iGEM 2024 — Team shsbnu-china

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# Engineering

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* Engineering

![Image](static/picture/innar-page-shape2.png)

### Overview

L-theanine is a non-protein amino acid found in tea leaves that can be
used to alleviate anxiety and improve sleep quality. However, conventional
production methods suffer from severe drawbacks. The plant extraction method
is low in yield and purity, high material costs, and a risk of contamination,
which can lead to significant loss of theanine. extracting theanine from
tea leaves involves high costs, complex procedures, and the risk of low
yields. Current chemical synthesis methods require extracting theanine
from mixtures containing toxic substances, which is not only harmful to
the environment but also costly.

This year, SHSBNU-China aims to use synthetic biology techniques to genetically
edit the E. coli strain BL21 to produce L-theanine. Our experiment is divided
into four stages. First, we designed a plasmid containing five genes (BsAld,
CsAlaDC, GMAS, PPK, and GNP1) responsible for the production and transport
of theanine. In the second stage, we assembled the five genes from four
plasmids onto pET28a through Golden Gate Assembly, which included the design
of Golden Gate primers and assembly. In the third stage, we transformed
the plasmid with the five genes into the E. coli strain BL21 and induced
expression with IPTG, followed by HP-LC testing of the supernatant to determine
the yield. Finally, we used the yield measured by HP-LC and our human practice
interviews to determine what steps we should take next to increase theanine
production.

In our project, we successfully achieved a production level of 38 mg/L
by genetically editing the BL21 strain. We hope to achieve higher yields
in the future through batch fermentation and by improving the fermentation
medium!

### Building: Construction of Plasmid

We used five genes in total: BsAld, CsAlaDC, GMAS, PPK, and GNP1.

-          BsAld, CsAlaDC, and GMAS are responsible
for the de novo synthesis of theanine, starting from glucose.

-          PPK provides ATP to aid the synthesis
pathway.

-          GNP1 is responsible for transporting
the synthesized theanine out of the bacterial cells.

In the enzymes synthesizing theanine, BsAld and CsAlaDC carry out the
conversion of pyruvate to ethylamine, and GMAS synthesizes theanine from
ethylamine and glutamate, which is produced by the generic bacterial TCA
cycle. The coding sequence of all five enzyme were first learned from NCBI
genebank, and sent for chemical synthesis by BGI genomics. The as-synthesized
DNA fragments were inserted in pUC vectors. The heads and tails of each
gene segment, as well as the empty vector pET28a, are designed with BsaI
enzyme binding sites, allowing all gene splicing and insertion to be conducted
within the same Golden Gate reaction system.

![](https://static.igem.wiki/teams/5235/new/nc30.jpg)

**Fig 1. Part of the de novo reaction pathway of L-Theanine synthesis in E. coli
utilizing BsAld, CsAlaDC, and GMAS to convert glucose into theanine [1].**

BsAld, from *Bacillus subtilis*, is the first step in theanine synthesis,
converting pyruvate to alanine. BsAld first binds with NADH to form an
enzyme-cofactor complex. Then, pyruvate binds to the active site of the
enzyme-cofactor complex, and NADH transfers its electrons to the pyruvate,
reducing the carbonyl group (C=O) to an imine (C=NH). The imine intermediate
reacts with ammonium ions to form a Schiff base intermediate, which is
then hydrolyzed to produce alanine, releasing NAD⁺ and water.

CsAlaDC, from *Camellia sinensis*, is the second step in theanine synthesis,
converting alanine to ethylamine. CsAlaDC requires the cofactor pyridoxal
phosphate (PLP), which is the active form of vitamin B6. Initially, the
amino group of alanine forms a Schiff base intermediate with the aldehyde
group of PLP. In the Schiff base intermediate, electrons on the α-carboxyl
group are redistributed, subsequently removing α-hydrogen as carbon dioxide,
forming a stable resonance structure. The resulting carbanion accepts a
proton, forming the PLP-bound form of ethylamine. Ethylamine dissociates
from PLP, restoring PLP to its original state, and the enzyme can proceed
to the next catalytic cycle.

GMAS, from *Methyloversatilis universalis*, is the final step in theanine
synthesis, converting ethylamine and glutamate into L-theanine, but this
step requires energy supplied by ATP. Glutamate is produced by the generic
bacterial TCA cycle. Therefore, this pathway is a de novo pathway, generally
independent of input substrate, needing only the basic nutrients glucose
for bacterial growth and intermediates synthesis.

![](https://static.igem.wiki/teams/5235/new/nc31.jpg)

**Fig. 2 The auxiliary functions of PPK and GNP1 during the production of
L-theanine [2].**

As mentioned, the synthesis of L-theanine by GMAS requires energy from
ATP. PPK, originating from *E. coli*, can transfer a phosphate group from
the end of a polyphosphate chain to ADP, generating ATP, thereby powering
GMAS. Polyphosphate is a long-chain molecule composed of multiple inorganic
phosphates linked by high-energy phosphate bonds. In many bacteria and
microorganisms, polyphosphate chains serve as storage forms of phosphate
groups and energy.

GNP1 is responsible for transporting theanine out of *E. coli*. As a membrane
protein, GNP1 has specific substrate binding sites. Since the structure
of theanine closely resembles that of glutamine, with similar amino structures,
GNP1 can recognize and bind theanine molecules. GNP1 is a proton gradient-dependent
transporter, meaning that its process of transporting theanine depends
on the transmembrane proton electrochemical gradient. Experimental studies
have also shown that other amino acids, such as lysine and basic amino
acids, cannot be transported by GNP1, indicating its strict structural
requirements for specific substrates.

These five genes are located on four plasmids, and we have designed BsaI
binding sites before each gene's RBS and after the stop codon. We also
designed BsaI restriction sites on the pET28a expression plasmid. The table
below describes the sticky ends produced by BsaI digestion of pET28a and
the four gene fragments.

![](https://static.igem.wiki/teams/5235/engineering/image5.png)

**Table 1. Sticky ends of BsaI Golden Gate assembly design.**

According to the principle of base-pair complementarity, the specificity
of these fragments allows them to automatically assemble together. Ultimately,
the five gene fragments were inserted into the empty vector pET28a between
the lactose promoter and the T7 terminator, as shown in the following figure.

![](https://static.igem.wiki/teams/5235/new/nc22.jpg)

**Fig 3. Golden Gate digestion of the parts and the pET28a vector.**

![](https://static.igem.wiki/teams/5235/new/nc21.jpg)

**Fig 4. Final vector using Golden Gate assembly**

In the Golden Gate assembly, we added four pUC plasmids each containing
five genes, the empty pET28a plasmid, and the Golden Gate Kit into the
same reaction system. The content of the reaction system is as follows:
200 ng of plasmids containing GMAS, PPK, GNP1, BsAld, and CsAlaDC each,
1 μL of PGGA, 2 μL of T4 DNA ligase, 2 μL of Golden Gate Mix enzymes (which
included BsaI), and the total reaction volume was adjusted to 14 μL with
the addition of 9 μL of water. The entire Golden Gate assembly was carried
out on ice. We performed two sets of Golden Gate Assembly following the
method described above.

![](https://static.igem.wiki/teams/5235/new/nc45.jpg)

**Fig 5. Schematic illustration of the gene circuit using T7 promoter and LacO
for controlled expression of the enzymes.**

After the Golden Gate Assembly, we conducted DNA electrophoresis to verify
whether the five gene fragments were successfully assembled. The results
indicated that the Golden Gate Assembly was successful:

![](https://static.igem.wiki/teams/5235/new/nc40.jpg)

**Fig 6. PCR and DNA electrophoresis verified the successful construction of the
expression plasmid.**

The verification of the success of Golden Gate assembly was performed
by PCR amplification of the target connected composite coding sequence.
The forward and reverse primers for the verification PCR run were design
to cross two neighboring coding sequence segments and only cover about
220 bp for faster PCR cycles. Therefore, only when the coding genes are
successfully connected, we can obtain a PCR product. If the assembly failed,
there will be no PCR product.

Finally, we also verified the success of Golden Gate via sending the plasmid
for sequencing.

### Test: Enzyme Expression in BL21 and Fermentation for L-Theanine

After successfully obtaining the complete pET28a plasmid containing five
genes via Golden Gate assembly, we transformed it into BL21 competent cells
and performed plate screening. Subsequently, we divided the transformed
cells into 12 tubes for fermentation in the following fermentation medium
composition: 10 g/L tryptone, 5 g/L yeast extract, 10 mmol/L MgCl2·6H2O,
150 mmol/L (NaPO3)6, 15 g/L glucose. The fermentation conditions were set
at 20°C and 200 rpm. We also added IPTG to eight of these tubes according
to a concentration gradient of 0 mM/L, 0.25 mM/L, 0.5 mM/L, and 1 mM/L,
with three tubes for each concentration gradient.

After overnight fermentation, we extracted the precipitates from two of
the tubes to perform protein electrophoresis for verification. Based on
the gene sequences, the estimated sizes of the proteins were approximately
39.7 kDa for BsAld, 53.6 kDa for CsAlaDC, 48.8 kDa for GMAS, 73.6 kDa for
GNP1, and 80.4 kDa for PPK. Our protein electrophoresis sequencing confirmed
the successful transformation of the target plasmid, and the following
figure showed the successful expression of one of the enzymes, GNP1.

![](https://static.igem.wiki/teams/5235/new/nc41.jpg)

**Fig 7. SDS-Page verified the expression of GNP1.**

Following the fermentation process, we separated the supernatant and the
precipitate from the remaining tubes of bacterial culture and used HPLC
(High Performance Liquid Chromatography) to check if the engineered strains
produced L-theanine. The HPLC results indicated that we produced L-theanine.
The results are shown in the graph below:

![](https://static.igem.wiki/teams/5235/new/nc6.jpg)

**Fig 8. HPLC results of the standard L-theanine,
the control group (no added glucose, no IPTG induction),
and the sample group (15g/L added glucose, 0.25mM IPTG)**

In the HPLC data, the standard L-Theanine sample used analytical grade
L-Theanine purchased from Aladdin Chemicals with greater than 98% purity.
The standard sample solution was prepared at a concentration of 200mg/L.
The HPLC result showed a single peak of standard L-Theanine around retention
time 2.4s. The control group was prepared using the E. coli culture with
the correct expression plasmid, but with no iPTG induction, nor added glucose.
There is a small hump observed in the HPLC results for the control group,
suggesting possible leaking expression of the designed enzymes. For the
sample group, proper iPTG induction was conducted at OD600 around 0.6 –
0.8, and 15g/L glucose was added upon iPGT induction. The HPLC result demonstrated
a clear rising around retention time 2.4s, indicating the production of
L-Theanine.

### Learn: Analysis of Results and Plan for Further Developments

![](https://static.igem.wiki/teams/5235/engineering/hplc.png)

**Fig 9. Standard curve of L-theanine is used to estimate the yield of
theanine upon varying the added glucose in the culture medium. The
bacteria culture of 20mL in 50mL container was used to collect the sample.
The highest yield reach 38.84 mg/L theanine.**

When analyzing our experimental results, one of our key focuses is on
how to enhance the production of L-theanine through a de novo biosynthetic
pathway in future experiments. This could further reduce the cost and
environmental impact of L-theanine production, allowing for better utilization of
L-theanine in anxiety mitigation.

According to our research, one of the advantages of using a de novo synthesis
method for L-theanine production is the ability to use a single strain of
microorganism instead of enzymatic preparations. This approach is more robust,
especially if we can significantly improve the conversion efficiency. Indeed,
should we achieve a high enough enhancement in the transformation efficiency, the
use of a single strain for whole-cell fermentation will likely become more
cost-effective in the future.

In terms of improving conversion efficiency and yield, our analysis of the
L-theanine synthetic pathway reveals that the entire process is glucose-dependent:
the two precursors for L-theanine, ethylamine, and glutamate, are generated
through two distinct pathways that rely on glucose input. Therefore, carbon source
selection and metabolic pathway optimization will be crucial factors.
Consequently, we tested various concentrations of glucose in the fermentation
culture medium, ranging from 0 g/L to 20 g/L, and estimated the final L-theanine
yield. Using standard L-Theanine at 0 mg/L, 50 mg/L, and 200 mg/L to produce a
standard curve, our results indicate that added glucose concentrations between
15 g/L and 20 g/L could yield approximately 38 mg/L of L-theanine. The findings
clearly demonstrate a strong dependence of yield on glucose supplementation levels.

Moving forward, we could consider employing both glucose and glycerol as dual
carbon sources to optimize the metabolic pathway. This would allow glycerol to be
independently converted into Acetyl-CoA, preventing competition during the
metabolic process.

Secondly, we could delve deeper into optimizing the enzymes within the synthetic
pathway by researching and testing enzymes with similar functions from different
species, selecting those with the highest production yields.

Thirdly, due to time and experimental capacity constraints, our current project
only attempted L-theanine production using E. coli for fermentation at the
laboratory scale. For future yield enhancement, we could optimize the fermentation
system further, such as considering alternative host strains (chassis) that
naturally accumulate higher levels of amino acids, particularly glutamate, like
actinobacteria. Additionally, we should consider experiments at larger reaction
volumes for production.

Fourthly, there is room for optimization in the expression system, including
aspects such as fermentation temperature, auxiliary additives (such as sodium
hexametaphosphate used in this fermentation), and the pH of the reaction system.

Furthermore, another significant advantage of designing L-theanine synthesis
using a de novo approach is the potential application of the produced metabolites
as probiotics for anxiety mitigation, should our production method pass safety
evaluations. This represents a potential application area that traditional
enzymatic production methods cannot achieve.

### References

[1] Hagihara R, Ohno S, Hayashi M, Tabata K, Endo H. Production of l-Theanine by
Escherichia coli in the Absence of Supplemental Ethylamine. Appl Environ
Microbiol. 2021 May 11;87(11):e00031-21.

<https://doi.org/10.1128/AEM.00031-21>

[2] Cao, R., Hu, S., Lu, Y., Wang, W., Fu, Z., & Cheng, J. (2023). Fermentative
Production of L-Theanine in Escherichia coli via the Construction of an Adenosine
Triphosphate Regeneration System. Fermentation, 9(10), 875–875.

<https://doi.org/10.3390/fermentation9100875>

[3] Hidese S, Ogawa S, Ota M, Ishida I, Yasukawa Z, Ozeki M, Kunugi H. Effects of
L-Theanine Administration on Stress-Related Symptoms and Cognitive Functions in
Healthy Adults: A Randomized Controlled Trial. Nutrients. 2019 Oct 3;11(10):2362.

<https://www.mdpi.com/2072-6643/11/10/2362>

[4] Anas Sohail A, Ortiz F, Varghese T, Fabara SP, Batth AS, Sandesara DP,
Sabir A, Khurana M, Datta S, Patel UK. The Cognitive-Enhancing Outcomes of
Caffeine and L-theanine: A Systematic Review. Cureus. 2021 Dec 30;13(12):e20828.

<https://doi.org/10.7759/cureus.20828>

[5] Kakuda, T., et al. (2002). "Inhibitory effect of theanine on the
binding of [3H]AMPA, [3H]kainate, and [3H]MDL 105,519 to glutamate receptors in
rat cortical neurons." Biosci Biotechnol Biochem. 66 (12): 2683-2686.

<https://pubmed.ncbi.nlm.nih.gov/12596867/>

[6] Nathan, P. J., et al. (2006). "The neuropharmacology of L-theanine
(N-ethyl-L-glutamine)." J Herb Pharmacother. 6 (2): 21-30.

<https://pubmed.ncbi.nlm.nih.gov/17182482/>

[7] Kakuda, T., et al. (2002). "Neuropsychopharmacological effects of theanine."
Nutrition. 18 (9): 843-846.

<https://www.sciencedirect.com/science/article/pii/S0899900702900114>

[8] Kimura, K., et al. (2007). "L-Theanine reduces psychological and physiological
stress responses." Biol Psychol. 74 (1): 39-45.

<https://pubmed.ncbi.nlm.nih.gov/16930802/>

[9] Wang, L., Brennan, M., Li, S., Zhao, H., Lange, K. W., Brennan, C., et al.
(2022). How does the tea L-theanine buffer stress and anxiety. Food Science and
Human Wellness, 11(1), 467-475.

<https://doi.org/10.1016/j.fshw.2021.12.004>

### Catalogue

* [Overview](#p0-5)
* [Building: Construction of Plasmid](#p1-5)
* [Test: Enzyme Expression in BL21
   and Fermentation for L-Theanine](#p2-5)
* [Learn: Analysis of Results and
   Plan for Further Developments](#p3-5)
* [References](#p4-5)

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# Experiments

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* Protocols

![Image](static/picture/innar-page-shape2.png)

### Extraction of Plasmids

1. Take 1.5-5.0 mL of overnight bacterial culture, add it to a new 2 mL
centrifuge tube, and centrifuge at 6000 rpm for 5 minutes at room temperature to
pellet the bacteria. Discard the supernatant.
2. Add 250 µL of Buffer K1 to the tube and vigorously vortex the bacterial pellet
to completely disperse the cells.
3. Add 250 µL of Buffer K2 to the tube and gently invert the tube 5-10 times to
mix. Let it stand at room temperature for 2-5 minutes.
4. Add 350 μL of Buffer K3 to the tube, and gently invert the centrifuge tube
10-20 times until a white flocculent precipitate forms.
5. Centrifuge at 12,000 rpm for 10 minutes at 4°C, or centrifuge at room
temperature at 12,000 rpm for 3-4 minutes.
6. Carefully transfer the supernatant from the centrifuged cells into the
adsorption column, and centrifuge at 12,000 rpm for 30 seconds at room temperature
to allow the lysate to completely pass through the column. Discard the filtrate in
the collection tube.
7. Place the column back into a 2mL collection tube, add 700 μL of Buffer KW to
the column, centrifuge at 12,000 rpm for 1 minute at room temperature, and discard
the flow-through.
8. Insert the column back into a 2 mL collection tube. Centrifuge at 14,000 rpm
for 1 minute at room temperature to remove any residual liquid from the column
matrix.
9. Place the column into a clean 1.5 mL centrifuge tube, add 50 µL of Buffer KE
to the center of the column membrane, leave it at room temperature for 5 minutes,
and centrifuge at 13,000 rpm for 1 minute to elute the DNA.
10. Plasmid DNA is stored at 4°C.

### Cultivation of Bacteria

1. Transform the plasmids BsAld-CsAlaDc, gnp1, PPK, and gmas into BL21 E.coli
competent cells, and the empty plasmid into DH5α E.coli competent cells.
2. Place the bacteria with plasmids BsAld-CsAlaDc, gnp1, PPK, and gmas on solid
LBs which contain amplify to culture them, and place the bacteria with the empty
plasmid on solid LBs which contain kanamycin to culture them.
3. Pick the best monoclonal colony of each kind of bacteria, and put the picked
colony of bacteria with plasmids BsAld-CsAlaDc, gnp1, PPK, and gmas into liquid LB
which contain amplify, the picked colony of the bacteria with empty plasmid into
liquid LB which contain kanamycin.

### PCR

1. Use SnapGene to design primers for the empty plasmid and plasmids
BsAld-CsAlaDc, gnp1, PPK, and gmas:

![](https://static.igem.wiki/teams/5235/experiments/image1.png)

2. Add 8μl ddH2O, 10μl mix, 0.5μl forward primer, 0.5μl reverse primer,
and the template of the empty plasmid and plasmids BsAld-CsAlaDc, gnp1, PPK,
and gmas into tubes respectively.
3. Put these tubes into PCR machine with the temperature of 58℃. After
the process of PCR, check if the results match the expected ones, BsAld-CsAlaDc
with 2636 bp, gnp1 with 215 bp, PPK with 2095 bp, and gmas with 1395 bp.

### Golden Gate Assembly

1. Add 1μl PGGA, 2μl T4 DNA Ligase Buffer (10X), 2μl NEBridge Golden Gate
Enzyme Mix, and 9μl H2O into a tube.
2. Put the tube into a thermal cycler, setting “(37 C, 1 min to 16 C, 1 min)
times 30 to 60 C, 5 min.”
3. Transform 2μl into 50μl BL21 E. coli competent cells.
4. Use SnapGene to design primers of this full plasmid:

![](https://static.igem.wiki/teams/5235/experiments/image2.png)

5. Test if this full plasmid can have the expecting base pair by DNA
electrophoresis, with 214 bp.

### Fermentation

1. Prepare the fermentation medium with tryptone 10 g/L, yeast extract 5 g/L,
MgCl2·6H2O 10 mmol/L, (NaPO3)6 150mmol/L, and glucose 15g/L.
2. Place the bacteria with full plasmid in the fermentation medium.
3. Add different amount of IPTG, 0μl, 0.25μl, 0.5μl, 0.75μl, and 1μl,
to each bacteria culture tube.
4. Send these samples to commercial laboratories to have a test of HPLC:
Centrifuge the fermented broth at 12,000 rpm, collect the supernatant,
pass it through a 0.22 µm filter membrane, and use a ZORBAX Extend 80A C18
4.6x250mm, 5µm column for HPLC with the following program:

![](https://static.igem.wiki/teams/5235/new/nc5.png)

### Catalogue

* [Extraction of Plasmids](#p0-5)
* [Cultivation of Bacteria](#p1-5)
* [PCR](#p2-5)
* [Golden Gate Assembly](#p3-5)
* [Fermentation](#p4-5)

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# Results

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* Results

![Image](static/picture/innar-page-shape2.png)

### 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)

### Catalogue

* [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)

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