iGEM 2023 — Team scie-pearldelta

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**Dry Team
Members:** pure
dry team members are Sheldon and Jerry. All other members from Wet Team also took over dry lab tasks.

**Wet Team
Members:** Joe,
John, Lyra, Clinton, Alan, Arnold, Lili, Jeff, Ulrica, Ivy, Emily, Osben, Anna

**Art
team:** Ulrica,
Ivy, Emily, Lyra, Lili, Anna

**Timeline**

April 6th to
April 21st:
Recruitment of members (All
team).

This
time is to find people of the same passion working together as a cohesive group and to proceed as a
compact (All
team).

April
24th to
May 12th:
Brainstorming and reading on regarding fields, deciding project aim is to design a POC strategy to detect
Cerebrospinal Fluid Rhinorrhoea. (All
team)

May
15th to
June 2nd :

Listing
out activities that can be done. (All
team)

Designing
of experiment protocol, contacting with experts to do Human Practise activities, contacting with
communities and school to carry out Education Activites.(Wet Team)

June
26th to
July 6th, Intensive Work in Shanghai:

WetLab---Production
of bTF-mCherry protein, production of Siglec-1

DryLab:
Interviews with experts, set up of information platforms e.g. WeChat Platfroms and video Channels, field
trips visiting Zhongshan Hospital and EMEH, Busineess plan 1st draft, Questionnaire release and
analysis.

July
14th:
Synbiogen visit (Clinton, Jeff, Joe, John)

July
16th: Group
meeting in Guangzhou Medical College  (John)

July
16th to
August 3rd:
Promotional Video filming, planning of Experiment in Shenzhen

August
4th to
September 6th:
Producing materials for Education programmes, e.g. boardgames cards (Art
Team)

August
21st:
Extracirricular acitivities fair (Joe,
John, Clinton, Lili, Osben)

September
12th:
Neuoscience and physiology club Extracirricular acitivity (Lyra)

September
16th to
17th,
23rd to
24th:
Experiment in Shenzhen, testing our test kit design. (Joe,
John, Lyra, Clinton, Alan, Ivy, Jeff)

September
21st onwards,
every Thursday: Education of iGEM extracirricular activities programmes (John, Joe)

September
22nd:
Wiki Building (Dry
Team)

September
27th:
Education in Assembly (Mini-Drama:
John, Joe; Speech: Lili, Lyra)

September
14th to
September 28th:
Release of interactive posters in school

October
2nd:
Final draft of business plan (John,
Clinton)

October
4th:
Final draft for presentation video completed

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Overview

In this project, we aim to
provide a test kit that detect beta-2-Transferrin, which is the specific biomarker that only present in
Cerebrospinal fluid (CSF). However, sialo Transferrin (sTF) is a major component that present in both
serum and CSF. The minor differences in the Transferrin (TF) based glycol chains make it difficult to
distinguish from sTF, which may lead to sTF binding with anti-TF antibody to create a false positive
result.

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**PCR
amplification**

|  |  |
| --- | --- |
| Reagent | Volume (Unit: μL) |
| Mixer (2 x buffer) | 25 |
| Template (targeted genes: Siglec-1/ beta-2-Transferrin/ mCherry) | 2 |
| Primer (F) | 1 |
| Primer (R) | 1 |
| ddH2O | 21 |

**Procedure**

1. Add
mixer, ddH2O,
targeted genes and primers respectively (with the volume denoted above) to the 0.2 mL PCR tube using a
micropipette. Label the top of the tube with “Siglec-1” which stands for Siglec-1 genes.
“bTF” stands for the beta-2-Transferrin genes. “mCherry” stands for mCherry
genes.

2. Put
the tube into the PCR machine/thermo cycler, using the following data.

|  |  |  |
| --- | --- | --- |
| Name of Process | Temperature (Unit: ℃) | Time (Unit: s) |
| Denaturation | 95 | 30 |
| Annealing | 66 | 30 |
| Extention | 72 | 120 |

Number
of cycles: 30

**Bacteria
cultivation**

|  |  |
| --- | --- |
| Reagent | Volume (Unit: μL) |
| LB | 4,000 |
| Bacteria sample | 20 |
| Antibiotics (Kanamycin) | 4 |

**Composition of LB in 1 L
ddH****2****O**

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

LB
then undergoes high temperature sterilisation.

**Agarose****Gel
preparation**

|  |  |
| --- | --- |
| Reagent | Mass/Volume |
| Agarose | 1 g |
| TAE Buffer | 100 mL |

**Gel
electrophoresis**

1. After
the reagents above are added, use a microwave oven to heat the solution and cool the solution. Then add 5
μL of nucleic acid dye (add it before the solution solidifies).

2. Pour
the gel into the mould. Insert the mould to form the wells on the gel where DNA samples can be injected.
Wait until it is solidified.

3.
3 μL of Loading buffer is added to each sample of DNA. Carefully using a micropipette, add DNA marker
on the first hole, and then add all the DNA sample into the gel with the corresponding order (noting down
the order is recommended).

4.
Start the cell. Wait for 15 minutes.

5.
Using ultraviolet. Take the photo of the result.

**Gel****Extraction**

1. Cut
the gel with the DNA strands off.

2. Weight
the mass of the gel.

3. Add
buffer B2 (3 times the mass of the sampled gel).

4. Add
the gel and the solution into the absorption column. Centrifuge for 30 s (8,000 x g), remove the liquid
collected at the bottom.

5. Add
50 μL
of wash solution to the absorption column. Centrifuge for 30 s (9,000 x g), remove the liquid collected at
the bottom.

6. Repeat
step 5.

7. Centrifuge
the empty absorption column for one minute (9,000 x g).

8.
Put the absorption column in a clean 1.5 mL centrifuge tube. Add 15 μL
of elution buffer. Stand for 1 minutes at room temperature. Centrifuge for 1 minute (7,000 x g). Remove
the absorption column and keep the DNA solution in the centrifuge tube. Label it with the corresponding
DNA (Siglec-1/bTF-mCherry).

**Plasmid
Extraction**

1. Put
the absorption column in the collection tube. Then add 500 μL of buffer S (into the absorption column).
Centrifuge for 1 minute (12,000 x g) and remove the liquid in the collection tube.

2. Add
1.5 mL of solution containing bacteria to the collection tube. Centrifuge for 2 minutes (8,000 x
g).

3. Add
250 μL of buffer SP1 to suspend the bacteria.

4. Add
250 μL of buffer SP2 (a strong base). Shake the tube upside down gently for 10 times, then stand the
tube for 2 minutes.

5. Add
350 μL of buffer SP3. Gently shake the tube upside down for 10 times.

6. Centrifuge
for 5 minutes (12,000 x g). Move the supernatant into the absorption column (inserted into the collection
tube). Centrifuge again for 30 s (8,000 x g). Remove the liquid in the collection tube.

7. Add
500 μL of Wash Solution. Centrifuge for 30 s (9,000 x g). Remove the liquid in the collection tube.

8. Repeat
step 7 and then centrifuge the empty absorption column for one minute (9,000 x g).

9. Put
the absorption column in a clean centrifuge tube (volume of 1.5 mL). Add 50 μL of Elution
buffer. Stands it for one minute, and then centrifuge it for one minute (7,000 x g). Keep the solution
containing DNA in the centrifuge tube and label it with “pET28a” (name of the plasmid we
used).

**Double****E****nzyme****Digestion**

|  |  |
| --- | --- |
| Reagent | Volume (Unit: μL) |
| Targeted genes (Siglec-1/bTF/mCherry) | 1 |
| Restriction endonuclease enzyme 1 (Nhe 1 for Siglec-1 and bTF, Xho 1 for mCherry) | 1 |
| Restriction endonuclease enzyme 2 (Sac 1 for Siglec-1, BamH for bTF and mCherry | 1 |
| CutSmart buffer | 5 |
| ddH2O | 42 |

Overnight
enzyme digestion at 37 ℃

**Ligation**

|  |  |
| --- | --- |
| Reagent | Volume (Unit: μL) |
| T4 ligase buffer | 2 |
| T4 ligase | 1 |
| Genes | 1 |
| Vector | 3 |
| ddH2O | 13 |

**Transformation**

1. Carefully
add the recombinant
plasmids
to the E.coli (DH5α/BL21)
in
the tube at the Super clean bench.

2. After
sealing the tube. Put the tube in an ice bath for 30 minutes.

3. Put
the tube on a water bath (42 ℃)
for 90 s.

4. Put
the tube above the ice bath for 5 minutes.

5. Add
LB culture medium (add until there is a total of 1 mL of solution in the tube).

6.
Put the tubes in the shaker (220 rpm, 37 ℃)
for 1 hour.

7.
Centrifuge
the tubes (9,000 turns per minute), and then absorb 950 mL of the LB solution (at the super clean
bench).

8.
Carefully
and gently mix the bacteria with the solution. Smear the bacteria on the growth medium.

9.
Put
the petri dish in the incubator (37 ℃) overnight.

**IPTG
induction**

1. Add
antibiotics (kanamycin), E. coli to the culture medium (with the given ratio).

2. Shake
the mixture for 3-4 hours until
OD600 reaches
0.6.

3. Add
IPTG to achieve a final concentration of 0.25mM and incubate overnight in a 18℃ shaker.

**Nickel Column
Purification**

His-A
solution composition (0.5 L, pH = 7.4)

|  |  |
| --- | --- |
| Reagent | mass |
| Na2HPO4 | 20 mM (1.78 g) |
| NaCl | 500 mM (14.6 g) |
| Imidazole | 20 mM (1.02 g) |

His-B
solution composition (0.25 L, pH=7.4)

|  |  |
| --- | --- |
| Reagent | Mass |
| Na2HPO4 | 20 mM (0.89 g) |
| NaCl | 500 mM (7.3 g) |
| Imidazole | 500 mM (8.5 g) |

\*The
sample (proteins) need to be kept in the ice box

Procedure

1. Add
10 mL of His A solution to wash the nickel column.

2. Add
10 mL of solution (proteins), filter it. Label the 10 mL collection tube with
“through.”

3. Add
10 mL His
A solution, filter it. Use the 10 mL filter pipe to collect the solution and label
“wash.”

4. Add
1 mL His B. Close the lids at the bottom of the nickel column. Shake the column and then
collect the solution with a 2 mL EP tube.

5. Add
1 mL of His B solution again. Collect the solution filtered to the 2 mL
tube (from step 4)

6. Add
5 mL of His B solution to wash the nickel column.

7. Add
5 mL of His A solution to wash the nickel column.

8.
Add 10 mL of methyl alcohol solution to wash the nickel column.

**SDS-PAGE
electrophoresis**

1. Insert
the prepared gel (12% BT) into the electrophoresis chamber.

2. Add
running buffer until the chamber is submerged.

3. Mix
the protein sample and the loading buffer.

4. Add
1.5 μL
of the marker to the first hole of the gel. Add 10 μL of protein sample in the following holes.

5. Set
up the electrophoresis program.

Step
1: 80 V, 30 minutes

Step
2: 120V, 60 minutes

6. Start
the program.

**Coomassie
Brilliant Blue staining and destaining**

1. Prepare
a Coomassie Brilliant Blue staining solution by mixing an appropriate volume of Coomassie Brilliant Blue
dye with methanol and acetic acid and water. The final staining solution contains 40% methanol, 10% acetic
acid.

2. Carefully
transfer the gel into a container filled with the staining solution. Gently shake the container or use a
rocking platform to ensure even staining.

3. Incubate
the gel in the staining solution for 1 hours.

4. Prepare
a destaining solution by mixing an appropriate volume of methanol, acetic acid, and water. The exact
composition may vary based on the destaining kit used.

5. Transfer
the gel into a container filled with the destaining solution.

6. Gently
shake the container or use a rocking platform to facilitate destaining.

7. Replace
the destaining solution every 30 minutes until satisfactory background clarity is achieved. This may take
several hours or overnight.

**Evaluation of Deletion
Chamber**

Deletion
Chamber Production and Usage

**Making Nickel beads for
deletion chambers**

Materials:
Nickel beads, PBS, sTF and bTF solution at 100 ng/mL and 10 ng/mL respectively.

Steps:

1. 262.5
μL Siglec-1 (10 μg/mL) is added to solution of Nickel beads

2. Wash
the Nickel beads 3 times with 1 x PBS solution. The removal of supernatant is by centrifugal of 50
x g
for 30 s.

3. Total
of 525 μL of overnight cultured Nickel beads were centrifuged at 50 x g for 30 s. PBS was used to wash
the beads three times.

4. After
the final wash, the beads were diluted in approximately 340 μL of 1x PBS solution (expected total
volume of about 350 μL) and shaken well.

5. The
magnetic beads were divided into 11 EP tubes, and each tube was filled with 90 μL of 1x PBS. Add 20
μL Nickel beads after thorough shaking.

A
total of 11 Deletion Chambers (DC) were made, each with 110 μL Nickel beads in PBS.

**Preparation of the solution
concentration in following experiment**

1. Prepared
700 μL sTF solution (1000 ng/mL, 100 ng/mL, 10 ng/mL, 1 ng/mL, 0.1 ng/mL) respectively, which will be
used ELISA experiment.

2. Prepared
500 μL 100 ng/mL sTF solution and 500 μL 10 ng/mL bTF solution.

3. Discharged
and mix 250 μL 100 ng/mL sTF solution with 250 μL 10 ng/mL bTF solution.

**Test the Function of****Deletion Chamber**

1. Add
190 μL of sample to the tube with 10 μL centrifuged Nickel beads, and there is totally 200 μL
solution in tube.

2. Wait
for 30 minutes for deletion to perform.

3. For
ELISA, transfer 100 μL of solution. For the same concentration solution that used colloidal gold (AuNP)
experiment, transfer the rest of 90 μL of solution.

Totally,
8 solutions are passed through Deletion Chamber (DC), which includes:

sTF:
1000 ng/mL, 100 ng/mL, 10 ng/mL, 1 ng/mL, 0.1 ng/mL

bTF:
10 ng/mL (Elisa + AuNP)

sTF:
100 ng/mL (Elisa + AuNP)

sTF
+ bTF: 100+10 ng/mL (Elisa + AuNP)

**ELISA standard
curve**

Reagent
consumables: ELISA kits, sTF, Nickel beads.

1.
Samples of sTF concentration is set in maximum of 1,000 ng/mL is because the ELISA assay can only provide
result in range 0 nmol/L to 1.5 nmol/L of Transferrin.

2.
Diluent for sTF is exactly the same with bTF, provided by the ELISA producer.

Sample
preparation for ELISA evaluation：

B-samples
containing bTF; S-samples containing sTF

450
μL or 500 μL solution for sTF for ELISA and further experiments, i.e. colloidal gold
experiment.

1. Make
1 mL of standard solution of sTF (2 mg/mL), take 250 μL of this solution, dilute it to 1,000 ng/mL to a
total volume of 500 μL by adding diluent of 250 μL. Label it as S1.

2. S2
consists of 450 μL diluent and 50 μL S1 to obtain 100 ng/mL solution.

3. Repeat
step 3 for making S3-S5 and then by Deletion
Chamber.
The solution sTF concentration is as following:

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| Tube: sTF | S1 | S2 | S3 | S4 | S5 |
| ng/mL | 0.1 | 1 | 10 | 100 | 1000 |

The
dilution process for the standard curve goes as following, to obtain bTF concentration in

|  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Tube: bTF | B 1 | B2 | B3 | B4 | B5 | B6 | B7 |
| nmol/L | 0 | 0.047 | 0.094 | 0.187 | 0.375 | 0.75 | 1.5 |

The
Standard solution contains concentration of B1. Transfer 300 μL of the solution to S1. Use 150 μL
from B1 to B2 and add 150 μL diluent. Repeat this procedure until B6, where the procedure is x2
dilution each time. B7 is empty, consisting solely diluent.

The
test samples named sTF, sTF + DC (which through the Deletion Chamber), bTF, bTF + DC

ELISA
(Enzyme Linked Immunosorbent Assay)

1. Prepare
the micro-plate

Plate
1:

|  |  |
| --- | --- |
| B1 | sTF |
| B2 | bTF |
| B3 |  |
| B4 |  |
| B5 |  |
| B6 |  |
| B7 |  |
|  |  |

Plate
2:

|  |  |
| --- | --- |
| B1 | S1 |
| B2 | S2 |
| B3 | S3 |
| B4 | S4 |
| B5 | S5 |
| B6 |  |
| B7 |  |
|  |  |

Plate
3:

|  |  |
| --- | --- |
| B1 | bTF |
| B2 | sTF |
| B3 | bTF + DC |
| B4 | sTF + DC |
| B5 |  |
| B6 |  |
| B7 |  |
|  |  |

2. Add
100 μL of solution to the well in the order stated on the diagram above. Cover the plate with adhesive
membrane. Incubate it at 37 ℃ for
60 minutes. Remove the liquid and add 200 μL of wash buffer. Wait for 2 minutes, and then wash off the
liquid.

3. Repeat
the above step for 2 times. Invert the ELISA plate on the filter paper. Tap the plate gently to remove the
remaining liquid.

4. Add
100 μL of HRP-conjugate (1x) to each well. Cover the plate with adhesive
membrane. Incubate
the plate at 37 ℃ for
60 minutes.

5. Remove
the liquid and add 200 μL of wash buffer. Wait for 2 minutes, and then wash off the liquid. Repeat the
washing step stated previously for 4 times.

6. Add
90 μL of TMB (the chromogenic
substrate adopted
in this experiment). Incubate the plate at 37 ℃ for
20 minutes in a darkroom.

7. Add
50 μL of stop solution to each cell to terminate the reaction. Tap the plate gently to ensure that it
is mix adequately.

8. Within
5 minutes. Measure the plate at a wavelength of 450 nm.

**Evaluation of the Test
Strip with Deletion Chamber**

Colloidal gold-antibody evaluation

Materials:

Linked
streptavidin-gold nanoparticles (referred to as streptavidin-AuNP in the content below),
1 x PBS, 0.1 M
borate buffer solution, silk protein
saver,
bTF
solution, Tween 20, TX 100, anti-TF antibody (CSB-PA00250H0Rb) conjugated by biotin (referred to as
sandwich secondary antibody below).

Preparation of reagents:

Adapter
solution: Prepare 1 mL of adapter solution A by adding 10 mg of silk
protein
saver,
4 mg
of
Tween 20, and 50 mg of TX 100.

10
mM borate buffer solution: Prepare 10 mM borate buffer solution by dissolving 10 mL of 0.1 M borate buffer
in 90 mL of ddH2O.

Protein
protectant: Prepare silk protein saver at a concentration of 50 mg/mL by dissolving 1 g in 20 mL.

**Coupling of Colloidal Gold and
Antibodies**

Purpose
of the experiment: preparation of results demonstrating bTF/sTF detection on NC membranes.

**Preparing Colloidal
Gold-Antibod****y**

Materials:
nanogold OD1 solution, 0.1 M borate buffer solution and 10 nM borate buffer solution (diluted 10 times
with 0.1 M, approximately 5 mL), protein protective agent solution (50 mg/mL dissolved in ddH2O,
200 μL), adapter solution (1 mL 1 x PBS, containing 50 mg/mL trehalose, 2 mg Tween 20, 5 mg protein
protective agent, 10 mg Triton-X 100), 1 x PBS (1 mL), anti-TF polyclonal antibody (liquid phase) (1
mg/mL, 10 μL)

Steps:

1. 1,000
μL of streptavidin-AUNP was added to an EP tube, followed by the addition of 100 μL of borate
buffer, and then 2 μL of anti-TF polyclonal antibody (1 mg/mL).

2. The
mixture was incubated at room temperature for 30 minutes, and then 100 μL of protein protective agent
was added.

3. The
mixture was incubated at 4 ℃ for
1 hour, and then centrifuged at 13,475 g for 20 minutes at 10 ℃.
Using
a pipette, remove the supernatant.

4. 1
mL of 10 nM borate buffer solution was added, and the mixture was centrifuged again at 13,475 g for 20
minutes at 10 ℃.

5. Repeat
step 4 for two times.

6. 550
μL of adapter solution was added to prepare 2x colloidal gold-antibody solution.

7. The
working concentration was 1x (550 μL of 1 x PBS was added to obtain approximately 1,100 μL of 1 x
colloidal gold-antibody solution).

**Further preparation of gold
nanoparticles**

(Performed
in a 2 mL EP tube)

1. Mix
100 μL of streptavidin-AuNP, 100 μL of borate buffer, and 10
μL
 (1 mg/mL)
solution.

2. Incubate
at 25 ℃ for
30 minutes.

3. Add
100 μg
(50 mg/mL) protein saver.

4. Incubate
at 4 ℃ for
60 minutes.

5. Centrifuge
at 13,475 g for 20 minutes at 10 ℃.

6. Resuspend
in 10 mM borate buffer solution and centrifuge again.

7. Discard
the supernatant and repeat steps 4-5 for 2 times.

8. Add
550 μL of 1 x PBS and 550 μL of adapter solution.

**Preparation of the NC
membrane Test strip**

1. Cut
six strips of nitrocellulose (NC) membrane, each measuring 5 cm in length and 1cm in width.

2. Align
and vertically add a 40 μL anti-Transferrin antibodies to each NC membrane using micropipette, 1 cm
away from the right edge.

3.
Wait until the Antibodies are drying.

**Evaluation of colloidal gold
test**

Samples
that are going to react:

sTF:
100 ng/mL

bTF:
10 ng/mL

sTF+bTF:
respectively 100 ng/mL, 10 ng/mL

All
three will have 90 μL of it untreated, 90 μL are from the three solutions that has been treated by
Deletion Chamber.

1. Mix
50 μL
colloidal gold solution, then 50 μL sample is added into the tube to make a 200 μL mixture.

2. React
in room temperature for 30 minutes.

3. When
waiting, drop 2 μL antibody (0.25 μg/mL) in a line ten times on the NC membrane.

4. Apply
the solution to 0.1 cm to the line of antibody.

5. Wait
and see whether there is an existence of color bands or not after waiting 5 minutes.

**Fluorescence tracing**

1. Prepare
your sample and mount it onto a glass slide using an appropriate mounting medium. Place the prepared slide
onto the stage of the fluorescence inverted microscope.

2. Turn
on the microscope and adjust the light source to the desired excitation wavelength of 587 nm.

3. Adjust
the focus using the coarse and fine adjustment knobs until the sample comes into view. Set the appropriate
filter cube for the excitation wavelength of 587 nm.

4. Adjust
the exposure time and gain settings on the camera to optimize image quality. Acquire the images by
activating the camera or software capture system.

5. Switch
to the emission wavelength of 610 nm by changing the appropriate filter cube.

6. Adjust
the focus and image settings accordingly for the emission wavelength. Acquire the images of the NC
membranes.

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Overview

The strategy for our
Cerebrospinal Fluid (CSF) Rhinorrheas detection consists of two essential parts. First, to avoid the false positive result from
the sample, the sialo Transferrin (sTF) in the sample needs to be removed by Siglec-1 lectin. Second, for detecting the presence of
beta-2-Transferrin (bTF), the conjugated colloidal gold nanoparticles
(AuNP)-bTF complex need to be captured by the
anti-TF antibodies immobilized on nitrocellulose (NC)
membrane. To verify the capability of CSF detection, the
following experiment is taken place.

For the first part of the
experiment, we used BL21 E. coli to synthesize the Bioactive Siglec-1 lectin-His tag. The His tag
connected with Siglec-1 lectin ensures that the lectin can bind to Nickel beads in the Deletion Chamber.
We then pass the bTF samples, and sTF samples with different
concentrations through the Deletion Chamber, and the concentration of each sample before and after the
Deletion Chamber is verified by Enzyme-Linked Immunosorbent Assay (ELISA).

For the second part of the
experiment, we also used the BL21 E.coil to synthesize bTF-mCherry complex (bTF-mC), which is used in the following NC membrane
test. First, Colloidal gold-antibody (AuNP-antibody) is
mixed with bTF-spiked solutions and sTF-spiked solutions, and
the mixed solutions are added to the test strip. The presence of the red line indicates that the
anti-TF antibody is not able to distinguish between
bTF and sTF, which substantiates the necessity
of a Deletion Chamber. Next, the bTF-mC complex synthesized from the bacteria is
spiked into to solution and mixed with the sTF respectively. After conjugated with AuNP-antibody solution, the
samples are added to the NC membrane. By using the spectrometer, the trace of
bTF on the NC membrane can be visualized due to
the connected mCherry, and we therefore prove that the presence of the red line after the Deletion Chamber
is induced by bTF, not sTF.

The result of the two parts
all shows that our Point of Care (POC) strategy for CSF Rhinorrheas test kit is essential, and
capable.

![](https://static.igem.wiki/teams/4847/wiki/results/1.png)

**Figure 1.****Function of the
AuNP and the Test Strip**

Construction of recombinant plasmids

![](https://static.igem.wiki/teams/4847/wiki/results/2.png)

**Figure****2.****Construction of****four****plasmids**

We designed 4 recombinant
plasmids: pET28a-bTF, pET28a-mCherry, pET28a-bTF-mCherry and pET28a-Siglec-1. In order to construct four recombinant
plasmids, we first amplified bTF (2094bp), mCherry (711bp), and Siglec-1 (5130bp) fragments using PCR. As shown in
Figure 2A-B, compared to the 15K maker, these three genes were successfully amplified. Through a
double
enzyme digestion, we
successfully obtained a linearized plasmid pET28a (Figure 2C), which was then
linked to bTF, mCherry, and Siglec-1 fragments, respectively. The map of the
recombinant plasmid after ligation is shown in Figure 2D-G.

![](https://static.igem.wiki/teams/4847/wiki/results/3.png)

**Figure 3. Results of
plasmid transformation, identification, and sequencing**

Next, the four plasmids were transformed into Escherichia coli BL21
(DE3), respectively. After overnight cultivation, the transformants successfully grew on LB plates (Figure
3B). We conducted colony PCR identification on the
transformants, and the results are shown in the Figure 3A and 3C. From Figure 3A, it can be seen that the
pET28a-beta-2-Transferrin and
pET28a-mCherry plasmid transformants were positive. Then we found that 9 out of 16 samples were
found to be positive in pET28a-beta-2-Transferrin-mCherry E.coli (DH5α) and only 1 positive
sample in pET28a-beta-2-Transferrin-mCherry E.coli (BL21) by PCR identification (Figure
3C). To further validate the
construction of the plasmid, we sequenced the positive transformants, and the sequencing results showed
that the gene sequences were all correct, indicating the
successful construction of the plasmid (Figure 3D).

Protein expression and purification

After centrifugation, the
bacteria samples are hypersonic-lysed for breaking the cell structures. The samples then go through filter
tubes containing nickel columns. Due to the double 6 His tag on the proteins, they bind to the nickel web
on the column. Next, the proteins are washed by His A solution, His B solution respectively. The His B
solution that washed the protein are then collected.

We purified the target protein using His tags and
validated the protein expression and purification results through SDS-PAGE. As shown in Figure 4, we successfully expressed the beta-2-Transferrin (79 kD), mCherry(30 kD), beta-2-Transferrin-mCherry(107 kD) and Siglec-1 protein (183 kD).

![](https://static.igem.wiki/teams/4847/wiki/results/4-1.png)
![](https://static.igem.wiki/teams/4847/wiki/results/4-2.png)

**Figure****4.****SDS-PAGE results of****four****protein expression and
purification**

**(P represents
“Precipitation”, S represents “Supernatant”, T represents “Flow
through”, and E represents “Eluent”)**

Deletion Chamber Evaluation

In order to deplete sTF which
is the protein that would affect the testing of the target protein bTF, a deletion chamber is constructed. Siglec-1
is immobilized with Nickel beads and its function is tested with ELISA and test strips.

The ELISA assay allows for
the detection of a specific antigen, thereby indicating the presence of a particular protein. However, it
is unfortunate that the presence of antigens in sTF and bTF cannot be distinguished by ELISA
testing as shown in figure 5. Thus, this underscores the critical importance
of utilizing the deletion chamber, as the presence of sTF would likely lead to false-positive results and
compromise the accuracy of diagnosing CSF rhinorrhea.

![](https://static.igem.wiki/teams/4847/wiki/results/n-5.png)

**Figure 5. ELISA
could use same antibody to detect sTF and
bTF**

In order to evaluate the
depletion capability of the deletion chamber on sTF concentration, solutions with varying concentrations
of sTF are utilized to assess the performance of the chamber. A concentration gradient is established
using this approach, and the result is presented in figure
6. The concentration of sTF
exhibits a substantial reduction and approached near-zero levels after undergoing reaction within the
deletion chamber. This outcome serves as compelling evidence supporting the effectiveness of the deletion
chamber in depleting sTF concentration.

![](https://static.igem.wiki/teams/4847/wiki/results/6-1.png)
![](https://static.igem.wiki/teams/4847/wiki/results/6-2.png)

**Figure 6. sTF
concentration before (x-axis) and after (y-axis)
deletion chamber**

To assess the impact of the
deletion chamber on both bTF and sTF, bTF and sTF solutions are passed through the deletion chamber
separately.  The concentration of these solutions before the deletion and after the deletion are
measured via ELISA, as shown in figure 7.

From the result in figure
7, we can see that there is
no significant change on the concentration of bTF. On the contrary, the concentration of sTF has a
substantial reduction. This confirms that bTF is barely affected by the deletion chamber.

![](https://static.igem.wiki/teams/4847/wiki/results/7-1.png)
![](https://static.igem.wiki/teams/4847/wiki/results/7-2.png)

**Figure 7.
concentration of sTF and bTF before and after (sTF+DC,
bTF+DC), respectively**

Nitrocellulose membrane test

In order to assess the
practical efficacy of the deletion chamber, a simulated test was conducted using six nitrocellulose
membranes in equal size. Anti-TF antibodies are applied to the test membranes.
Solutions containing bTF-mC, sTF, and a combination of bTF-mC and sTF are prepared, with half of each
solution being processed using the Deletion Chamber. Equal volumes of each solution are then mixed with an
AuNP solution and added to the six separate membranes prepared for testing.

According to our hypothesis,
the unprocessed solutions are expected to react with the antibodies, yielding a red band on the test
strip, whereas no reaction is anticipated for the sTF solution that has been processed. Approximately 10
minutes after the time of solution added to the membrane, the appearance of red bands confirms our
hypothesis, as depicted in the accompanying image. This experiment will provide
strong evidence on
the efficiency and
availability of our product, while also further verifying the effectiveness of the deletion
chamber.

When the antibody on the NC membrane captures
collodial gold-TF complex, the complex of colloidal gold moves
slower/doesn’t move as antibodies applied on NC membrane are immobilized. On the contrary, those
colloidal gold that does not bind with TF goes continuously. Thus, when there is a successful capture of
TF, there will be two bands visible, the solvent
front band, and the antigen band. The antigen band, i.e., the band after the band on the solvent front,
proves a successful capture of sTF by the antibody on NC membrane and on the colloidal gold. By measuring
the color intensity of the mCherry of the secondary band, we can therefore determined the cause of the band**.**

![](https://static.igem.wiki/teams/4847/wiki/results/8.png)

**Figure****8****:** **bTF****-mC****sample,** **bTF****-mC****sample with deletion
chamber****processed (bTF****-mC****+DC)**

As we can see in figure
8, there are antigen bands
on the test strip, which proves that
antibodies and colloidal gold
can be
used for visualization of the detection of bTF.
Furthermore, the spectrometer
experiment also proves that the Deletion Chamber barely affects the result
for bTF detection. Under the spectrometer, the color intensity of
mCherry presented in both of the bands are visibly similar, which proves that the appearing red line is
caused by bTF, and the Deletion Chamber has a minor
effect on the depletion of
bTF.

![](https://static.igem.wiki/teams/4847/wiki/results/9.png)

**Figure****9****: sTF sample, sTF sample****with deletion
chamber****processed**

In figure 9,
the presence of a antigen band in Strip sTF shows that sTF
can factually affect the test to cause a false positive result.

![](https://static.igem.wiki/teams/4847/wiki/results/n-10.png)

**Figure****10****: Result of the test on the bTF-mC solution
mixed with sTF after going through the deletion chamber**

Comparing to
figure 8 and 9, in figure 10 we can see that the sample bTF with sTF will result in the same antigen
band which echoes the concern of false positive results caused by sTF. After DC, sTF shall be removed
per the figure 9 result while the bTF can be still detected by presenting a positive antigen band,
therefore, we can conclude that the deletion chamber can successfully eliminate the effect from sTF in
the real context. With this conclusion, we believe our upgraded CSF Rhinorrheas detection kit has
a very promising future.

Future plan

As we have proved that the strategy works for the CSF rhinorrhoea
test kit, improvement and industrialization of making the test kit is possible.

Firstly, as we must ensure that all reactants react
(*e.g.* colloidal gold-antibody binds with bTf; sTf is immobilized by nickel beads), we might be assigned longer
times for reactants to react than what is really required. Regarding reducing manual inaccuracies, the common to immobilize
antibodies on an NC membrane is by use of a dispenser machine, which in our experiment, we find not as
necessary as using a pipettor directly applying on the NC membrane to demonstrate the success of the
experiment is enough.    Regarding the system, every system listed, e.g. the volumetric
ratio between colloidal gold solution, PBS, and sample, can be readjusted to lower the cost for a test
and to maintain, even improve the accuracy of the test.

Regarding making it into a product, we can consider assembling the
test kit into hardware, which can be achieved by simply adapting our NC membrane on absorption pads,
etc., just like a common COVID-19 Rapid Antigen Test’s non-biological components. Then, we may think about the test in the market, which must pass
through testing real clinical samples to apply a Receiver Operating Characteristic (ROC) analysis, and a
calculation of Youden's index may be required. Stringent clinical examinations and assessments then will be
carried out.

Then, as the product is proven to be acceptable to the public,
industrial large-scale production can then be considered.

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