iGEM 2018 — Team CDHSU-CHINA

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# Team:CDHSU-CHINA/Notebook

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TEAM

[Team Members](https://2018.igem.org/Team%3ACDHSU-CHINA/Team)
[Collaborations](https://2018.igem.org/Team%3ACDHSU-CHINA/Collaborations)

PROJECT

[Description](https://2018.igem.org/Team%3ACDHSU-CHINA/Description)
[Design](https://2018.igem.org/Team%3ACDHSU-CHINA/Design)
[Experiments](https://2018.igem.org/Team%3ACDHSU-CHINA/Experiments)
[Notebook](https://2018.igem.org/Team%3ACDHSU-CHINA/Notebook)
[InterLab](https://2018.igem.org/Team%3ACDHSU-CHINA/InterLab)
[Model](https://2018.igem.org/Team%3ACDHSU-CHINA/Model)
[Results](https://2018.igem.org/Team%3ACDHSU-CHINA/Results)
[Demonstrate](https://2018.igem.org/Team%3ACDHSU-CHINA/Demonstrate)
[Improve](https://2018.igem.org/Team%3ACDHSU-CHINA/Improve)
[Attributions](https://2018.igem.org/Team%3ACDHSU-CHINA/Attributions)

PARTS

[Parts Overview](https://2018.igem.org/Team%3ACDHSU-CHINA/Parts)
[Basic Parts](https://2018.igem.org/Team%3ACDHSU-CHINA/Basic_Part)
[Composite Parts](https://2018.igem.org/Team%3ACDHSU-CHINA/Composite_Part)
[Part Collection](https://2018.igem.org/Team%3ACDHSU-CHINA/Part_Collection)

[SAFETY](https://2018.igem.org/Team%3ACDHSU-CHINA/Safety)

HUMAN PRACTICES

[Human Practices](https://2018.igem.org/Team%3ACDHSU-CHINA/Human_Practices)
[Education & Engagement](https://2018.igem.org/Team%3ACDHSU-CHINA/Public_Engagement)

AWARDS

[Applied Design](https://2018.igem.org/Team%3ACDHSU-CHINA/Applied_Design)
[Entrepreneurship](https://2018.igem.org/Team%3ACDHSU-CHINA/Entrepreneurship)
[Hardware](https://2018.igem.org/Team%3ACDHSU-CHINA/Hardware)
[Measurement](https://2018.igem.org/Team%3ACDHSU-CHINA/Measurement)
[Model](https://2018.igem.org/Team%3ACDHSU-CHINA/Model)
[Plant](https://2018.igem.org/Team%3ACDHSU-CHINA/Plant)
[Software](https://2018.igem.org/Team%3ACDHSU-CHINA/Software)

[JUDGING FORM ⇗](https://igem.org/2018_Judging_Form?team=CDHSU-CHINA)

# NoteBook

**February, 2018**, our team established

**May, 2018**, we began our first stage experiments, including collection of miracle fruit’s RNA and design of primer of PCR

**June, 2018**, the primer we designed was available. Then we did digestion and reconstruction of plasmid experiments, and we transfer the reconstructed plasmid into E.coli. The preparation for stage 2 experiment was finished.

**July and august, 2018**, we had more time to do the main experiment

**July, 2018**, we bought pve5523, a vital part of our main experiment. Then we began our stage 2 experiment. We doubly digested the reconstructed plasmid and DNA of miracle fruit, and we connected those two together to get a reconstructed expressing plasmid

**The end of July, 2018**, we began our interlab experiments, including calibration 1, calibration 2, and calibration 3.

**The beginning of August, 2018**, we did cell measurement. At the middle of August, we improved our interlab experiment and the new data was accepted by IGEM authority

**August and September**, we finished the experiment that transferring the reconstructed expressing plasmid into E.coli. After we confirmed that DNA of miracle fruit can be expressed in E.coli, we transferred the reconstructed plasmid into lactic acid bacteria and fostered the trans-genetic lactic acid bacteria in September

**October, 2018**, we examined that miracle fruit’s DNA was successfully expressed in trans-genetic lactic acid bacteria.


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# Team:CDHSU-CHINA/Experiments

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TEAM

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PROJECT

[Description](https://2018.igem.org/Team%3ACDHSU-CHINA/Description)
[Design](https://2018.igem.org/Team%3ACDHSU-CHINA/Design)
[Experiments](https://2018.igem.org/Team%3ACDHSU-CHINA/Experiments)
[Notebook](https://2018.igem.org/Team%3ACDHSU-CHINA/Notebook)
[InterLab](https://2018.igem.org/Team%3ACDHSU-CHINA/InterLab)
[Model](https://2018.igem.org/Team%3ACDHSU-CHINA/Model)
[Results](https://2018.igem.org/Team%3ACDHSU-CHINA/Results)
[Demonstrate](https://2018.igem.org/Team%3ACDHSU-CHINA/Demonstrate)
[Improve](https://2018.igem.org/Team%3ACDHSU-CHINA/Improve)
[Attributions](https://2018.igem.org/Team%3ACDHSU-CHINA/Attributions)

PARTS

[Parts Overview](https://2018.igem.org/Team%3ACDHSU-CHINA/Parts)
[Basic Parts](https://2018.igem.org/Team%3ACDHSU-CHINA/Basic_Part)
[Composite Parts](https://2018.igem.org/Team%3ACDHSU-CHINA/Composite_Part)
[Part Collection](https://2018.igem.org/Team%3ACDHSU-CHINA/Part_Collection)

[SAFETY](https://2018.igem.org/Team%3ACDHSU-CHINA/Safety)

HUMAN PRACTICES

[Human Practices](https://2018.igem.org/Team%3ACDHSU-CHINA/Human_Practices)
[Education & Engagement](https://2018.igem.org/Team%3ACDHSU-CHINA/Public_Engagement)

AWARDS

[Applied Design](https://2018.igem.org/Team%3ACDHSU-CHINA/Applied_Design)
[Entrepreneurship](https://2018.igem.org/Team%3ACDHSU-CHINA/Entrepreneurship)
[Hardware](https://2018.igem.org/Team%3ACDHSU-CHINA/Hardware)
[Measurement](https://2018.igem.org/Team%3ACDHSU-CHINA/Measurement)
[Model](https://2018.igem.org/Team%3ACDHSU-CHINA/Model)
[Plant](https://2018.igem.org/Team%3ACDHSU-CHINA/Plant)
[Software](https://2018.igem.org/Team%3ACDHSU-CHINA/Software)

[JUDGING FORM ⇗](https://igem.org/2018_Judging_Form?team=CDHSU-CHINA)

# Experiments

The following is our experimental procedure:
1.The isolation of RNA of Miracle fruit:
First, we are going collect the RNA of Miracle fruits we bought from online shop. The RNA of miracle fruits could only be expressed in the mature miracle fruit.
![](https://static.ouorz.com/%E5%B1%8F%E5%B9%95%E5%BF%AB%E7%85%A7%202018-10-16%2010.18.32%20PM.png)
2.Then we transcribe the RNA to cDNA

3.Design the PCR primer according the sequence of miracle fruit’s DNA on NCBI
![](https://static.ouorz.com/%E5%B1%8F%E5%B9%95%E5%BF%AB%E7%85%A7%202018-10-16%2010.18.44%20PM.png)
4.Use PCR method to amplify the miracle fruit’s DNA
![](https://static.ouorz.com/%E5%B1%8F%E5%B9%95%E5%BF%AB%E7%85%A7%202018-10-16%2010.18.53%20PM.png)
5.Do the DNA sequencing and compare the result to the standard DNA sequence

6.Digest the the amplified Miracle fruit’s DNA, connect the digested part to T-carrier. Then transfer the re-constructed plasmid to competent cell of E.coli
![](https://static.ouorz.com/%E5%B1%8F%E5%B9%95%E5%BF%AB%E7%85%A7%202018-10-16%2010.19.03%20PM.png)
![](https://static.ouorz.com/%E5%B1%8F%E5%B9%95%E5%BF%AB%E7%85%A7%202018-10-16%2010.18.58%20PM.png)
7.Smear the E. coli with re-constructed plasmid on the plate with Ampicillin, select the E. coli that could grow on the plate normally. Then we do colony PCR of the selected E. coli. After the PCR operation, the DNA of miracle fruits that shows amplification is confirmed to be positive clone. Compare the sequence of miracle fruit’s DNA gained through amplification to standard one to check if there is a mutation
![](https://static.ouorz.com/%E5%B1%8F%E5%B9%95%E5%BF%AB%E7%85%A7%202018-10-16%2010.19.16%20PM.png)
8.Examine the pve5523(the expressing vector of lactobacillus) to make sure it is usable

9.Collect the plasmid of pve5523 and E. coli with re-constructed plasmid, do the double-digestion, recycle the fragments of MF’s DNA located in the E.coli plasmid with re-constructed plasmid and fragments of pve5233 after the digestion individually
![](https://static.ouorz.com/%E5%B1%8F%E5%B9%95%E5%BF%AB%E7%85%A7%202018-10-16%2010.19.44%20PM.png)
![](https://static.ouorz.com/%E5%B1%8F%E5%B9%95%E5%BF%AB%E7%85%A7%202018-10-16%2010.19.54%20PM.png)
10.Use T4 ligase to connect fragments of MF’s DNA to the fragments of pve5233(both of fragments gained from recycle), then we get a new plasmid involves those two fragments

11.Transfer the new re-constructed plasmid into the competent cell of E.coli, smear the new E.coli above on the plate with clarithromycin. Then we could select the E.coli that could normally grow, and do the colony PCR of the normal growth E.coli. After the colony PCR, the E.coli has amplified DNA of MF is comfirmed to be positive clone. Compare the amplified DNA of MF to the standard one to check if there is a mutation

12.Digest the pve5233, connect the fragment to DNA of MF, so we have the re-constructed expressing plasmid
![](https://static.ouorz.com/%E5%B1%8F%E5%B9%95%E5%BF%AB%E7%85%A7%202018-10-16%2010.20.02%20PM.png)
13.Transfer the re-constructed plasmid to the competent cell of E.coli

14.Smear the E.coli with re-constructed expressing plasmid on the plate with clarithromycin, select the the normally growing E.coli and do colony PCR. After PCR, the E.coli with production is confirmed to be positive clone. Compare the result to the standard DNA sequence to check if there is a mutation

15.Foster the E.coli with re-constructed expressing plasmid, use Sds-page method to check there is miraculin
![](https://static.ouorz.com/%E5%B1%8F%E5%B9%95%E5%BF%AB%E7%85%A7%202018-10-16%2010.20.06%20PM.png)
16.Collect the plasmid of E.coli with re-constructed plasmid, and transfer the plasmid into the competent cell of lactic acid bacteria

17.Smear the lactic acid bacteria with re-constructed E.coli plasmid on the plate with clarithromycin select the normally growing bacteria and do colony PCR. After PCR, the bacteria with MF’s DNA is confirmed to be positive clone. Then compare the amplified MF’s DNA sequence to the standard sequence to check if there is mutation

18.Foster the lactic acid bacteria with re-constructed plasmid, use Sds-page method to check if there is miraculin expressing

19.The lactic acid bacteria expresses miraculin

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# Team:CDHSU-CHINA/Results

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[HOME](https://2018.igem.org/Team%3ACDHSU-CHINA)

TEAM

[Team Members](https://2018.igem.org/Team%3ACDHSU-CHINA/Team)
[Collaborations](https://2018.igem.org/Team%3ACDHSU-CHINA/Collaborations)

PROJECT

[Description](https://2018.igem.org/Team%3ACDHSU-CHINA/Description)
[Design](https://2018.igem.org/Team%3ACDHSU-CHINA/Design)
[Experiments](https://2018.igem.org/Team%3ACDHSU-CHINA/Experiments)
[Notebook](https://2018.igem.org/Team%3ACDHSU-CHINA/Notebook)
[InterLab](https://2018.igem.org/Team%3ACDHSU-CHINA/InterLab)
[Model](https://2018.igem.org/Team%3ACDHSU-CHINA/Model)
[Results](https://2018.igem.org/Team%3ACDHSU-CHINA/Results)
[Demonstrate](https://2018.igem.org/Team%3ACDHSU-CHINA/Demonstrate)
[Improve](https://2018.igem.org/Team%3ACDHSU-CHINA/Improve)
[Attributions](https://2018.igem.org/Team%3ACDHSU-CHINA/Attributions)

PARTS

[Parts Overview](https://2018.igem.org/Team%3ACDHSU-CHINA/Parts)
[Basic Parts](https://2018.igem.org/Team%3ACDHSU-CHINA/Basic_Part)
[Composite Parts](https://2018.igem.org/Team%3ACDHSU-CHINA/Composite_Part)
[Part Collection](https://2018.igem.org/Team%3ACDHSU-CHINA/Part_Collection)

[SAFETY](https://2018.igem.org/Team%3ACDHSU-CHINA/Safety)

HUMAN PRACTICES

[Human Practices](https://2018.igem.org/Team%3ACDHSU-CHINA/Human_Practices)
[Education & Engagement](https://2018.igem.org/Team%3ACDHSU-CHINA/Public_Engagement)

AWARDS

[Applied Design](https://2018.igem.org/Team%3ACDHSU-CHINA/Applied_Design)
[Entrepreneurship](https://2018.igem.org/Team%3ACDHSU-CHINA/Entrepreneurship)
[Hardware](https://2018.igem.org/Team%3ACDHSU-CHINA/Hardware)
[Measurement](https://2018.igem.org/Team%3ACDHSU-CHINA/Measurement)
[Model](https://2018.igem.org/Team%3ACDHSU-CHINA/Model)
[Plant](https://2018.igem.org/Team%3ACDHSU-CHINA/Plant)
[Software](https://2018.igem.org/Team%3ACDHSU-CHINA/Software)

[JUDGING FORM ⇗](https://igem.org/2018_Judging_Form?team=CDHSU-CHINA)

# Results

The left graph shows the standard RNA of miracle fruits, and the right graph shows the RNA of miracle fruits that we gain. The DNA molecular weight showed in two graphs matches each other, confirming that we actually collect the authentic RNA of miracle fruit.

![](https://static.ouorz.com/%E5%B1%8F%E5%B9%95%E5%BF%AB%E7%85%A7%202018-10-17%208.11.09%20AM.png)
![](https://static.ouorz.com/%E5%B1%8F%E5%B9%95%E5%BF%AB%E7%85%A7%202018-10-17%208.11.36%20AM.png)

We designed the PCR primer of miracle fruit’s cDNA according to primer principle and our seqeuncing result.

* **F**  tcccccggg ATGAAGGAAT TAACAATGCT CTC
* **R**  ggggtaccTTAGAAGTATACGGTTTTGTTG

![](https://static.ouorz.com/3107D22D3EE6690AAF23B642F958C9F8.jpg)
This is the result of RT-PCR. There were 7 tubes of PCR products, but only two tubes are usable. The seqeunce result of this PCR operation is matched to the standard miracle fruit’s seqeunce, which means the RT-PCR operation is successful, and there is no mutation of base pair.
![](https://static.ouorz.com/1A6E8F13871702DFD6FDD965530FFDD8.jpg)
We digested the DNA we gained, then connect it to T-vector. After that, we transfer the re-constructed plasmid into the competent cell of E.coli.

![](https://static.ouorz.com/60937EF82D514B7ACBCAA48BFA7D4B6F.jpg)
![](https://static.ouorz.com/%E5%B1%8F%E5%B9%95%E5%BF%AB%E7%85%A7%202018-10-17%208.18.43%20AM.png)
![](https://static.ouorz.com/%E5%B1%8F%E5%B9%95%E5%BF%AB%E7%85%A7%202018-10-17%208.19.44%20AM.png)

Smear the E. coli with re-constructed plasmid on the plate with Ampicillin, select the E. coli that could grow on the plate normally. Then we do colony PCR of the selected E. coli. After the PCR operation, the DNA of miracle fruits that shows amplification is confirmed to be positive clone.

![](https://static.ouorz.com/90318B461FD7D3AC108C847D275989D9.jpg)
![](https://static.ouorz.com/%E5%B1%8F%E5%B9%95%E5%BF%AB%E7%85%A7%202018-10-17%208.20.47%20AM.png)

Connect the competent cell of lactic acid bacteria to green fluorescent protein. After fostering, the green fluorescent is expressing, confirming that competent cell of lactic acid bacteria is usable.

![](https://static.ouorz.com/6094B350433E564D8E38E8BBB7EC5A8A.jpg)
![](https://static.ouorz.com/97162CE9DE7244EAB40F90C754753AF1.jpg)

We collected the plasmid of E.coli. After the digestion, we reconstructed the digestive fragment of pve5523 with the plasmid of E.coli.

![](https://static.ouorz.com/13646E098B4C0008FB0895C02E439775.jpg)
![](https://static.ouorz.com/%E5%B1%8F%E5%B9%95%E5%BF%AB%E7%85%A7%202018-10-17%208.25.29%20AM.png)
![](https://static.ouorz.com/FC00807163C41ACB779CECC32DBE37AE.jpg)

We transferred the reconstructed plasmid into E.coli and foster the E.coli. After that, we collected the plasmid of that E.coli and transferred the plasmid into the competent cell of lactic acid. Finally, we got the trans-genetic lactic acid bacteria.
The Sds-page examine for lactic acid bacteria

![](https://static.ouorz.com/ABB60D4F3A534D2A5E4A4C3CD7429A9A.jpg)
![](https://static.ouorz.com/A9622FA27AB1D797BE4194D0D8E59245.jpg)