iGEM 2024 — Team bwya

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Notebook

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Engineering

### Overview

Our project addresses the critical need for enhanced risk screening and
early detection of gynecological diseases. Early diagnosis significantly
improves treatment success rates and quality of life after treatments.
An array of biomarkers should be studied and included to tackle this challenge,
and as a proof-of-concept, we concentrated on two biomarkers, CA125 and
IL-6, which are associated with various gynecological diseases. They are
intended to be used in biosensors that utilize the antigen-antibody binding
mechanism, employing colloidal gold and lateral flow assay (LFA) technology
for a visual read-out. We believe this approach will lead to portable,
non-invasive, and thus user-friendly gynecological care products.

To learn more about the project background, please refer to our Description
[<https://2024.igem.wiki/bwya/description>]
wiki page.

### **Design**

### **Biomarker Selection**

CA125 and IL-6 were chosen based on their diagnostic relevance and the
availability of research supporting their use. CA125 and IL-6 are proteins
that, when present in abnormal concentrations, can indicate the presence
of disease.

CA125, in particular, is mostly known as an indicator of ovarian cancer,
while at the same time, its concentration shows abnormal elevations in
many gynecological diseases, such as Endometriosis, Endometrial cancer,
Adenomyosis, and etc. The diseases indicated by CA125 covers a wide range
of population group.

![Figure 1](https://static.igem.wiki/teams/5234/engineering/image1.png)

IL-6 is a common inflammation marker. In fact, gynecological inflammation
is significantly more common than cancers in female patients. Such inflammations
and infections are also hard to cure and often transform into chronicle
conditions, posing a long-term burden on females. Besides, IL-6 is not
only associated with inflammations but also indicative of various other
conditions, including some cancers.

![Figure 1](https://static.igem.wiki/teams/5234/engineering/image2.png)

**Detection**

We plan to use the lateral flow assay method for a visual read-out upon
antigen detection. This is a well-established technique mostly used in
point-of-care diagnostics. It is known for its simplicity, requiring only
a small sample and providing results within minutes.

![Figure 1](https://static.igem.wiki/teams/5234/engineering/image3.jpeg)

Figure. Principle of lateral flow assay test strips. Referenced from Aydin
et al, 2020.

The antigen-antibody binding principle is at the heart of our detection
method. When the target biomarkers, such as CA125 and IL-6, are present
in a sample, they bind to specific antibodies, triggering a visual signal
via colloidal gold particles.

### **Antibody Selection**

We selected specific CA125 and IL-6 antibodies from the literature that
have demonstrated high affinity and specificity. These antibodies are crucial
for the effects of our diagnostic tool.

For CA125 detection, we selected humanized 4H11 (h4H11) antibody against
the specific CA125 ectodomain. The ability of the 4H11 to recognize CA125
was proven experimentally by previous works in Fluorescence Activated Cell
Sorting (FACs),

CAR-T cell application, and blocking of matrigel invasion [MUC-2024] .

For IL-6 detections, in a newly published work, Biozzi mice were immunized
with IL-6 emulsified in sodium chloride and Freund's adjuvant, followed
by booster injections, and a final intraperitoneal boost. Splenocytes were
harvested and fused with mouse myeloma cells to create hybridomas, which
were screened by ELISA. Positive clones were cloned and injected into mice
for ascites production. [Celik-2023]

### **scFv Design**

Producing full-length antibodies using biomedical methods usually involve
animal agents, which incurred animal welfare concerns and raising production
cost. To address this challenge, we opted for single-chain variable fragment
(scFv) antibodies. These scFv antibodies are minimal antigen-binding fragments,
approximately 25 kDa, and they are composed of the variable domains from
both the heavy (VH) and light (VL) chains bridged by a linking sequence.
These fragments offer multiple benefits over their full-length monoclonal
counterparts. The scFv antibodies can be expressed in simple eukaryotic
organisms like yeast and prokaryotic bacteria due to their simplified architecture,
and they are not dependent on glycosylation for function. In our experiments,
we aim to express the scFv in E. coli chassis.

![Figure 1](https://static.igem.wiki/teams/5234/engineering/image4.png)

Fig. This scheme illustrates the principles of scFv design from an antibody,
referenced from Monnier et al, 2013.

### **Build**

### **Expression Optimization**

To enhance the expression of our scFv antibodies, we employed various
strategies, including codon optimization for E. coli expression. The antibody
originates from mammalian protein productions, and codons corresponding
to the designed amino acids do not reflect the best expression in E. coli.
We used the online tool TI signer (https://tisigner.com/tisigner) to optimize
the coding DNA.

![Figure 1](https://static.igem.wiki/teams/5234/engineering/image5.png)

We also composite the coding sequence with a strong T7 promoter. It is
derived from the T7 bacteriophage and is recognized by the T7 RNA polymerase,
which transcribes the gene at a much faster rate than the host cell's polymerase.
Aided with a lac operator (LacO), the expression is muted without external
induction of iPTG, which allows the E. coli to grow to the best condition
of the exponential phase. Upon induced expression, T7 promoter can facilitate
5 times heterogenous expression than generic proteins in E. coli. We chose
the BL21 strain of E. coli for its robust expression capabilities. The
pET28a expression vector was selected for its efficiency in producing recombinant
proteins under the control of the T7 promoter.

![Figure 1](https://static.igem.wiki/teams/5234/engineering/image6.png)

### **Test**

Fermentation Conditions

We selected fermentation conditions based on literature empirical conditions,
including temperature (20 C), media (common LB media) and induction timing
(OD600 0.6-0.8), and induction strength (0.3 mM). These mild conditions
allow more time so that the protein can properly fold into the desired
structures.

SDS-PAGE Analysis

SDS-PAGE was used to confirm the successful expression of the scFv antibodies.
The gel electrophoresis results provided visual evidence of protein expression
levels and purity. The anti-IL-6 scFv is anticipated to be 27.09 kDa, and
the anti-CA125 scFv is anticipated to be 28.38 kDa.

![Figure 1](https://static.igem.wiki/teams/5234/engineering/image7.png)

Protein Purification

We purified the protein using Tris-HCl buffer, guanidine hydrochloride
and imidazole denaturing solution, as well as a column packed with nickel
resin that resists denaturation. The fermented bacteria were resuspended,
sonicated, and the supernatant and precipitation were separated, with the
precipitation being redissolved. Samples of both the supernatant (sup.
) and the precipitation (prec.) were taken for analysis.

![Figure 1](https://static.igem.wiki/teams/5234/engineering/image8.png)

### **Learn**

The SDS-Page results from our TEST showed the successful expression of
the designed scFv. One of the main challenges we faced was the production
of insoluble proteins. This is often the case when the protein originates
from mammalian origins. The lack of complex cell functions in E. coli undermines
the synthesis and folding of these proteins. There exist various strategies
to improve solubility, including changes in expression conditions and the
choice of new expression chassis.

To further enhance protein solubility and expression, we considered using
the Shuffle T7 strain, which is designed to improve the solubility of recombinant
proteins. BL21 and Shuffle T7 are both E. coli strains used for recombinant
protein expression, but they have distinct features. BL21 is suitable for
general protein expression, especially those without disulfide bonds. In
contrast, Shuffle T7 is genetically engineered for efficient disulfide
bond formation in the cytoplasm, making it ideal for proteins requiring
proper folding. It harbors mutations in trxB and gor, essential for a redox
environment conducive to disulfide bond formation, and expresses DsbC,
which aids in correct folding. Additionally, Shuffle T7 has a chromosomal
copy of T7 RNAP for high-level expression and lacks lon and ompT proteases,
reducing proteolytic degradation. In summary, for proteins that need correct
disulfide formation, Shuffle T7 is preferred, while BL21 is fit for easily
soluble proteins.

![Figure 1](https://static.igem.wiki/teams/5234/engineering/image9.png)

#### **Solubilisation of Inclusion Bodies**

![](https://static.igem.wiki/teams/5234/engineering/purification-1.webp)

**Figure 4.** Using NLS to resolubilize the inclusion bodies can
result in the purification of the anti-CA125 scFv and anti-IL-6 scFv.

The previous experimental results showed that after the production of anti-IL-6
and anti-CA125 scFv, a lot of proteins existed in the form of inclusion bodies.
In order to solubilize the protein within the inclusion bodies,
we attempted purification with non-denaturing agents.

Experiment procedure:
1、Collect cell pellets after bacteria culture fermentation, resuspend the pellets
with 20 mM Tris-Cl, and lysis with ultrasonication;
2、Separate supernatant and precipitations, and dissolve the precipitations with
non-denaturing agents.
3、Pass the above re-dissolved solution through Ni column and wash with 5 column volume.
4. Elute with 2 to 3 column volumes of elution buffer.
5. Collect the protein eluate and concentrate it.
Non-denaturing solvent/wash buffer:
1% (w/v) N-lauroyl sarcosine (NLS) (Celik et al., 2023) ，
300 mM NaCl ，50 mM Tris-Cl ，pH 8.0

Elution Buffer：
0.1% (w/v) NLS ，300 mM NaCl, 50 mM Tris-Cl ，300 mM Imidazole ，pH 8.0

The SDS-Page run after purification and elution shows a clear band for the
anti-CA125 scFv and anti-IL-6 scFv, proving they can be properly solubilized.

We estimated the production quantity of
anti-IL-6 scFv to be 10mg/L (0.5 mg protein from 50 mL bacteria culture) and
anti-CA125 scFv to be 4 mg/L (0.2 mg protein from 50 mL culture).

#### **Binding Interaction Verification**

![](https://static.igem.wiki/teams/5234/engineering/native-page.webp)

**Figure 5.** Native PAGE results showed down-pulled protein bands
of CA125 antigen after inoculation with anti-CA125 scFv,
indicating binding interactions between CA125 and our designed scFv antibody.

To verify whether the designed scFv antibodies can bind to their corresponding antigens,
we used a simpler method, native PAGE, instead of more complex techniques such as ELISA
or co-immunoprecipitation. We incubated the purified scFv antibodies with the purchased
IL-6 and CA125 antigen proteins at room temperature for 2 hours
and then performed native-PAGE under non-denaturing conditions.

In the native PAGE results, it is evident that the position of the CA125 protein bands
shifted after incubation of the CA125 antigen with our anti-CA125 scFv.
The CA125 antigen protein has an original size of approximately 80 kDa.
Due to native PAGE being a non-denaturing electrophoresis,
the protein is not linearized and appears at a position
slightly higher than its expected size.

The anti-CA125 scFv, on the other hand, is a much smaller protein,
with a size of about 28 kDa.
We believe that due to the binding between the two,
the CA125 antigen protein was pulled down by the smaller scFv,
causing the shift in band position.
Therefore, we infer that our designed scFv can effectively bind to the target antigen.

On the other hand, the results with IL-6 are nonconclusive.
The IL-6 antigen protein we purchased was reported to be around 20 kDa.
The antigen protein showed a consistent band on the PAGE results.
However, after inoculation with the scFv antibodies,
the resulting bands showed no discernible shifts.
It could be that the scFv antibody does not have a high enough affinity for the antigen,
or due to inappropriate electrophoresis conditions such as pH values.
The conditions can affect protein migration and interactions.
It invites us to further study and characterize the scFv antibodies.

### References

[MUC-2024] Lee, K., Perry, K., Xu, M. et al. Structural basis for antibody
recognition of the proximal MUC16 ectodomain. J Ovarian Res 17, 41 (2024).

[Celik-2023] Şahinbaş, D., & Çelik, E. (2023). Enhanced production and
single-step purification of biologically active recombinant anti-IL6 scFv
from Escherichia coli inclusion bodies. Process Biochemistry, 133, 151-157.

[scFv-1] Monnier PP, Vigouroux RJ, Tassew NG. In Vivo Applications of
Single Chain Fv (Variable Domain) (scFv) Fragments. Antibodies. 2013; 2(2):193-208.

[Liu-2017] Liu D., Zhang L., Indima N., Peng K., Li Q., Hua T., Tang G.
CT and MRI findings of type I and type II epithelial ovarian cancer. Eur.
J. Radiol. 2017;90:225–233.

[Karimi] Karimi-Zarchi M, Dehshiri-Zadeh N, Sekhavat L, Nosouhi F. Correlation
of CA-125 serum level and clinico-pathological characteristic of patients
with endometriosis. Int J Reprod Biomed. 2016 Nov;14(11):713-718.

[Zou-2006] Zou, S., & Yu, H. (2006). Clinical application of serum CA-125
level detection in gynecological diseases. Medical Clinical Research, 23(2),
151-152

[Zhang-2012] Zhang, D., Hu, Y., Zhang, D., Xu, K., & Lu, J. (2012). Variations
in serum CA125, CA15-3, and CA19-9 levels during the menstrual cycle in
women of reproductive age. Journal of Radioimmunology, (6), 690-690.

[Munezz-2020] Munezza AK and Mohammad M. Recent Advances in Electrochemical
and Optical Biosensors Designed for Detection of Interleukin 6. Sensors
2020, 20, 646.

[Bellone-2005] Bellone S, Watts K, Cane' S, Palmieri M, Cannon MJ, Burnett
A, Roman JJ, Pecorelli S, Santin AD. High serum levels of interleukin-6
in endometrial carcinoma are associated with uterine serous papillary histology,
a highly aggressive and chemotherapy-resistant variant of endometrial cancer.
Gynecol Oncol. 2005 Jul;98(1):92-8.

[Aydin-2020] Aydın, Beyza & Kul, Yaren. (2020). DESIGN OF ANTIGEN DETECTING
IMMUNOCHROMATOGRAPHIC RAPID TEST KIT AGAINST SPIKE GLYCOPROTEIN OF SARS-CoV-2.
10.13140/RG.2.2.16968.96001.]

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Experiments

## Contents

* [Extracting plasmids](#p0-5)
* [Nanodrop Test:](#p1-5)
* [Nanodrop test results:](#p2-5)
* [Picking Colony:](#p3-5)
* [Bacteria Preservation:](#p4-5)
* [Competent cell preparation and maintenance:](#p5-5)
* [Transformation](#p6-5)
* [Protein electrophoresis-ScFv expression verification](#p7-5)
* [Magnetic Agarose Beads for Protein Purification:](#p8-5)

The purpose of our experiment is to verify that the proteins we chose (CA125 and IL-6) are able to react with the antibodies.

To achieve this aim, we started by building plasmids. We built two plasmids based on fragments of the two antigens, CA125 and IL-6. After the construction is complete, the plasmid is injected into the E. coli and therefore, the plasmids are going to replicate.

After adding IPTG, and the plasmid expresses more protein, we will then break the E. col to make the protein flow out. The protein electrophoresis experiment will be conducted to verify whether the molecular content of the protein was what we needed (25Kda).

If the result of the protein electrophoresis experiment shows the molecular content of the protein is what we need, we will know that the protein we chose can react with the antibodies. It will tell us the feasibility of our product.

### Extracting plasmids

1. Using 1000µl micropipette added 1ml of various bacteria solution into EP tube (total 4 types of bacteria, each type 3 tubes, total 12 tubes)
2. Place all 12 EP tubes into centrifuge for 3 minutes
 3. Extract supernatant
 4. Repeat 3 times of above steps
 5. Added 250µl of K1 reagent, using micropipette to aspirate and dispense until bacteria pallet has disappeared
 6. Added 250µl of K2 reagent in all EP tubes then slightly shake 3 times
7. Added 350µl of K3 reagent in all EP tubes then aggressively shake
 8. Put all EP tubes inside centrifuge for 10 minutes
 9. Extract supernatant (around 750µl) into a collection tube and place into centrifuge for 30 seconds
 10. Pour out supernatant inside collection tube
 11. Add 750µl of KW reagent, centrifuge for 1 minute, pour out supernatant in collection tube
 12. Step 11 repeat 2 times
 13. Place into centrifuge for 1 minute
 14. Pour out the liquid inside the collection tube, place collection column into new EP tube, add 50µl of KE reagent
 15. Place into centrifuge for 1 minute
 16. Throw out collection column, low temperature storage of liquid inside EP tube

### Nanodrop Test:

- Take 1µl sample plasmid from each tube and place on Nanodrop to test the plasmid concentration and purity.

### Nanodrop test results:

![](https://static.igem.wiki/teams/5234/experiments/table.webp)

### Picking Colony:

1. Use small pipette head to pick out a small amount of bacteria from each tray, place in 15ml centrifuge tube, place on shaking incubator and shake for 1 night. Shaking incubator criteria 37°C, 180 rpm, 4 hours

2. Add 1ml iptg(concentration 50mg/ml) into four of the tubes (one tube for each strain) and mark the tubes.

### Bacteria Preservation:

In the laminar flow hood add 500µl of bacteria solution and 500µl of 50% glycerine in all 11 tubes and shake slightly

### Competent cell preparation and maintenance:

- Operate on a super clean table
- Draw approximately 1.5ml of bacterial fluid from each tube and add it to two 2ml centrifuge tubes (1.5ml each)
- Centrifuge for two minutes at 12000rpm
- Remove the supernatant and add the remaining bacteria (about 1.5ml per tube)
- Centrifuge for two minutes at 120,00rpm
- remove the supernatant
- Add 400µl of 0.1M CaCl, blow until the fungus disappears, and place in ice for 30 minutes
- Take 2ml bacterial solution from a large ice bath centrifuge and put it into a 2ml centrifuge tube at 4℃,12000rpm, for 2 minutes.
- Remove the supernatant and add 2ml of bacterial solution
- Centrifuge, remove supernatant
- Blot out residual culture fluid
- Add 400ml of Cacl2 of ice to each small centrifugal tube and repeatedly suck until the bacteria are suspended
- Ice bath for 30 minutes
-4 ℃,12000rpm,2 minutes, remove the supernatant, put the centrifugal tube upside down on the absorbent paper for 1 minute
- Add 100-200ul ice CaCl2, repeatedly suck until bacteria are suspended, and put into a 4°C ice bath

### Transformation

- Take the centrifuge tube containing the competent cells and place it in an ice bath
- Add 10µl of target DNA into the competent cell suspension, mix it with a whisk, label it, and leave it in an ice bath for 30 minutes.
- Place the centrifuge tube in a 42 ° C water bath for 90 seconds, then quickly transfer the tube to the ice bath and cool it for 3 minutes, keeping the centrifuge tube stable during the process.
- 900µl sterile SOC or LB medium (without antibiotics) was added to each centrifuge tube, mixed, and oscillated in a 37℃ shaker for 45 minutes (150 rpm).

### Protein electrophoresis-ScFv expression verification

*1) Preparation of DNA gel (25g agarose gel (1%)):*
- Put 0.25g agarose into the conical bottle
- Add 25ml 1XTAE
- Microwave until bubbling, clear and clear
- Add 2.5µl nucleic acid staining solution
- Pour into container and insert comb to prevent air bubbles
- Let stand until the glue sets

*2) Preparation of the tris buffer:*
- Preparation of protein electrophoresis requires 100ml 20mM tris buffer
- Calculated using the formula C1\*V1=C2\*V2
- Add 100ml of water and 1.335ml of 1.5M tris buffer to the beaker

*3) Sample preparation:*
- There are eight tubes of bacterial solution, each about 5ml, of which four tubes have IPTG added and four tubes have no IPTG added. (the four tubes with IPTG are the experimental group, and the four tubes without IPTG are the control group)
- Remove 4ml bacterial solution from each tube with a pipette and inject into a 50ml centrifuge tube. (eight tubes in total)
- Centrifuge 10000-12000rpm for 5-8 min.
- Remove the supernatant and add 20mM tris buffer
- Ultrasonic crusher for 10 minutes for each tube of bacterial liquid

*Experimental group:*
- Pack the experimental group bacterial solution broken by ultrasound, take 1.5ml out of each tube, and put it into 2ml centrifuge tube
- Centrifuge for 5 minutes at 12000rpm, 4˚C
- Place in an ice bath
-Pack the supernatant and precipitation separately, take out 1ml of supernatant and add it into a new centrifuge tube, label the tube, and add 1ml of 20mM tris buffer into the remaining precipitation for re-suspension and label the tubes

*Control group:*
- The bacterial solution of the control group broken by ultrasound was divided into four tubes and three ep tubes were divided into each tube, each tube was 1.5ml
- Centrifuge for 8 min, 10000rpm, 4˚C
- Take 1ml supernatant and label it

*4) Heating the protein:*
- Select one of each sample, a total of 12 centrifugal tubes (each bacteria has three tubes: supernatant of the control group, supernatant of the experimental group, and precipitation of the experimental group)
- Remove 160µl per tube and add 40µl loading buffer
- Heat in a constant temperature metal field at 95˚ C for ten minutes

*5) Electrophoresis:*
- Take 10µl of liquid per tube, inject it into the vertical electrophoresis apparatus, start the instrument, the voltage is 70 volts (the lane on the far right is marker)
- When the instrument is finished, remove the lane, scrape off the concentrated glue at the top, remove the remaining part, and put it in the petri dish

*6) Dyeing the glue:*
- Pour Coomassie brilliant blue stain into the petri dish, put the petri dish on to the shaker and wait 15 minutes
- Remove the dyed gel from the dye solution and rinse it off with deionized or distilled water to remove residual dye solution from the surface.
- Place the rinsed gel in the decolorizing solution, ensuring that the decolorizing solution completely covers the surface of the gel.
- Use a shaker to distribute the decolorizing liquid evenly on the gel surface and accelerate the decolorizing process. The speed and time of the oscillation or shaker should be adjusted according to the actual situation to avoid excessive decolorization or damage to the gel.
- As the decoloring process progresses, the color of the decoloring solution will gradually become darker. When the color of the decolorization solution is similar to the color of the gel, a new decolorization solution should be replaced to ensure the decolorization effect.
- The above decolorization steps can be repeated as needed until the blue background on the gel has largely faded and the protein bands are clearly visible.

### Magnetic Agarose Beads for Protein Purification:

*1) Sample preparation:*
- Add the bacterial solution to the centrifuge tube and centrifuge at 4ºC 4000xg for 20 minutes or at 4ºC 15000×g for 1 minute
- remove the supernatant to collect the precipitation.
- Re-suspension precipitation with Binding/Wash Buffer.
- Ultrasonic cracking of bacteria on ice. Ultrasonic power 200-300W, each ultrasonic treatment 10 seconds, each interval 10 seconds, a total of 6 ultrasonic treatment.
*2) Preparation of magnetic beads:*
- Gently blow the heavy suspended magnetic beads with pipette, and take the appropriate amount of magnetic beads into a clean centrifuge tube according to the dosage of 1ml magnetic bead suspension for every 5mg of target protein (molecular weight of about 60kD), and magnetic separation removes the supernant; Add Binding/Wash Buffer with the same volume as the original magnetic bead suspension or appropriately larger volume to wash the magnetic bead.
- Gently blow the suspended magnetic bead with the pipette. Place on magnetic rack and separate for 10 seconds to remove supernatant.
- Repeat the above steps twice.
*3) The target protein binds to the magnetic bead:*
- Add magnetic beads and incubation: add magnetic beads at the ratio of 1ml magnetic bead suspension per 5mg target protein (molecular weight of about 60kD) sample (usually about 10ml), place in a shaker, incubate at room temperature for 20-30 minutes.
- Magnetic separation: After incubation, place on magnetic rack for 10 seconds to remove supernatant. Part of the supernatant was retained for testing the effect of purification.
- Wash: Add 2ml Binding/Wash Buffer and gently blow the resuspended magnetic bead with the pipette. Place on magnetic rack and separate for 10 seconds to remove supernatant. Repeat washing three times.
*4) Elution:*
- According to the concentration of the target protein and the requirements of subsequent experiments, 0.2-1ml Elution Buffer can be added, the centrifuge tube can be turned over gently several times, and the eluent can be shaken in the shaking table for 10 minutes for elution, magnetic separation, and the eluent can be collected into the new centrifuge tube, which is the purified His-Tag protein.
**5) Cleaning and regeneration of magnetic beads:**
- Add 2ml Elution Buffer to the centrifuge tube, turn the centrifuge tube up and down several times, make the magnetic bead re-suspended, vortex for 10 seconds, magnetic separation, remove the supernatant. Repeat 3 times.
- Add 2ml 20% ethanol to the centrifuge tube, turn the centrifuge tube up and down several times, so that the magnetic bead is re-suspended, the vortex is rotated for 10 seconds, the magnetic separation is done, and the supernant is removed. Repeat 3 times.
- The magnetic beads are stored in 20% ethanol so that the total volume is equal to the initial suspension volume at 2-30ºC (long-term storage at 2-8ºC), which can be used for the next purification of the same protein.

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{% block title %}Results {% endblock %}
{% block lead %}Collaborate throughout the year with at least one other 2022 iGEM team on a set of shared objectives related to both of your projects.{% endblock %}
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Results

## Contents

* [The Seven (7) components of SynFlora7](#p0-5)
* [Sequence verification](#p1-5)
* [Plasmid vector expression benchmark](#p2-5)
* [Putting everything together](#p3-5)
* [Expression the whole pathway](#p4-5)
* [Anthocyanin: delphinidin color changes with pH](#p5-5)
* [Naringenin test](#p9-5)
* [Hardware Product](#p6-5)
* [1.Delphinidin dyed contact lens](#p7-5)
* [2.Eye masks](#p8-5)

### The Seven (7) components of SynFlora7

Anthocyanins give plants different colors. A long time ago, scientists had been curious about the production way of anthocyanins and conducted in-depth studies on their synthesis molecular pathway. Different anthocyanins are synthesized from different substrates and catalyzed by different enzymes. In our project this year, we chose the blue anthocyanin delphinidin as the target product. We found that delphinidin synthesis depends on a continuous reaction catalyzed by seven enzymes:

![](https://static.igem.wiki/teams/4255/wiki/poc1.png)

### Sequence verification

After determining the sequences, we asked the biological company to synthesize. This year, we used pETDuet-1 and pRSFDuet-1 plasmids for bacteria expression.

##### F3H: Naringenin to Dihydrokaempferol

In order to first detect the expression of plasmids, F3H gene, which is the first gene we received, was constructed on the pRSFDuet-1 vector through Gibson cloning. F3H gene was of most importance since it catalyze a reaction rate limiting step according to the interviews with Prof. Geng.

After transforming to E.coli and IPTG induction, we lysed the bacteria for SDS-PAGE and Coomassie brilliant blue staining at last. The result showed our plasmids pRSFDuet-F3H worked pretty well. We also studied the effect of induction time on the expression level, and set the induction time as 16 h, 20 h and 24 h at 16℃. Three parallel experiments were performed at each induction time. There was an obvious expression band (shown by red arrow).

![](https://static.igem.wiki/teams/4255/wiki/poc2.png)

![Figure 1](https://static.igem.wiki/teams/4255/wiki/poc3.png)

![Figure 1](https://static.igem.wiki/teams/4255/wiki/poc4.png)

To quantitatively measure the expression level for the optimal conditions, we converted the SDS-Page bands into grey scale pixel figures and used imageJ to read off the integrated grey scale of each band. Error bars were obtained from the three parallel experiments. The above figure indicates that 16 hours and 20 hours have statistically similar expression efficiency, while moving up to 24 hours the express level drops. We assume the bacteria culture might need more nutritional supply after that many hours of inoculation.

Incubation temperature could make a difference as well. In our next experiment, the incubation temperatures were set to be 16℃, 20℃, 30℃ and 37℃, respectively. There are also three repeats for each temperature. The result told us the expression level was higher at 30℃.

![Figure 1](https://static.igem.wiki/teams/4255/wiki/poc6.png)

![Figure 1](https://static.igem.wiki/teams/4255/wiki/poc7.png)

The effect of iPTG concentration was also considered. We setup an IPTG concentration gradient from 0.05mM, 0.1mM, 0.2mM, 0.5mM, 1mM, to 2mM.

![Figure 1](https://static.igem.wiki/teams/4255/wiki/poc8.png)

![Figure 1](https://static.igem.wiki/teams/4255/wiki/poc9.png)

Gel electrophoresis showed that, IPTG concentration of 0.2-0.5 mM resulted in a better protein yield, although the difference is not significant.

### Plasmid vector expression benchmark

Furthermore, although we have successfully expressed F3H gene on the plasmid, the expression level on pETDUET plasmid was not satisfactory. Because the pathway to produce anthocyanins is long, we want each enzyme to be expressed as efficiently as possible. So we talked with experts and decided to adjust the expression methods. Here we used GFP as a reporter gene, CK1-3 serve as control,(IPTG concentration of 0.5mM, 16℃,200rpm,16h), and the result showed that the fluorescence is 5 orders of magnitude brighter than the control group, indicating good expression capacity of the vector.

![Figure 3](https://static.igem.wiki/teams/4255/wiki/poc10.png)

### Putting everything together

Having found the optimized parameters for gene expression, we started to construct the complete anthocyanin bio-synthesis pathway.

We divided the seven enzymes into two parts, 4CL, CHS and CHI catalyzed the production of naringenin one by one, and naringenin was further transformed into anthocyanins in the presence of F3H, F3'5'H, DFR and ANS. We constructed the first three enzymes on the pETDUET-1 vector, in which 4CL was linked to CHS and CHI via linkers to form a fusion protein. We constructed the last four enzymes on the pRSFDuet-1 vector. F3H was fused to F3'5'H to express a larger protein. DFR and ANS were expressed separately.

The results of plasmid construction are shown in the figure below:

![Figure 4](https://static.igem.wiki/teams/4255/wiki/poc11.png)

![Figure 4](https://static.igem.wiki/teams/4255/wiki/poc12.png)

### Expression the whole pathway

Next, we tested whether bacteria could express the anthocyanin synthesis pathway we constructed. We took two large plasmids and transformed into to E.coli. The two kinds of bacteria were cultured overnight in shake flask and grown to logarithmic stage. IPTG with a final concentration of 0.2 mM was added and induced at 16℃ for 16 h, 20 h and 24 h, respectively. Three repeats for each induction time. Under proper IPTG concentration induced for different times, we lysed the bacteria for SDS-PAGE and Coomassie brilliant blue staining at last.

![Figure 4](https://static.igem.wiki/teams/4255/wiki/poc13.png)

![Figure 4](https://static.igem.wiki/teams/4255/wiki/poc14.png)

pETDUET-4CL-CHS-CHI was expressed as we saw a clear band shown by red arrow. The size of the band is slightly above 100kD which matches our expectations. Induction time analysis showed that with longer expression time, the expression increased significantly beyond 16 hours, but 24 hours and 20 hours are not significantly different.

![Figure 4](https://static.igem.wiki/teams/4255/wiki/poc15.png)

pRSFDuet-F3H-F3'5'H-DFR-ANS didn’t show a clear band on the SDS-PAGE results. We were expecting a band near 95 kD.

Then we moved on to change expression temperature.

Temperature gradient optimization experiments were suggested by Dr. Yong Zu, from our Human Practices interview. You can refer to our HP page for more details. The induction temperatures were set to be 16℃, 20℃, 30℃ and 37℃, respectively. There are also three repeats for each induced temperature.

![Figure 4](https://static.igem.wiki/teams/4255/wiki/poc16.png)

![Figure 4](https://static.igem.wiki/teams/4255/wiki/poc17.png)

pETDUET-4CL-CHS-CHI can be well expressed, indicated by a clear band near the top of 100kD, which is exactly what we had expected. When the temperature rises, the expression level goes unstable, lower at 20 degree C and higher at 30 degree C. The expression dropped significantly at 37 degree C. At the same time, there is a band near 120 kD showing clear expression. This band doesn’t correspond to a designed protein, and at the same time, it has hint amount of expression at 16 degree C and 20 degree C. We determined this is not a band of our design and disregard it. The lower temperature 16 degree C seems to be the best condition here. We think the reason might be the lower temperature slows down the folding process, and the plant-originated proteins have a better chance to be correctly folded and properly dissolve in the solution.

![Figure 4](https://static.igem.wiki/teams/4255/wiki/poc18.png)

![Figure 4](https://static.igem.wiki/teams/4255/wiki/poc19.png)

![Figure 4](https://static.igem.wiki/teams/4255/wiki/poc20.png)

On the SDS-Page figure, pRSFDuet-F3H-F3’5’H-DFR-ANS didn’t show a band near the expect position around 95 kD. After image analysis, we suspect that our naked-eyes are not accurate enough. The grey-scale inverted image showed possible expression near 95KD bands at 20 and 30 degree Celsius. The impression is backed-up on the numeric analysis from integrating the grey scales. At 30 degree C, the expression is the highest in average level, but not significantly better than 20 degree due to the large error bar. When temperature is higher at 37 degree C, the expression dropped to the background level, indicating too high a temperature is not helping.

The heavier band (at > 100 kD position) at higher temperatures at 20, 30, 37 degree C are clear, but do not correspond to any protein we designed. We determine this is a polluted band and disregard its appearance.

Next, we set 0.05mM、0.1mM、0.2mM、0.5mM、1mM、0.2mM (O2 short) IPTG concentration to give a expression test as well. “O2 short” means the oxygen supply is limited by sealing the culture container.

![Figure 4](https://static.igem.wiki/teams/4255/wiki/poc21.png)

![Figure 4](https://static.igem.wiki/teams/4255/wiki/poc22.png)

For the expression of pETDUET-4CL-CHS-CHI, we picked the previously determined temperature of 30 degree C. We can observe a clear band on SDS-Page results around the expected band of 100kD. The quantitative readings do not suggest a best iPTG concentration in this case. It seems 0.2 to 0.5 mM worked the best. At 1.0 mM, the expression level is remarkably higher, but given the diminishing error bar, we would treat this as an outlier.

![Figure 4](https://static.igem.wiki/teams/4255/wiki/poc23.png)

pRSFDuet-F3H-F3'5'H-DFR-ANS is experimented with the above iPTG concentration gradient at the previously found optimal temperature, i.e., 30 degree C. This time, we did not observe any band around expected 95kD. After image processing and readings, there is still no hint of the expression. This doesn’t mean the plasmid is not working, but for this experimental trial, we failed to observe a nice result. Given the complex experiment conditions, the plasmid will be produced clearly in other rounds of experiments.

### Anthocyanin: delphinidin color changes with pH

The second plasmid contained last four enzymes on the pRSFDuet-1 vector, in which F3H was fused to F3'5'H and DFR was fused to ANS. We added naringenin to the culture and induced the expression of bacteria, and the next morning, we were surprised to see pink bacteria. We immediately found the relevant experts and ask whether this is the target product delphinidin, expert suggested that anthocyanins have a significant characteristics, which shows different colors at different pH, so we went back to the lab for pH adjustment, and successfully in different pH conditions to see the different color, this suggests that we eventually succeeded in producing anthocyanins. (IPTG with a final concentration of 0.5 mM was added and induced at 16℃ for 16 h). The color tone is too bright for delphinidin, which indicates the substance we obtained is not pure enough.

![Figure 7](https://static.igem.wiki/teams/4255/wiki/poc24.png)

### Naringenin test

Synthesizing delphinidin involves a very long pathway. If something goes wrong, we need a proper signal to debug. We therefore planned to build a sensor to detect the product from the first plasmid, which expressed three enzymes on pETDUET-1 to produce naringenin. In the previous iGEM project, we found a naringenin detector, which produced GFP fluorescence upon receiving naringenin signal, but unfortunately, after we synthesized this naringenin detector and constructed it on the PETDuet-1 plasmid, we failed to see GFP fluorescence signal even using 50 μM standard naringenin. PETDuet-1 -GFP was used as a control to show at the right panel.

![Figure 7](https://static.igem.wiki/teams/4255/wiki/poc30.png)

This result indicated that the naringenin detector uploaded earlier was incorrect, so we reviewed the original literature and constructed a new naringenin sensor. The sensor contains a constitutive promoter to initiate the expression of a protein FdeR. This protein can specially bind to naringenin and then the compound will attach to a promoter in the opposite direction and activates the expression of a GFP.

![Figure 7](https://static.igem.wiki/teams/4255/wiki/poc31.png)

![Figure 7](https://static.igem.wiki/teams/4255/wiki/poc99.png)

We synthesize the GFP-FdeR sensor and construct it into pETDuet vector. T7 promoters remained on the vector but are assumed to be non-interfering with the sensor.

![Figure 7](https://static.igem.wiki/teams/4255/wiki/poc33.png)

The result showed upon introduction of naringenin into the bacteria culture, the sensor will successfully express GFP proteins at the expected band. However, our experiments are limited in that the GFP is not bright enough. We think either it is our GFP is not correctly expressed, or the FdeR protein is not sufficiently produced to give a strong GFP expression.

### Hardware Product

In order to test if delphinidin can be used to make end-user product, we carried out the following experiments. Our lab experiments are not capable of producing large amounts of delphinidin yet, and we purchased food-grade delphinidin powder from specialty suppliers. These are extracted from fruits and plants, and therefore they contain a mixture of color substances. When we pick what powder to buy, we studied the ingredients and colors. All powder we purchased have greater than 80% verified to be delphinidin.

### 1.Delphinidin dyed contact lens

In the interview with Dr. Ligong Shao, we received the advice that it is promising to satisfy the requirements for the safety, market reception and medical efficacy of our delphinidin-dyed contact lenses, and we were happy to experiment with the viability make this product and investigate possible problems. Therefore we conducted the following experiments:

![Figure 16](https://static.igem.wiki/teams/4255/wiki/poc25.png)

##### Observations

The above image shows our delphinidin-dyed contact lenses. We set 3 colors for 5 concentration gradients and 6 time periods. Among the different molar concentrations, bilberry extract has the darkest color and blueberry extract has the lightest color. In the experimental comparison of three colors with different concentrations, we found that the staining time of 10 min is difficult to make the color attached, and the staining results of 24h and 48h are basically the same.

In the comparison of experimental results of the same time and the same plant extract, we conclude that there is no apparent difference between the results of 2.5mol and 5mol is , and the there is apparent difference between 7.5mol and 10mol.

From the perspective of cost, it can be concluded that the best effect of dyeing bilberry extract in a concentration of 7.5 mol for 24 hours has the best effect and the lowest cost.

The light transmittance of our contact lenses is from 100% to 20%, which means that we can create all shades from colorless to darkest (like sunglasses) by molar concentration and staining time. We have proved the breadth of our contact lens staining range, which can be applied to the vast majority of scenes and meet the needs of the vast number of logarithms.

![Figure 16](https://static.igem.wiki/teams/4255/wiki/poc26.png)

The light transmittance of contact lenses after 10 min, 3 h, 6 h, 12 h, 24 h, 48 h after staining is shown in the figure above. A lower light transmittance indicates deeper staining. Our data shows that anthocyanins can color contact lenses very well.

Color fastness should be considered next. Absorbance data were obtained by negative logarithmic conversion of the transmittance data. Let the absorbance data after staining be A\_(initial), the data after washing (de-coloring) be A\_(final), A\_(final)/A\_(initial) can define a color fastness data.

![Figure 16](https://static.igem.wiki/teams/4255/wiki/poc27.png)

It can be seen that the color fastness of mulberry extract is relatively optimal. But all pigments have a color of 20%-50%. This indicates that the problem of color loss does occur. We believe that in the future, we should further test the bioaffinity, irritation and safety of pigments for the cornea, and find ways to improve color fastness.

For color fastness, we also tested coloring different cloth materials with delphinidin. We intend to dye cloth and make clothes, canvas bags and other related dyeable products. This dye does not cause allergies or some other adverse reactions. Coloring was done by soaking the cloth overnight a high concentration of the delphinidin dye. The next day, the cloth is collected and air-dried. Decoloring was done by water-soaking the cloth overnight. The results showed that the bilberry extracted delphinidin dye can keep the color after washing.

![Figure 16](https://static.igem.wiki/teams/4255/wiki/poc28.png)

##### Safety Considerations

The product is manufactured and experimented entirely in a laboratory setting. Our contact lenses are only used for color fastness experiments and nobody has ever worn the samples. After the experiment, we strictly followed the safety regulations and properly treated the used contact lens test products.

### 2.Eye masks

In our interview with Dr. Dan Ding, we learned that in addition to daily eye wash and eye care for the eyes, commonly used and sold products include eye protection patches (eye masks) that can contain plant-based medicine. We contacted Good Vision Company, a company with many years of experience and properly licensed in the R&D of eye patch, to try out the idea of mix delphinidin into the eye patch product.

For safety concerns, we do not dare to be careless. We learned from the research and development department of Good Vision Company about quality inspections before the manufacturing. They helped us verified our delphinidin material meet their safety requirement. After production, further safety and quality inspections will be carried out to ensure that all products have no safety problems when they are delivered to customers. Refer to safety page for more details on eye patch safety inspection rules.

The product production of delphinidin eye patch is completed by Good Vision Company, We also combined with the HP interview with Minister Qingli Wang for safety confirmations

We received the product and evaluated the appearance and possible use of it. Its overall appearance is brownish yellow, the taste of menthol is refreshing, the eye patch is wrapped in food-grade tin foil, and the interior has a waterproof layer, so the overall weight is easy to carry. After inspecting this batch of goods, we sealed it into the laboratory refrigerator, everyone wore masks and gloves throughout the process, there was no skin contact, and in the follow-up experiments, we will conduct safety experiments through regular companies and formal processes supervised by the health department.

![](https://static.igem.wiki/teams/4255/wiki/poc29.png)

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