iGEM 2020 — Team GDSYZX

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:GDSYZX/Notebook

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

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

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:GDSYZX/Engineering

[GDSYZX](https://2020.igem.org/Team%3AGDSYZX)

* PROJECT
  + [Description](https://2020.igem.org/Team%3AGDSYZX/Description)
  + [Design](https://2020.igem.org/Team%3AGDSYZX/Design)
  + [Results](https://2020.igem.org/Team%3AGDSYZX/Results)
  + [Contribution](https://2020.igem.org/Team%3AGDSYZX/Contribution)
* EXPERIMENT
  + [Implementation](https://2020.igem.org/Team%3AGDSYZX/Implementation)
  + [Engineering](https://2020.igem.org/Team%3AGDSYZX/Engineering)
  + [Proof of concept](https://2020.igem.org/Team%3AGDSYZX/Proof_Of_Concept)
  + [Parts](https://2020.igem.org/Team%3AGDSYZX/Parts)
* HP
  + [Human Practices](https://2020.igem.org/Team%3AGDSYZX/Human_Practices)
  + [Education](https://2020.igem.org/Team%3AGDSYZX/Education)
  + [Collaborations](https://2020.igem.org/Team%3AGDSYZX/Collaborations)
  + [Safety](https://2020.igem.org/Team%3AGDSYZX/Safety)
* TEAM
  + [Attributions](https://2020.igem.org/Team%3AGDSYZX/Attributions)
  + [Members](https://2020.igem.org/Team%3AGDSYZX/members)
* [PROTOCAL](https://2020.igem.org/Team%3AGDSYZX/protocal)

* [Project Background Investigation](#section1)
* [STEP 1 : Expression of Key Enzymes for Chlorogenic Acid Synthesis In Rice](#section2)
* [STEP 2 : HQT Expression Increases The Content of Chlorogenic Acid](#section3)
* [STEP 3 : Specific Expression of HQT In Rice Seeds](#section4)
* [Conclusion](#section5)
* [Further Research Directions](#section6)
* [References](#section7)

![](https://static.igem.org/mediawiki/2020/2/25/T--GDSYZX--chatu7.png)

# ENGINEERING

## Project Background Investigation

For more information and references, please see our [PROJECT DESCRIPTION](https://2020.igem.org/Team%3AGDSYZX/Description).

↓ The application of traditional Chinese medicine in the prevention and treatment of COVID-19.

↓ Commonness of traditional Chinese medicine prescriptions for COVID-19-Honeysuckle.

↓ The main active ingredient in honeysuckle-chlorogenic acid.

↓ Chlorogenic acid has clinical antiviral effects.

↓ Honeysuckle is not cheap and the production area is scarce.

↓ The price of rice is relatively cheap and the acceptance is high.

↓ Rice is a good choice for food tonic as a Chassis creature.[1]

## ---------------------------------------------------------

## STEP 1 : Expression of Key Enzymes for Chlorogenic Acid Synthesis In Rice

Research → Imagine → Design → Build → Test → Learn → Improve → Research...

### Research

HQT is a key enzyme for the synthesis of chlorogenic acid in honeysuckle, and rice contains precursor
substances required for the synthesis of chlorogenic acid.[2]

### Imagine

The key enzyme HQT for chlorogenic acid synthesis can be expressed heterologously in rice.

### Design

Use 35s as a promoter, pUC19-HA as a vector to express HQT heterologously in rice protoplasts.

[https://2020.igem.org/Team:GDSYZX/Design](https://2020.igem.org/Team%3AGDSYZX/Design)

### Build

HQT (BBa\_K3458001);

35S Promoter + HQT (BBa\_K3458003)

[https://2020.igem.org/Team:GDSYZX/Parts](https://2020.igem.org/Team%3AGDSYZX/Parts)

### Test

Use Western Blot to detect the expression level of HQT in rice protoplasts.

### Learn

Western Blot results showed that the target band of HQT could be detected in rice. HQT can be
expressed heterologously in rice.

[https://2020.igem.org/Team:GDSYZX/Results](https://2020.igem.org/Team%3AGDSYZX/Results)

### Improve

Confirm whether the expression of HQT can actually increase the content of chlorogenic acid.

## ---------------------------------------------------------

## STEP 2 : HQT Expression Increases The Content of Chlorogenic Acid

→ Imagine → Test → Learn → Improve → Research...

### Imagine

The expression of HQT gene can actually increase the content of chlorogenic acid in rice protoplasts.

### Test

Use the HPLC to detect the content of chlorogenic acid in p35S::HQT transfected rice protoplasts, and
compared with the chlorogenic acid standard and wild-type.

### Learn

The p35S::HQT transfected rice protoplasts can detect the same peak value as the standard product,
but the wild type does not. The expression of HQT can actually increase the content of chlorogenic
acid in rice.

[https://2020.igem.org/Team:GDSYZX/Results](https://2020.igem.org/Team%3AGDSYZX/Results)

### Improve

The HQT gene is specifically expressed in rice seeds.

## ---------------------------------------------------------

## STEP 3 : Specific Expression of HQT In Rice Seeds

Research → Imagine → Design → Build → Test → Learn → Improve → Research...

### Research

The GluD-1 promoter can promote gene specific expression in rice endosperm.[3]

### Imagine

GluD-1 promoter can promote the specific expression of HQT gene in rice endosperm.

### Design

Use GluD-1 as a promoter, pUC19-HA as a vector to transfected rice protoplasts.

### Build

GluD-1 Promoter(BBa\_K3458002)；

HQT (BBa\_K3458001)；

GluD-1 Promoter + HQT (BBa\_K3458004)

[https://2020.igem.org/Team:GDSYZX/Parts](https://2020.igem.org/Team%3AGDSYZX/Parts)

### Test

Use Western Blot to detect the expression level of HQT in rice protoplasts and use the HPLC to detect
the content of chlorogenic acid in pGluD-1::HQT transfected rice protoplasts, compare with the
p35S::HQT transfected rice protoplasts and wild-type.

### Learn

Western Blot results showed that the HQT couldn't be detected in pGluD-1::HQT transfected rice
protoplasts. HPLC results showed that the pGluD-1::HQT transfected rice protoplasts cannot detect the
same peak value as the standard product. Due to the endosperm specificity of the GluD-1 promoter, in
rice protoplasts, pGluD-1::HQT transfection has the same characteristics as wild-type. The GluD-1
promoter is specific.

[https://2020.igem.org/Team:GDSYZX/Results](https://2020.igem.org/Team%3AGDSYZX/Results)

### Improve

Do further testing in rice seeds.

## ---------------------------------------------------------

## Conclusion

We successfully expressed the HQT gene heterologously in rice protoplasts, and verified that the
expression of the HQT gene can indeed increase the content of chlorogenic acid in rice. In order to
express HQT specifically in rice seeds, we introduced the endosperm-specific promoter GluD-1 and
confirmed the specificity of GluD-1 promoter.

## ---------------------------------------------------------

## Further Research Directions

Due to the COVID-19, the laboratory has temporarily closed. Also, considering the safety of GMO
crops, our experimental plan stops at the stage of protoplasts. Although it is a pity that we have not
been able to further verify our ideas in rice seeds and conduct clinical tests, we have some ideas for
future research directions, which may be helpful.

**·** Use pGluD-1::HQT transfected protoplasts to obtain transformation seedlings;

**·** Detect the expression in other stages and parts of the transformed seedlings to verify the
specificity of expression;

**·** Cultivate transformed seedlings to obtain rice seeds;

**·** Test the HQT expression and chlorogenic acid content in rice seeds;

**·** Testing medicinal value;

**·** Edible safrty test;

**·** …………

## ---------------------------------------------------------

## References

[1]Engineering plants with increased levels of the antioxidant chlorogenic acid Ricarda
Niggeweg1,3,Anthony J Michael2 & Cathie Martin

[2]Cloning and Characterization of a cDNACoding a Hydroxycinnamoyl-CoAQuinate Hydroxycinnamoyl
Transferase Involved in Chlorogenic Acid Biosynthesis in Lonicera japonica Xiaoxiao Peng, Weidong Li,
Wenquan Wang, Genben Ba

[3]Characterization of a new rice glutelin gene GluD-1 expressed in the starchy endosperm Taiji
Kawakatsu1, Masayuki P. Yamamoto1,\*, Sakiko Hirose1, Masahiro Yano2 and Fumio Takaiwa1,

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:GDSYZX/Experiments

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:GDSYZX/Results

[GDSYZX](https://2020.igem.org/Team%3AGDSYZX)

* PROJECT
  + [Description](https://2020.igem.org/Team%3AGDSYZX/Description)
  + [Design](https://2020.igem.org/Team%3AGDSYZX/Design)
  + [Results](https://2020.igem.org/Team%3AGDSYZX/Results)
  + [Contribution](https://2020.igem.org/Team%3AGDSYZX/Contribution)
* EXPERIMENT
  + [Implementation](https://2020.igem.org/Team%3AGDSYZX/Implementation)
  + [Engineering](https://2020.igem.org/Team%3AGDSYZX/Engineering)
  + [Proof of concept](https://2020.igem.org/Team%3AGDSYZX/Proof_Of_Concept)
  + [Parts](https://2020.igem.org/Team%3AGDSYZX/Parts)
* HP
  + [Human Practices](https://2020.igem.org/Team%3AGDSYZX/Human_Practices)
  + [Education](https://2020.igem.org/Team%3AGDSYZX/Education)
  + [Collaborations](https://2020.igem.org/Team%3AGDSYZX/Collaborations)
  + [Safety](https://2020.igem.org/Team%3AGDSYZX/Safety)
* TEAM
  + [Attributions](https://2020.igem.org/Team%3AGDSYZX/Attributions)
  + [Members](https://2020.igem.org/Team%3AGDSYZX/members)
* [PROTOCAL](https://2020.igem.org/Team%3AGDSYZX/protocal)

* [1 Sequencing and comparison of gene HQT synthesis](#section1)
* [2 Cloning, recovery and purification of target gene HQT and promoter GluD-1](#section2)
  + [2.1 HQT electropherogram](#section21)
  + [2.2 GluD - 1 electropherogram](#section22)
* [3 Screening of positive clones-colony PCR & sequencing](#section3)
  + [3.1 Colony PCR electrophoresis](#section31)
  + [3.2 Sequencing results](#section32)
* [4 Western Blot detects the expression of the target protein](#section4)
  + [4.1 35s::HQT](#section41)
  + [4.2 GluD-1::HQT](#section42)
* [5 Extraction and detection of target product](#section5)

![](https://static.igem.org/mediawiki/parts/c/ca/T—GDSYZX—chatu2.jpg)

# RESULTS

## 1 Sequencing and comparison of gene HQT synthesis

After obtaining the genes synthesized by Tianyi Huiyuan Company, we conducted gene sequencing and
compared it with the previously designed gene sequences. The result showed that the HQT gene sequences
synthesized by tianyi Huiyuan Company were consistent with those designed by us, which was in line
with our expectation.

![](https://static.igem.org/mediawiki/parts/7/74/T—GDSYZX—result1.png)

fig.1

## ---------------------------------------------------------

## Cloning, recovery and purification of target gene HQT and promoter GluD-1

### 2.1 HQT electropherogram

The fig.2 is the DNA gel electrophoresis of the HQT gene after PCR. From it we can see that there is
clearly a bright band between 1000bp and 2000bp, which is basically the same size as our target gene
(the length of the HQT gene is 1320bp ), so we speculate that the band amplified by PCR is the HQT
gene.

![](https://static.igem.org/mediawiki/parts/e/ea/T—GDSYZX—result2.png)

fig.2

### 2.2 GluD - 1 electropherogram

The fig.3 is the DNA gel electrophoresis of the GluD-1 promoter after PCR. From it we can see that
there is clearly a bright band between 750bp and 2000bp, which is basically the same size as our
target gene (the length of the HQT gene is 1228bp ), so we speculate that the band amplified by PCR is
the GluD-1 promoter.

![](https://static.igem.org/mediawiki/parts/d/d7/T—GDSYZX—result3.png)

fig.3

## ---------------------------------------------------------

## 3 Screening of positive clones-colony PCR & sequencing

Colony PCR can help us eliminate false positive colonies and screen out colonies that can be
sequenced.

### 3.1Colony PCR electrophoresis

#### 3.1.1 35s::HQT

The fig.4 shows the electrophoresis of the colony PCR with the promoter of P35S::HQT vector. The gene
we designed to amplify is HQT. The length of the HQT gene is 1320bp. The electropherogram shows that
the first, second, third, and fifth wells have bands between 1000-2000bp (the size of HQT is about
1320). Therefore, this result indicating that the colony may contain our target gene. Next, these
colonies were expanded and cultured to extract plasmids for later sequencing verification.

![](https://static.igem.org/mediawiki/parts/2/21/T—GDSYZX—result4.png)

fig.4

#### 3.1.2 GluD-1::HQT

The fig.X shows the electrophoresis of the colony PCR with the promoter of PGluD-1::HQT vector. The
gene we designed to amplify is GluD-1. The length of the GluD-1 gene is 1228bp. The electropherogram
shows that all wells have bands between 1000-2000bp. Therefore, this result indicating that the colony
may contain our target gene. Next, these colonies were expanded and cultured to extract plasmids for
later sequencing verification.

![](https://static.igem.org/mediawiki/parts/0/01/T—GDSYZX—result5.png)

fig.5

### 3.2 Sequencing results

Sequencing results showed that colonies 1 and 2 picked from P35S::HQT positive colonies contained
the correct HQT gene sequence (fig.6), and colonies 1 and 3 picked from PGluD-1::HQT positive
colonies contained the correct GluD-1 gene sequence (fig.7). （Because we use the GluD-1 gene to
replace the 35S promoter on the P35S::HQT vector, we do not need to sequence the HQT gene.）

#### 3.2.1 35s::HQT

![](https://static.igem.org/mediawiki/parts/6/6d/T—GDSYZX—result6.png)

fig.6

#### 3.2.2 GluD-1::HQT

![](https://static.igem.org/mediawiki/parts/5/5c/T—GDSYZX—result7.png)

fig.7

## ---------------------------------------------------------

## 4 Western Blot detects the expression of the target protein

### 4.1 35s::HQT

The results of Western Blot showed that a large amount of HQT expression could be detected in rice
protoplasts transfected with 35s::HQT(fig.8). And the expression level of HQT increased with the
extension of the expression time. The expression level was the highest at 15h, and then the expression
level began to decline. It may be due to the death of some cells during the cell culture time. We
selected the protoplasts cultured for 15 hours and their culture medium for HPLC detection.

![](https://static.igem.org/mediawiki/2020/0/0c/T--GDSYZX--zu_3hqt.png)
![](https://static.igem.org/mediawiki/parts/3/3c/T—GDSYZX—result9.png)

fig.8

### 4.2 GluD-1::HQT

The results of Western Blot showed that the target band could not be detected in the rice protoplasts
transfected with GluD-1::HQT, which indicated that the HQT gene was not expressed (or the expression
level was too low to be detected), which further proved that GluD-1 promoter was endosperm specificity
(fig.9).

![](https://static.igem.org/mediawiki/2020/3/37/T--GDSYZX--zu_Ghqt.png)

fig.9

## ---------------------------------------------------------

## 5 Extraction and detection of target product

![](https://static.igem.org/mediawiki/2020/b/b2/T--GDSYZX--zu_hplc.png)

fig.10

We use HPLC to compared the wile type of Oryza sativa L. protoplast, chlorogenic acid standard
samples and our product sample obtained from the transfected protoplast (fig.10). The peak value of
the product measured in our product sample was basically consistent with the peak value of chlorogenic
acid samples. However, no overlapping peak with the protoplast standard sample was found in the
wild-type liquid chromatography, which proved that wild protoplasts do not produce chlorogenic
acid，and we can determine that the transfected protoplast can produce chlorogenic acid.