclease-free water | to 20 μl |
| T4 DNA Ligase | 1 μl |

3.
Gently mix the reaction by
pipetting up and down and microfuge briefly.

3. For cohesive (sticky) ends,
incubate at 16°C overnight or room temperature for 10 minutes.

4. Heat inactivate at 65°C for
10 minutes.

4.
**Extraction**

Apparatus: Centrifuge

1.
PCR reaction solution extraction

Use Vazyme DC301 and recycle
4
 samples

1
)
Transient centrifuge PCR
products
.
Measure the body with a pipette
gun
.
 Accumulate and transfer to
sterilized 1.5 ml or 2 ml centrifuge tubes
,
 refill with sterilized
water to 100 µl.

2
)
 Add 5 times the volume of
Buffer GDP and mix upside down or vortex. If less than 100 bp DNA fragments need to be recovered, add
1.5 times the volume (the volume of sample +Buffer GDP) of anhydrous ethanol.

3
)
 Enclose the adsorption
column in the collection tube. Transfer solution to the adsorption column. 12,000 rpm (13,800 x g)
centrifuge 30-60 sec.

4)
Discard the filtrate and place
the adsorption column in the collection pipe. Add 700 µl Buffer GW(with anhydrous ethanol added) to
the adsorption column. 12,000 rpm (13,800 × g) centrifuge 30-60 sec.

Repeat Step 5.

5)
Discard the filtrate and put the
adsorption column back into the collection tube. Centrifuge at 12,000 rpm (13,800 × g) for 2 min.

6)
Place the adsorption column in a
1.5ml sterilized centrifuge tube and add 20-30µl Elution Buffer to the center of the adsorption
column.

Leave for 2 minutes. Centrifuge
at 12,000 rpm (13,800 × g) for 1 min. The adsorption column was discarded and the DNA was stored at -20
° C

7)
Measure the
concentration

2.

Gel extraction

Apparatus: Microwave Oven, UV transilluminator, Electro,
Bainmarie

Use
Vazyme
 DC301 and recycle
1
samples

1)
Agarose gel
Electrophoresis

2
）
After DNA electrophoresis,
quickly cut the gel containing the target DNA fragment under the ultraviolet lamp.Weigh the gel weight
(remove the empty tube weight), 100 mg gel equal tothe same volume as 100 μl,
as a gel volume.

3
)
Add equal-volume Buffer GDP.
50-55
℃
 water bath 7-10 min, according to the size of the gel appropriate
adjustment time, indeed
t
he adhesive block is completely
dissolved. Inversely mix the accelerative sol twice during the water bath.

4
)
Centrifuge briefly to collect
droplets on the tube wall. The FastPure DNA Mini Columns-Gadsorption column was placed in the Collection
Tubes 2 ml collection tube, transfer liquid into the adsorption column, centrifuge 30 -60 secat 12,000
rpm (13,800 × g)
.

5
)
 Discard the filtrate and
place the adsorption column in the collection pipe. Add 300 µl Buffer GDP to the adsorption column.
Let stand for 1 minute. 12,000 rpm (13,800 × g) centrifuge 30-60 sec.

6
)
 Discard the filtrate and
place the adsorption column in the collection pipe. Add 700 µl Buffer GW(with anhydrous ethanol
added) to the adsorption column. 12,000 rpm (13,800 × g) centrifuge 30-60 sec.

Repeat Step 5.

7
)
 Discard the filtrate and
put the adsorption column back into the collection tube. Centrifuge at 12,000 rpm (13,800 × g) for 2
min.

8
)
Place the adsorption column in a
1.5ml sterilized centrifuge tube and add 20-30µl Elution Buffer to the center of the adsorption
column.

Leave for 2 minutes. Centrifuge
at 12,000 rpm (13,800 × g) for 1 min. The adsorption column was discarded and the DNA was stored at -20
° C

9
)
Measure the
concentration

5.
**Transformation**

A
pparatus: Bainmarie, oscillations incubator

1）
Remove 100ul of Escherichia coli receptor cells
(
DH5α
)
from the -80
℃
 refrigerator and thaw them on ice;

2）
Add 1ul plasmid (generally no more than 50ng), gently shake well, and place on ice
for 10min;

3）
42
℃
 water bath 90s, quickly transferred to the ice for 2min;

4）
In the
 clean bench: add 100ul LB liquid medium
into the EP tube and mix with the bacterial solution;

5）
Apply the bacterial solution to the LB plate containing antibiotics, let the
bacterial solution dry, and then invert

Overnight at 37
℃
 (about 16h)

Ps：
A total of 4 samples were transformed: ①wuhan- pET-28a, ② Delta-pET-28a, ③
BQ.1.1-pET-28a, ④XBB.1.5- pET-28a
 ; Coating on 4 Kana/LB plates (4
plates required)
,
Overnight culture at
3.37
℃

**Part 2   Protein expression and purification**

1.
**IPTG inducible expression**

Apparatus: oscillations incubator , Centrifuge

When OD=0.6~0.8 (visual
turbidity), add the final concentration in the medium as 0, 0.1, 0.25, 0.5,
0.75, 1, 2mM
IPTG,

One group was cultured at
37
℃
 200 rpm for about 3h
.

Two groups were cultured
overnight at 16
℃
 at 200 rpm
.

(
 After induction: 10000g/2min centrifuge collection of bacteria
(packed with 50ml centrifuge tube), 10 times the volume (15ml)

The 50 mM Tris-HCl buffer (pH 8.0) is suspended (eventually combined into 7 tubes)
and temporarily stored in a 4
℃
refrigerator.)

**2. SDS-PAGE**

1) 15%
separation gel

|  |  |
| --- | --- |
| D istilled water | 2.4mL |
| 30% Acr-Bis(29:1) | 5.0mL |
| Gel buffer A | 2.5mL |
| 10% APS | 0.1mL |
| TEMED | 0.006mL |

A layer of isopropyl alcohol is
coated on the lower gel solution to keep the gel surface flat

2) 5% concentrating gel

|  |  |
| --- | --- |
| D istilled water | 1.0mL |
| 30% Acr-Bis(29:1) | 0.5mL |
| Gel buffer B | 1.5mL |
| 10% APS | 0.03mL |
| TEMED | 0.003mL |

3) Electrophoresis

Electrophoretic buffer:
Tris-Glycine-SDS buffer

Loading quantity: small hole
10ul/ large hole 30-40UL

Top glue 80V, 15min

Glue layer 120V,
45min

**3.****C****oomassie brilliant blue**

1)
Dyeing: Use directly with Koomas
Bright Blue -R250 dyeing solution

2)
D
estainin
g solution
:

|  |  |
| --- | --- |
| Ethanol | 400mL |
| Acetic acid | 100mL |
| Distilled water | 500mL |

2)
Dyeing: Soak in petri dish for
30min

Decoloration: Wash with water
first, and then use the
destaining
solution to decolorize
overnight.

3.
**Extend Culture**

Apparatus: : oscillations incubator

10ml of E. coli BL(DE3) cultured overnight were
added to 500 mL of LB with kana resistant liquid medium at
37
℃
 at 200 rpm
c
ulture for about 4h.

Added 312.5ul IPTG

4.
**Ultrasonic crushing----Collecting protein**

Apparatus
：
S
onicator

1)
Collection of inclusion body:
ultrasonic crushing on ice, power 500W (40%), on 3s off 3s, total duration: 10min. Centrifuge at
8000 rpm at 4
℃
 for 30 min and remove the precipitate (inclusion body).

2
)
 Us
ing
 10 times the volume
(15-20ml) of the washing buffer(50 mM Tris-HCl, pH 8.0, 1 M NaCl, 2 M

Urea) wash the inclusion body
twice. Centrifuge at 8000 rpm at 4
℃
 for 10min
.

3
)
 Dissolved inclusion body:
Dissolved buffer (50 mM Tris-HCl buffer, pH 8.0, 1 mM EDTA
,
15 mM DTT, 6 M GuHCl guanidine
hydrochloride)was added to the inclusion body.
A
fter the inclusion body is
completely or mostly dissolve
d
, centrifuge
(5000g/5min/4
℃
) to obtain supernatant.

4. The concentration of
superalbumin was determined by A280, and the concentration of superalbumin was adjusted to 0.5 mg/mL
with dissolved buffer.

**6. Dialyze**

1
)
Configure the solution

① 2% (W/V) sodium bicarbonate and 1 mmol/L EDTA-2Na (pH=8.0)
:

Weigh
1g sodium bicarbonate is added
water
to the beaker for dissolv
ing
, then
weigh
0.168g EDTA, and finally the
volume was set to 500ml;

② 500ml of 1 mmol/L EDTA-2Na (pH=8.0)
:

W
eigh
0.168g of EDTA, fixed volume to
500 mL.

2
)
 Take a dialysis bag of the
required length for the experiment, with 2% (W/V) sodium bicarbonate and 1 mmol/L EDTA-2Na in 500 mL
(pH=8.0)
.
Boil the dialysis bag for 10
min. After thoroughly cleaning with distilled water, place at 500 mL 1 mmol/L EDTA-2Na
(pH=8.0)
boil
for 10 min. After removal, the
dialysis bag was thoroughly cleaned with distilled water.

**7. Renaturation**

1
)
The inclusion body solution was
transferred to the dialysis bag and dialysis was performed at 4
℃
 with 10
X
 volume (300ml) of retaliated
buffe
r for
4h.

2) Replace the fresh dialysate
for the second dialysis for 4h, and put the
one last dialysate
in the refrigerator at
4
℃
 for overnight dialysis.

**8. Ni-NTA Purification**

1
)
Use a
pipette
 to suck out the
renaturated inclusion body from the dialysis bag

2
)
 Balance column: Use 5
times nickel column volume (5ml) balance buffer to wash nickel column

3
)
 Pass the column: add the
final concentration of 5mM imidazole (1ml-10UL of 500mM mi) to the inclusion body solution after
refolding
. T
he flow fluid was collected to
detect the ability of protein binding to the nickel column (
collect
sampl
e 1
).

4
)
 Washing: Wash the nickel
column using a
B
alance
B
uffer of 10 times the nickel
column volume (10ml) (collect sample 2)

5
)
 Wash: Wash the nickel
using a
W
ash
ing
 Buffer (
B
alance
B
uffer + 20 mM imidazole) of 3
times the nickel column volume (3 mL) and collect the flow fluid (collect sample 3)

6) Elution: Elution
B
uffer (
B
alance
B
uffer + 200 mM imidazole) with 5
times nickel column volume (5ml) is used to elute
protein. C
ollect eluent into EP tube.
(Collect sample 4)

7
)
A280 was used to determine
protein concentration (BCA quantification), and SDS-PAGE was used to detect protein purity

**Part 3   Protein expression and purification**

**1. RBD coating ELISA high binding plate**

The purified RBD protein was diluted with PBS to 0,0.0064, 0.032, 0.16, 0.8, 4, 20,
100, 500 ng/mL, 100µL per well, three multiple
w
ells per sample, incubation for 2 hours at 37
℃
(this step is done in advance to the day before, overnight incubation is used)

**2****. ELISA test**

1)
Wash: PBST (PBS-0.05% Tween/ 0.5mL), 350µl per well, wash three times (add the
solution, then pour it out, use absorbent paper inverted suction)

2)
Closure: 5%BSA dissolved in PBS, 100 µL per well, sealed at 37℃ for 2 hours (Do not
replace the sealing solution with skim milk powder, skim milk powder contains Biotin that will produce
high background)

3)
Wash: PBST, 350 µl per well, washed three times

4)
Incubate: Biotinylated-ACE2 (1mg/mL)1:2000 Diluted with PBST, 100 µL per well, and
incubated at 37℃ for 2h

5)
Wash: PBST, 350µL per well, wash three times

6)
Incubate: Streptavidin-HRP (1mg/mL) 1:2000 diluted with PBST, 100 µL per well, and
incubated at 37℃ for 1h

7)
Wash: PBST, 350 µl per well, wash five times

8)
Color rendering:TMB substrate solution, 100 µL per well, 10 min at room temperature
(time needs to be observed, and add when the substrate begins to turn yellow when viewed by the naked
eye end with concentrated sulfuric acid)

9)
Termination: 0.5 M H2SO4/termination solution, 100 µ L per well, detected within 15
minutes

**3****.****OD detection**

OD450 and OD630

**Appendix**

Apparatus: Electronic balance

1.
**IPTG**

After dissolving 1g IPTG in 4ml distilled water, it is filled with distilled water to 5ml
and filtered with a disposable needle sterilize, then divide into 1ml small portions and store at -20℃.
(Concentration 0.8mol/l)

2.
**Tris-HCl (500m****L****)**

Dilute 1M Tris-HCl solution at pH 8.0 (Take 50ml of 1M Tris-HCl and add 950ml
water)

3.
**Wash buffer (200m****L****)**

|  |  |
| --- | --- |
| Tris | 1.211g |
| NaCl | 11.7g |
| Urea | 24g |

4.
**Solution buffer (200m****L****)**

|  |  |
| --- | --- |
| Tris | 1.211g |
| EDTA-2Na | 0.067g |
| DTT | 0.46g |
| GuHCl | 114.636g |

5.
**Retaliating buffer****(1L)**

|  |  |
| --- | --- |
| Glycerol | 100ml |
| PB (0.2M) | 100ml |
| EDTA-2Na | 0.06g |
| l-arginine | 87.1g |
| GSH | 0.55g |
| GSSG | 0.55g |
| Urea | 120g |

6.
**PB (200m****L****, 0.2M)**

2.4g NaH2PO4 dissolved in 100ml water 5.68g

Na2HPO4 is dissolved in 200ml of water

Take 38ml of NaH2PO4, take 162ml of Na2HPO4 and mix

7.
**Balance buffer (500m****L****)**

|  |  |
| --- | --- |
| Glycerol | 50ml |
| PB | 50ml |
| Urea | 30g |
| Imidazole | 0.17g |

8.
**Imidazole mother liquor (50m****L****, 500mM)**

Dissolve 1.7g of imidazole in water and fill to 50ml

9.
**Nickel column washing buffer**

Add an additional 4ml of 500mM of imidazole mother liquid to the 100ml balance
buffer

10.
**Elution buffer**

Add an additional 33ml of 500mM of imidazole mother liquid to the 50ml balance
buffer

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/4861/wiki/results.png)

**Results**

# **Overview**

Our primary aim is to design a recombinant protein vaccine. In the
last
few days, we obtained four recombinant proteins targeting the four types of COVID-19 virus: Wuhan,
Delta, BQ1.1, and xBB1.5. Our experiment could be separated into three parts:

1. Plasmids construction

2. Protein expression and purification

3. ELISA test

![](https://static.igem.wiki/teams/4861/wiki/engineering-success/enginnering01.png)

We inserted the target fragment into the plasmid vector pET28a
through
enzyme cleavage and ligation. By cultivating in large quantities within E. coli BL21, we obtained a bacterial culture containing the target protein.
Finally, the target protein was extracted and purified using a nickel column, which gives us the pure
target
proteins. In order to test the validity of the proteins, we test it with ELISA.

# **Experiment** **Results**

## **Step 1: Plasmid Construction**

In the initial phase, we began with extracting the pET28a plasmid,
which served as the foundational framework. Following this, we conducted double enzyme cuts on both the
blank plasmid and the gene segments of four viral strains – Wuhan, Delta, BQ.1.1, and XBB.1.5 – using
NcoI and XhoI enzymes. After these cuts, we merged the target fragments from four viral strains with the
pET28a plasmid using T4 DNA ligase. This intricate process transported the target fragments onto the
blank plasmid.

Subsequently, we transferred the constructed plasmids into DH5α
cells
and subjected them to overnight cultivation in a culture medium. The objective behind this step was to
foster a vast yield of the constructed plasmids through DH5α cloning. The cultured DH5
α
 are shown in Figure 1

![](https://static.igem.wiki/teams/4861/wiki/results/1.png)

Figure
 1. LB medium
of
 DH5α for overnight culture

Following
 that, we isolated the plasmids from the well-cultivated DH5α
cells and subjected them to a second round of enzyme cuts, followed by gel electrophoresis to confirm
the success of our plasmid construction. As depicted in figure 2
.1
, the gel electrophoresis results displayed two distinct bands at
approximately 6000 base pairs and 760 base pairs. These bands corresponded respectively to the pET28a
plasmid and the viral target segments. This visual confirmation signifies the achievement of our plasmid
construction endeavor and sets the stage for the subsequent protein expression phase.

![](https://static.igem.wiki/teams/4861/wiki/results/2.png)

Figure 2
.1
 gel electrophoresis results

Enzyme digestion validation showed that we have got the correct
recombinant plasmid. Subsequently, for further confirmation, we sent the recombinant plasmid to a
biological company (Azenta) for sequencing, and the sequencing results are shown in the
figure 2.2. This visual confirmation signifies the achievement of our plasmid
construction endeavor and sets the stage for the subsequent protein expression phase.

![](https://static.igem.wiki/teams/4861/wiki/results/2-2.png)

Figure
2
.
2
. Sequencing results

## **Step 2: Protein Expression and Purification**

With the success of our plasmid construction confirmed in the
previous
phase, we moved forward to express and purify the proteins. As DH5α lacks the ability to express
proteins, we transferred the constructed plasmids into BL21 bacteria for protein expression. Following
an overnight cultivation (as shown in figure 3), we proceeded to the next steps.

![](https://static.igem.wiki/teams/4861/wiki/results/3.png)

Figure 3. LB medium for BL21 overnight culture

**Inducing Protein Expression**

We selected specific bacterial colonies and initiated
single
-
clone cultivation. We added a molecule called IPTG to encourage
these
bacteria to produce the protein. To figure out the best conditions, we carried out some
rigorous
experiment
s
. We played around with two variables: temperature and the
concentration of IPTG. Here's what our exploration looked like:

![](https://static.igem.wiki/teams/4861/wiki/results/1.jpg)

U
nfortunately, there was a little hiccup, and we lost some data for
the
37°C, 0.75mM group. Nevertheless, we moved on. After inducing the BL21 E. coli to express the
protein, we went through ultrasonic disruption and centrifugation. Then, we ran a protein gel (figure 4)
and used equipment to quantify our results (figure 5).

![](https://static.igem.wiki/teams/4861/wiki/results/4.png)

F
igure 4.
SDS-PAGE
gel results

![](https://static.igem.wiki/teams/4861/wiki/results/5.jpg)

F
igure 5. Protein concentration of each group

**Extended culture**

Using the results from our modeling analysis, we decided to go with
conditions involving 37°C and an IPTG concentration of 0.5mM for scaling up the culture. After
expanding the cultivation, we once again went through the steps of ultrasonic disruption and
centrifugation.

Consulting the scientific literature, we learned that the COVID-19
RBD
protein is an inclusion body protein. In simple terms, it's like a protein
cluster
that forms inactive solid particles within the cells. This can be seen in
figure
 6 with the red circle.

![](https://static.igem.wiki/teams/4861/wiki/results/6.png)

F
igure 6. inclusion body protein

**Renewing the Protein's
Structure**

Since our protein was in inclusion bodies, we went the extra mile
for
refolding. We used something called dialysis bags to gradually lower the concentration of an external
solution. This helped remove the denaturing agents and allowed the inclusion bodies to regain their
natural structure.

**Purification with a Nickel
Column**

For the next step, we went for purification using a nickel column.
Due
to a special "His" tag on the pET28a plasmid, our protein had a soft spot for nickel. After a couple of
thorough washes, we eluted the purified RBD protein from the nickel column.

**Final Measurements**

Wrapping things up, we measured the concentration using A280
readings.
Here's what we found for each strain – Wuhan, Delta, BQ1.1, and XBB1.5: 0.001mg/ml, 0.007mg/ml,
0.012mg/ml, and 0.001mg/ml respectively. These measurements set the stage for calculating the dilution
gradient for ELISA testing.

## **ELISA****test**

Having obtained the purified protein, we proceeded to a crucial
phase:
the ELISA test. Our objective was to scrutinize the interaction between the RBD protein and the human
ACE2 protein, a pivotal step in assessing the potential of our vaccine. This phase held the key to
unveiling whether our project was on the right track.

In a meticulous sequence, we finished the ELISA test within a
high-binding plate. Sequentially, we introduced the components – RBD protein, Biotinylated-ACE2,
Streptavidin-HRP, and the TMB substrate solution. As this chemical symphony unfolded, a discernible
change in solution color emerged, signifying the culmination of our experiment and its intrinsic
success.

![](https://static.igem.wiki/teams/4861/wiki/results/7.png)

Figure 7. Changing color in solution on the high-binging plate

Moving forward, we quantified our success by measuring the OD450
values. By subtracting the values of the control group (0mg/ml), we accentuated the true essence of the
experiment. The graphical representation of this processed data
in figure 8
not only shows the trend but also clarifies the impact of the
interaction.

![](https://static.igem.wiki/teams/4861/wiki/results/8.png)

Figure
8
.
Curves of the OD450 values of each group against the concentration of RBD

**A Definitive Achievement**

To encapsulate our findings, our endeavors bore a definitive
revelation. We not only successfully produced the RBD protein but also conclusively demonstrated its
binding affinity with the human ACE2 protein across the Wuhan, Delta, BQ1.1, and XBB1.5 strains. This
discovery holds profound implications, illuminating a potential avenue towards the development of a
vaccine.

# **Future****Plan**

While we acknowledge that our current capabilities and time
constraints
may have introduced a degree of experimental error, we aspire to advance our research in the future with
a sharper focus on precision. We aim to mitigate experimental inaccuracies by employing stricter
protocols and more sophisticated laboratory equipment. This pursuit is driven by our commitment to
enhancing the quality of experimental results, increasing protein yield, and elevating purity
levels.

Furthermore, we are acutely aware that vaccine development is an
arduous and protracted process, and our current work represents just a fraction of this extensive
journey. In the subsequent phases of our research, we intend to extend our efforts towards manufacturing
subunit vaccines, gradually progressing to safety testing in animals. Ultimately, our goal is to develop
a recombinant protein vaccine for COVID-19, bolstering humanity's defenses against this
virus.

Lastly, our experiments have illuminated the advantages of
recombinant
protein vaccines. These vaccines boast formidable safety profiles, cost-effectiveness, and the capacity
to induce humoral immunity while also activating cellular immunity. As a result, we envision that
recombinant protein vaccines may emerge as a versatile defense against a spectrum of infectious diseases
in the future.

In summary, our current work represents a stepping stone towards a
broader and more impactful journey. With a commitment to precision, a clear vision of the future, and an
appreciation for the versatility of recombinant protein vaccines, we remain steadfast in our pursuit of
safer and more effective defenses against infectious diseases. Our iGEM journey is a reflection of our
shared commitment to discovery and growth.

{% endblock %}