iGEM 2022 — Team ibowu-china

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## What should this page contain?

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* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

## Inspirations

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* [2018 Munich](http://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

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{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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# Engineering

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## Engineering success

At this year's iGEM competition, we focused on Alzheimer's disease (AD), a neurodegenerative disease, and identified astragalin as a potential drug for the treatment of AD. In the preliminary project of iGEM, we did not find any project related to astragalin. Therefore, we synthesized some new gene sequences and uploaded them. The whole pathway in plants to produce astragalin contains three enzymes, F3H, FLS and UGT. Every sequence we submitted has been verified by sequencing and protein expression, and all of them can work well.
Detailed results are listed below.

![](https://static.igem.wiki/teams/4236/wiki/en1.png)

## His-ATUGT + EGFP

UGT is an enzyme catalyzing kaempferol into astragalin, and is the largest one among the three enzymes we used in our pathway (F3H, FLS, UGT), so we firstly constructed a His-ATUGT + EGFP vector to test the expression of UGT gene.

### Protocol we used:

1. Verification of the sequence. The sequence we submitted here was come from (Arabidopsisthaliana). In order to get better field, we has done codon optimization for E.coli expression for this sequence. We contacted with a biology company to synthesize the sequence.
2. We constructed it into a EGFP tagged plasmid and transformed the plasmids into E. coli BL21(DE3).
3. After culture overnight, we picked a single colony added it into 4 ml LB medium with the corresponding antibiotic. The mix was then shook at 37℃ until OD = 600.
4. Dividing the liquid medium into 23 parts, adding 1 mM IPTG to two of them for inducing the expression 16℃ at 12 h or 36 h . The other one added nothing to serve as a control.
5. Centrifuged the bacterial solution at 12000 g, the precipitation was mixed with RIPA as a lysis buffer. Added loading buffer to heat at 96℃ for 10 min, underwent SDS-PAGE and Coomassie brilliant blue staining for expression test.
According to our SDS-PAGE result, we could see a band at around 52.04kD, which is in line with the molecular weight of UGT. This band only occurred in IPTG group, which confirmed with our induction. Besides, UGT expression at 12 h was higher than that at 36 h. We proposed that this may be due to the large number of bacteria at 36 h and the high survival pressure, which is not conducive to protein expression. Taken together, we have successfully expressed UGT in E.coli.

![](https://static.igem.wiki/teams/4236/wiki/en2.png)

## AtF3H+AtFLS

F3H is an enzyme of 41.36 kD, which catalyzes naringenin into dihydrokaempferol. FLS is of 39.41 kD to catalyze dihydrokaempferol into kaempferol, we constructed an intact plasmid to express AtF3H and AtFLS respectively.

### Protocol we used:

1. Verification of the sequence. The sequence we submitted here was come from (Citrus sinensis, Citrus unshiu). In order to get better field, we has done codon optimization for E.coli expression for this sequence. We contacted with a biology company to synthesize the sequence.
2. We constructed it into a pETDuet plasmid and transformed the plasmids into E. coli BL21(DE3).
3. After culture overnight, we picked a single colony added it into 4 ml LB medium with the corresponding antibiotic. The mix was then shook at 37℃ until OD = 600.
4. Dividing the liquid medium into 23 parts, adding 1 mM IPTG to two of them for inducing the expression 16℃ at 12 h or 36 h . The other one added nothing to serve as a control.
5. Centrifuged the bacterial solution at 12000 g, the precipitation was mixed with RIPA as a lysis buffer. Added loading buffer to heat at 96℃ for 10 min, underwent SDS-PAGE and Coomassie brilliant blue staining for expression test.
According to our SDS-PAGE result, we could see two band at around 41.36 kD and 39.41 kD, which are in line with the molecular weight of F3H and FLS. The two band only occurred in IPTG group, which confirmed with our induction. in keeping with the previous conclusion, the expression level of F3H and FLS after 12 h induction were slightly higher than that with 36 h induction. Taken together, we have successfully expressed FSH and FLS in E.coli.

![](https://static.igem.wiki/teams/4236/wiki/en3.png)

## F3H-FLS+UGT

The final try is to construct a big plasmid consisting of three genes we have introduced to produce astragalin. In order to better express each enzyme, we tried to connect the smaller F3H and FLS with a linker to form a fusion protein, and the larger UGT was expressed separately. Therefore, F3H-FLS +UGT expression vector was constructed.

### Protocol we used:

1. We constructed it into a pETDuet plasmid and transformed the plasmids into E. coli BL21(DE3).
2. After culture overnight, we picked a single colony added it into 4 ml LB medium with the corresponding antibiotic. The mix was then shaken at 37℃ until OD = 600.
3. Dividing the liquid medium into 23 parts, adding 1 mM IPTG to two of them for inducing the expression 16℃ at 12 h or 36 h . The other one added nothing to serve as a control.
4. Centrifuged the bacterial solution at 12000 g, the precipitation was mixed with RIPA as a lysis buffer. Added loading buffer to heat at 96℃ for 10 min, underwent SDS-PAGE and Coomassie brilliant blue staining for expression test.
According to our SDS-PAGE result, we could see two band at around 81 kD and 51kD, which are in line with the molecular weight of F3H-FLS fusion protein and UGT. The two band only occurred in IPTG group, which confirmed with our induction. in keeping with the previous conclusion, the expression level of F3H and FLS after 12 h induction weas slightly higher than that with 36 h induction. Taken together, we have successfully expressed the whole pathway to produce astragalin in E.coli.

![](https://static.igem.wiki/teams/4236/wiki/en4.png)

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{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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# Experiments

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### Molecular Cloning

【1】PCR Primers were designed by snapgene software and synthesized at BGI Company. PCR was performed by following the manuals of PrimeSTAR Max Premix (2X) (Taraka, R045) or 2 X EasyTaq PCR SuperMix (+ dye) (Transgene, M256). The former was used for construction and the latter was used for identification.
【2】Gel extraction
DNA fragment were separated by agarose gel electrophoresis and then recycled by TIANgeo Midi Purification Kit (TIANGEN, DP209-02) or AxyPrep DNA gel Extraction Kit (AXYGEN, AP-GX-250).
【3】Gibson
Gibson was performed by following the manual of pEASY- Basic Seamless Cloning and Assembly Kit (Transgene, CU201).
【4】Endonuclease digestion and ligation
The endonucleases and T4 ligase were bought from NEB Company and the experiments were performed by following instructions.
【5】Transformation
Transformation was performed by following the manuals of Trans5α Chemically Competent Cell (Transgene, CD201) or Trans1-T1 Phage Resistant Chemically Competent Cell (Transgene, CD501).
【6】Plasmid extraction
Bacteria were cultured in lysogeny broth (LB) at 37℃ overnight and plasmid extraction was performed by following the manuals of TIANprep Mini Plasmid Kit (TIANGEN, DP103-02) or AxyPrep Plasmid Miniprep Kit (AXYGEN, AP-MN-P-250).
【7】Sequencing
Sanger sequencing was done by BGI Company and snapgene software is used to align the sequences.

### Expression and Measurements

1. Transform the plasmids into E. coli BL21(DE3)
2. Pick a single colony by a sterile tip from each of the LB plates for all the experimental and control groups. Add the colony into 4ml LB medium with kanamycin. Add 1mM IPTG to all experimental groups as needed. Incubate at 37 degree Celsius in a shaker overnight.
3. Fluorescence. Acquire 1-3 ml bacteria culture after overnight inoculation. Took fluorescence picture under UV illumination.
4. SDS-PAGE.
4.1 Take 1ml of bacterial solution, centrifuge at 12000g for 1min at 4℃, discard the supernatant, add 100ul of pre-cooled RIPA lysate (high), vortex to mix, settle for 5min, centrifuge at 4℃, 12000g for 10min, and take the supernatant which contains the protein extract. After protein quantification by BCA method using commercial test box, adjust the concentration of the protein solution to 1mg/ml. Take 4 parts of the protein solution and 1 part of 5X protein loading buffer and mix it in a boiling water bath for 5 minutes, centrifuge at 12000g at 4°C for 10 minutes, take the supernatant, and store on ice. Use precast gel purchased from commercial company (Transgen, Precast Tris-Glycine Gel) for protein electrophoresis.
4.2 Add marker at 5μl, and add sample on all other lanes with 10ul sample on each lane. Use 100V for electrophoresis. After the electrophoresis, take out the gel, put it in the staining box, add Coomassie Brilliant Blue dye solution to about 3mm below the gel surface, shake on a horizontal shaker, dye for 30min at room temperature, discard the dye solution, wash off the floating color with distilled water, and add decolorization liquid, decolorize on a horizontal shaker until the result is in good condition for taking pictures.

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{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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# Results

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## Overview

This year, iBowu\_China team paid attention to the potential value of astragalin in the treatment of Alzheimer's disease (AD), and tried to produce astragalin by synthetic biology method. As a natural flavonoid compound widely existing in medicinal plants, we first inquired the synthesis pathway of astragalin from scientific papers. With naringenin as the substrate, astragalin is catalyzed by three enzymes, F3H, FLS and UGT. We considered whether the three enzymes can be expressed in E.coli to give them the ability to produce astragalin.

![](https://static.igem.wiki/teams/4236/wiki/en1.png)

### Sequence verification:

The first work we completed was to determine the sequence information to be used this year. Our astragalin synthesis pathway contains a total of three enzymes, and we searched many homologous proteins in different medicinal plants. After discussion with the gene synthesis company, we selected three sequences suitable for E. coli expression: F3H from Citrus sinensis, FLS from Citrus unshiu, UGT from Arabidopsis thaliana. And then we asked them to synthesize these three sequences.

### Expression test for UGT

1. Verification of the sequence. The sequence we submitted here was come from (Citrus sinensis, Citrus unshiu). In order to get better field, we has done codon optimization for E.coli exprThe three enzymes F3H, FLS and UGT used in this year were 41.36 kD, 39.41 kD and 52.04 kD, respectively. Considering that for E.coli, the higher the molecular weight of the protein, the more difficult it is to express. Therefore, we first detected the largest enzyme UGT. We constructed His-ATUGT +EGFP expression vector, and after induced expression in E.coli BL21 by IPTG, we successfully saw the expression product of UGT gene.
At the same time, we also tested whether there were differences in the expression level under different induction times. According to our results, UGT expression at 12 h was higher than that at 36 h. We proposed that this may be due to the large number of bacteria at 36 h and the high survival pressure, which is not conducive to protein expression.

![](https://static.igem.wiki/teams/4236/wiki/re1.png)

![](https://static.igem.wiki/teams/4236/wiki/en2.png)

### Expression test for F3H and FLS

When we detected the expression of UGT gene, we accidentally selected a vector with GFP tag. We found that both UGT expression bands and GFP expression bands were seen on SDS-PAGE, which suggested that we could construct the two genes on the same vector.
Therefore, we constructed the first two enzymes of the astragalin production pathway, F3H and FLS, on the same vector. after transforming to E.coli and IPTG induction, we lysed the bacteria for SDS-PAGE and Coomassie brilliant blue staining at last.
This result was in line with our expectation. We detected the expression bands of F3H and FLS enzymes respectively, which indicated that all enzymes in the astragalin production pathway could be expressed in E.coli.
Besides, in keeping with the previous conclusion, the expression level of F3H and FLS after 12 h induction were slightly higher than that with 36 h induction.

![](https://static.igem.wiki/teams/4236/wiki/re2.png)

![](https://static.igem.wiki/teams/4236/wiki/en3.png)

### Construction for the whole astragalin production pathway

After verifying the expression of each enzyme, we tried to construct the three enzymes on the same plasmid. In order to better express each enzyme, we tried to connect the smaller F3H and FLS with a linker to form a fusion protein, and the larger UGT was expressed separately. Therefore, CSF3H-CuFLS +AtUGT expression vector was constructed.
We took this plasmid and transformed into to E.coli, under proper IPTG concentration induced for 12 h or 36 h, we lysed the bacteria for SDS-PAGE and Coomassie brilliant blue staining.
The results showed that we indeed obtained a fusion protein of about 80 kD and a UGT of about 50 kD. Therefore, we constructed a complete astragalin synthesis pathway on this vector.

![](https://static.igem.wiki/teams/4236/wiki/re3.png)

![](https://static.igem.wiki/teams/4236/wiki/en4.png)

### Astragalin production test

Based on the previous experimental results, we constructed a plasmid for astragalin synthesis and successfully expressed it in E.coli. However, does E.coli generate astragalin?
Since astragalin is a colorless substance, we felt confused to identify the product. Later, we asked relevant experts and carried out HPLC experiments under the advice of experts. We could clearly saw that a peak appeared at 8.164 min. This is consistent with the peak of the standard product astragalin at 8.15 min, which proves that our E.coli is indeed capable of producing astragalin.

![](https://static.igem.wiki/teams/4236/wiki/re4.png)

![](https://static.igem.wiki/teams/4236/wiki/re5.png)

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