iGEM 2022 — Team shsbnu-china

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Notebook

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Engineering

### Overview

This year our project is focused on the synthesis of anthocyanin, which has a lot of research and reporting on this canonical pathway. But due to anthocyanins are ubiquitous in plants, therefore, each enzyme can find multiple homologous proteins. To get the better yeild, we first compared enzymes in different species and selected the most suitable sequence for E.coli expression, we constructed several new parts, which has been submitted this year.

![Figure 1](https://static.igem.wiki/teams/4255/wiki/eng1.png)

### 1.pETDUET- F3H

The first new part we uploaded this year is to express F3H, which is enzyme working to catalyze dihydrokaempferol into dihydromyricetin. The sequence we submitted here was come from Ginkgo biloba L. And we has done codon optimization for E.coli expression.

##### Major protocol we use:

We asked biological company to synthesize the sequence at first. And we constructed it into pETDUET plasmid. Next, we transformed the plasmids into E. coli BL21(DE3).
After the colony has grown up on the plate, we picked a single colony by a sterile tip and added it into 4 ml LB medium with the corresponding antibiotic.
Later, we added IPTG for induction and shook overnight. We also set a control group and didn’t add IPTG into it.
Finally, we centrifuged the bacterial solution at 12000 g, discard the supernatant, and used RIPA as a lysis buffer. we added loading buffer to the supernatant which contains the protein extract, and after heating at 96℃ for 10 min, we underwent SDS-PAGE and Coomassie brilliant blue staining for expression test.
We could see a clear band at the position of ~43kD, which only appeared in the group with IPTG. The molecular weight of this band was in line with the expectation, and it was under the condition of induction, so we believed that we had successfully expressed F3H gene.

![Figure 1](https://static.igem.wiki/teams/4255/wiki/e1.png)

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), but the expression levels were similar at the induction time of 16h, 20h and 24h.

![Figure 1](https://static.igem.wiki/teams/4255/wiki/eng3.png)

Besides of time, we do concern whether expression temperature could make a difference. In our next experiment, the induction temperatures were set to be 16℃, 20℃, 30℃ and 37℃, respectively. There are also three repeats for each induced temperature. The result told us the expression level was higher at 20℃.

![Figure 1](https://static.igem.wiki/teams/4255/wiki/eng4.png)

Based on the result before, we thought about if the concentration of IPTG could effect the expression level, so we set 0.05mM、0.1mM、0.2mM、0.5mM、1mM、0.2mM concentration to give a test.

![Figure 1](https://static.igem.wiki/teams/4255/wiki/eng5.png)

The result showed IPTG concentration of 0.1-0.2mM induced higher expression.

### 2.pETDUET-4CL, CHS-CHI

We divided the seven enzymes into two parts, 4CL, CHS and CHI catalyzed the production of naringenin one by one, and naringenin was further generated into anthocyanins in the presence of F3H, F3'5'H, DFR and ANS. The 4CL, CHS and CHI sequences come from Glycine max. We constructed the first three enzymes on the pETDUET vector, in which 4CL was linked to CHS and CHI via linkers to form a fusion protein.

![Figure 1](https://static.igem.wiki/teams/4255/wiki/engineering-1.jpg)

![Figure 1](https://static.igem.wiki/teams/4255/wiki/engineering-2.png)

The function of the enzymes can be listed below:
4CL: catalyze phenylalanine into 4-coumaryl coenzyme A
CHS: catalyze 4-coumaryl coenzyme A into chalcone
CHI: catalyze chalcone into flavanone

##### Major protocol we use:

We asked biological company to synthesize the sequence at first. And we constructed it into pETDUET plasmid. Next, we transformed the plasmids into E. coli BL21(DE3).
After the colony has grown up on the plate, we picked a single colony by a sterile tip and added it into 4 ml LB medium with the corresponding antibiotic.
Later, we added IPTG for induction and shook at overnight. We also set a control group and didn’t add IPTG into it.
Finally, we centrifuged the bacterial solution at 12000 g, discard the supernatant, and used RIPA as a lysis buffer. we added loading buffer to the supernatant which contains the protein extract, and after heating at 96℃ for 10 min, we underwent SDS-PAGE and Coomassie brilliant blue staining for expression test.
We could see a clear band at the position of 112kD, which only appeared in the group with IPTG. The molecular weight of this band was in line with the expectation, and it was under the condition of induction, so we believed that we had successfully expressed fusion protein combined by 4CL, CHS and CHI.
We studied the effect of induction time on the expression level, 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.
Here are our results:

![Figure 1](https://static.igem.wiki/teams/4255/wiki/eng8.png)

The pETDUET-4CL-CHS-CHI was expressed as we saw a clearly band shown by red arrow, but the expression level is similar between each time.
Then we moved on to change expression temperature. The induction temperatures were set to be 16℃, 20℃, 30℃ and 37℃, respectively. There are also three repeats for each induced temperature.

![Figure 1](https://static.igem.wiki/teams/4255/wiki/eng9.png)

As shown in the figure, 20℃ seems to be the worst condition for pETDUET-4CL-CHS-CHI.
Next, we set 0.05mM、0.1mM、0.2mM、0.5mM、1mM、0.2mM IPTG concentration to give a expression test as well.

![Figure 1](https://static.igem.wiki/teams/4255/wiki/eng10.png)

According to our result, there is not much difference between IPTG concentrations for pETDUET-4CL-CHS1-CHI expressing.
So we successfully expressed three of the enzymes in anthocyanin synthesis pathway.

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Experiments

## CONTENTS

* [Biosensor sensing naringenin](#p0-5)
* [Delphinidin Synthesis Operon (Naringenin Added)](#p1-5)
* [Naringenin synthesis operon (add 4-coumaric acid)](#p2-5)

### Biosensor sensing naringenin

a) Inductor BL21 was added to two liquid media (containing 3um naringenin), after cultured overnight at 16 ° C,
centrifuged, it is observed that the fluorescence intensity of the bacterial mass (in the morning).

b) Add 0.5mm IPTG to the two liquid culture media, induce culture at 16 ° overnight, centrifuge, and observe the
fluorescence intensity of the bacterial mass (in the afternoon).

c) Prepare two pieces of protein glue, centrifuge the preserved solution, ultrasonic wave, take the supernatant,
add it into the protein loading buffer, boil it, and electrophoresis (electrophoresis staining, afternoon).

### Delphinidin Synthesis Operon (Naringenin Added)

Prepare 50 ml LB culture solution containing 3um naringenin and 50ug / ml Kan, and prepare 50 ml LB culture
solution containing no naringenin and only 50ug / ml.

c) Select the BL21 transformed monoclonal containing plasmid 5, inoculate it in LB containing antibiotics, grow
it overnight, dilute it with fresh lb to OD600 of 0.1, and culture it in a shake flask. Incubate at 37 ° C until
OD600 is 0.8, add 0.5 mm isopropyl-d-mercaptogalactoside (IPTG) for induction, and grow at 250rpm-300rpm at 30 °
C for 3 h to overexpress the protein. The cells were collected by centrifugation and resuspended in M9 liquid
medium at pH5, containing necessary antibiotics, 1% glucose, 20mm naringenin, 0.5mm IPTG, 0.1 mm 2-oxoglutarate,
2.5 mM sodium ascorbate. The cells were incubated at 30 ° C for 48h (the supplement at the middle 24h can be
selected: 1% glucose (20ul), 2um naringenin). After that, the cells were centrifuged to observe the color of the
bacterial mass (adding HCl to make it pink, adding NaOH to make it grass green). d) BL21 solution of carrier 5
was cultured in LB medium of 3um naringenin and 50ug / ml Kan for 16h. Respectively take 1ml of culture,
12000rpm, 1min, observe the color of the bacteria at the bottom of the tube, and take photos.
![](https://static.igem.wiki/teams/4255/wiki/experiment-1.png)

##### IN THIS PICTURE

Plasmid 5 contains four fusion proteins, among these proteins, F3H catalyzes the conversion of naringenin to
dihydrokaempferol, F3 '5' H catalyzes the conversion of dihydrokaempferol to dihydromyricetin, DFR catalyzes the
conversion of Dihydromyricetin to colorless delphinidin, and ans catalyzes the conversion of colorless
delphinidin to colorless delphinidin.

The coding genes of the first two proteins are driven by the first T7 promoter of the vector for transcription,
and the coding genes of the last two proteins are driven by the second T7 promoter of the vector for
transcription, and finally translated into four corresponding proteins.

e) The BL21 bacterial solution of carrier 5 was cultured in LB medium of 0um naringenin and 50ug / ml Kan for
16h, and 1ml of culture was taken respectively at 12000rpm for 1min. The color of the bacterial mass at the
bottom of the tube was observed and photographed.

f) BL21 solution of carrier 5 was cultured in LB medium of 3um naringenin, 50ug / ml Kan and 0.5 mm IPTG for
16h. Respectively take 1ml of culture, 12000rpm, 1min, observe the color of the bacteria at the bottom of the
tube, and take photos.

g) BL21 solution of carrier 5 was cultured in LB medium containing 0 um naringenin, 50 UG / ml Kan and 0.5 mm
IPTG for 16 h. Respectively take 1ml of culture, 12000rpm, 1min, observe the color of the bacteria at the bottom
of the tube, and take photos.

### Naringenin synthesis operon (add 4-coumaric acid)

h) Prepare a plate containing 25ug / ml ampicillin and 12.5ug/ml chloramphenicol. The competent cells dh5a and
BL21 were co transformed with the receptor plasmid and plasmid 4.

i) Prepare 50ml LB liquid medium containing 0.2g/l coumaric acid, add 12.5ug/ml cm and 25ug/ml amp

j) Prepare LB liquid medium without coumaric acid, add 12.5ug/ml cm and 25ug/ml amp

k) Plasmid was extracted and transformed.

l) Prepare a receptive state.

m) Plasmid 5 and plasmid 4 were transformed into dh5a and BL21 competent cells, respectively.

n) The receptor plasmid and plasmid 4 were co transformed into BL21, expanded and shaken, and screened and
cultured in LB plates containing 25ug / ml ampicillin and 12.5ug/ml chloramphenicol. After 16 hours, monoclonal
antibodies were selected, cultured in LB medium containing coumarin and without coumarin, induced, centrifuged,
and observed the changes of green fluorescence of the bacteria. The bacterial fluid of receptor and BL21 were
selected for comparison.

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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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