iGEM 2018 — Team NAU-CHINA

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# Team:NAU-CHINA/Notebook

Template:2018\_NAU-CHINA

header

[![](https://static.igem.org/mediawiki/2018/4/4d/T--NAU-CHINA--MOSFET_LOGO.png)](https://2018.igem.org/Team%3ANAU-CHINA)

* TEAM
  + [Team members](https://2018.igem.org/Team%3ANAU-CHINA/Team_members)
  + [Collaborations](https://2018.igem.org/Team%3ANAU-CHINA/Collaborations)
  + [Attributions](https://2018.igem.org/Team%3ANAU-CHINA/Attributions)
* PROJECT
  + [Overview](https://2018.igem.org/Team%3ANAU-CHINA/Overview)
  + [Design](https://2018.igem.org/Team%3ANAU-CHINA/Design)
  + [InterLab](https://2018.igem.org/Team%3ANAU-CHINA/InterLab)
  + [Protocols](https://2018.igem.org/Team%3ANAU-CHINA/Protocols)
  + [Application Prospects](https://2018.igem.org/Team%3ANAU-CHINA/Application_prospects)
* RESULTS
  + [Demonstrate](https://2018.igem.org/Team%3ANAU-CHINA/Demonstrate)
  + [Basic Part](https://2018.igem.org/Team%3ANAU-CHINA/Basic_Part)
  + [Composite Part](https://2018.igem.org/Team%3ANAU-CHINA/Composite_Part)
  + [Improved Parts](https://2018.igem.org/Team%3ANAU-CHINA/Improve)
  + [Part Collection](https://2018.igem.org/Team%3ANAU-CHINA/Part_Collection)
* MODEL
  + [Overview](https://2018.igem.org/Team%3ANAU-CHINA/Model)
  + [Model Illustration](https://2018.igem.org/Team%3ANAU-CHINA/Model%20Details)
  + [Auxiliary Understanding](https://2018.igem.org/Team%3ANAU-CHINA/Auxiliary%20Understanding)
* [iGEMCloud](https://2018.igem.org/Team%3ANAU-CHINA/Software)
* HUMAN PRACTICES
  + [Overview](https://2018.igem.org/Team%3ANAU-CHINA/Human_Practices)
  + [Integrated HP](https://2018.igem.org/Team%3ANAU-CHINA/IntegratedHP)
  + [Public Engagement](https://2018.igem.org/Team%3ANAU-CHINA/Public_Engagement)
* [SAFETY](https://2018.igem.org/Team%3ANAU-CHINA/Safety)
* [NOTEBOOK](https://2018.igem.org/Team%3ANAU-CHINA/Notebook)

![](#)

notebook

NOTEBOOK

# February

## Week 1

18/02/2018-24/02/2018

On January 18th, the team held a meeting to arrange winter vacation learning tasks.

From January 18th to January 21st, Wu Yaxuan and former iGEM team members gave us experimental skills training.

## week 2

25/02/2018-03/03/2018

On February 27th, the team held a meeting and reported the results of the brain storm for the first time. In the following period, our respective teams analyzed and improved the results of the brain storm.

# March

## Week 5

18/03/2018-24/03/2018

On March 24th, the team held a meeting and reported the brainstorm results for the second time. Next, analyze and improve the ideas of each group. Some of the team members also went to the Shanghai Academy of Sciences to consult the relevant issues.

## week 6

25/03/2018-31/03/2018

On March 30th, The college instructor and other professors participated in our report, and we finalized the topic after the meeting.

# May

## Week 12

06/05/2018-12/05/2018

On May 12th, Fang Chengzhu connected TetO-miniCMV to pEGFP-N1 and verified by sequencing.

# June

## Week 17

10/06/2018-16/06/2018

On June 10th , Yu Yuan and Ge Jitao used the plasmid provided by Fudan University to replace the intracellular domain of Syn-Notch with TEV.
 On June 14th, Fang Chengzhu connected TetO-miniCMV-EGFP to V2 and verified by sequencing.

## Week 18

17/06/2018-23/06/2018

From June 14th to June 17th , Zhou Jiaxin and Wang Chunfeng successfully constructed the EF1α-SV40 plasmid.
From June 17th to June 22nd, Zhou Jiaxin and Wang Chunfeng successfully constructed the Ubc-SV40 plasmid.
On June 19th, Yu Yuan and Ge Jitao initially connected the modified synnotch to the lenticrisper V2 plasmid.

## Week 19

24/06/2018-30/06/2018

On June 25th, Fang Chengzhu connected TetO-UbC-EGFP and TetO-EF1α-EGFP into pEGFP-N1 and verified by sequencing.

# July

## Week 23

22/07/2018-28/07/2018

From July 12th to July 23rd, Zhou Jiaxin and Wang Chunfeng successfully constructed three standard parts of EF1α-EGFP, Ubc-EGFP and miniCMV-EGFP.
From July 15th to July 25th, some members went to the Shanghai Academy of Health Sciences for an interview.

![](https://static.igem.org/mediawiki/2018/8/85/T--NAU-China--notebook1.jpg)
![](https://static.igem.org/mediawiki/2018/4/41/T--NAU-China--notebook2.jpg)

From July 21st to July 25th, Ge Jitao used the homologous recombination to correct and recombine the anti-GFP-mnotch-tev fragment into the V2 plasmid.
On July 27th, we carried out popular science propaganda in Sun Yat-sen's Mausoleum.

![](https://static.igem.org/mediawiki/2018/6/6b/T--NAU-China--notebook5.jpg)

## Week 24

28/07/2018-04/08/2018

From July 23th to July 28th, We interviewed Nanjing residents at different locations in Nanjing.

![](https://static.igem.org/mediawiki/2018/e/ea/T--NAU-China--notebook3.jpg)
![](https://static.igem.org/mediawiki/2018/d/d9/T--NAU-China--notebook4.jpg)

On July 28th, the original lenticrisper V2 plasmid was modified to make it stable.
On July 29th, Fang Chengzhu connected TetO-EF1α-EGFP to V2 and verified by sequencing..
From July 30th to August 2nd, Some players participated in the Asia-Pacific exchange meeting..
On August 4th, Fang Chengzhu connected the recombination sites attB and attP of Bxb1 into pEGFP-N1 and verified by sequencing.

# August

## Week 25

05/08/2018-11/08/2018

From July 25th to August 5th, Zhou Jiaxin and Wang Chunfeng successfully constructed a reporter gene plasmid.
 On August 5th, Yu Yuan originally constructed IL6scfv on the PEGFP-N1 plasmid.
On August 9th, Fang Chengzhu connected the recombination sites attB and attP of TP901 and PhiC31 into pEGFP-N1 and verified by sequencing.

## Week 26

12/08/2018-18/08/2018

On August 13th, TetR was constructed on the Clastblast plasmid.
From July 26th to August 15th, Ge Jitao used the homologous recombination to construct the anti-GFP-mnotch-tev standard part, but there was no result.
From August 15th to August 18th, Ge Jitao constructed the anti-GFP-mnotch-tev standard part using homologous recombination.

## Week 27

19/08/2018-25/08/2018

On August 19th, Fang Chengzhu connected TetO-UbC-EGFP to V2 and verified by sequencing.
On August 20th, Fang Chengzhu connected mCherry into the recombination sites attB and attP of Bxb1 and TP901 and verified by sequencing.
On August 21st, Yuyuan built Surface-expressed GFP on lenticrisper V2 plasmid, but the sequencing was bimodal and was further sequenced.
From August 19th to August 25th, Ge Jitao used homologous recombination to construct Bxb1 site reporter, PhiC31 site reporter, and TP901 site reporter standard part.
On August 25th, Fang Chengzhu connected mCherry to the recombination site attB and attP of PhiC31 and verified by sequencing.
From August 26th to August 31st, Xu Sheng successfully constructed miniCMV-Bxb1-V2 (none, nls, flag), miniCMV-tp901-V2 (none, nls, flag), EF1α-Bxb1-V2 (none, nls, Flag), EF1α-tp901-V2 (none, nls, flag), ubc-tp901-V2 (none, nls, flag).

# September

## Week 29

02/09/2018-08/09/2018

From August 30th to September 3rd , Zhou Jiaxin and Wang Chunfeng successfully constructed miniCMV-phiC31-V2 (none, nls, flag), Ubc-phiC31-V2 (none, nls, flag).

From August 26th to September 5th, Ge Jitao used EcoR1 and Pst1 enzyme enzyme enzymes to construct the standard part of Bxb1, PhiC31 and TP901 coding sequences.
From September 1st to September 6th, Xu Sheng successfully constructed miniCMV-Bxb1-V2, miniCMV-tp901-V2, EF1α-tp901-V2
From September 3rd to September 6th, Zhou Jiaxin and Wang Chunfeng successfully constructed EF1α-phiC31-V2 (none, nls, flag)
From September 7th to September 8th, Xu Sheng successfully constructed EF1α-Bxb1-V2, ubc-tp901-V2, to be verified by sequencing.

## Week 30

09/09/2018-15/09/2018

From September 5th to September 15th, Ge Jitao used the homologous recombination to construct the Bxb1 site, PhiC31 site, and TP901 site standard parts, but there was no result.
On September 12th, all the team members reported on the summer work and assigned a wiki writing task.

## Week 32

23/09/2018-29/09/2018

On September 26th, all the team members reported on the summer work of the previous stage and collected wiki material.

footer

Acknowledgements

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![](https://static.igem.org/mediawiki/2018/1/13/T--NAU-CHINA--NJU.png)

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Useful Links

[Main Page](https://2018.igem.org/)

[Special Pages](https://igem.org/Special%3ASpecialPages)

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NAU-CHINA 2018 from


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# Team:NAU-CHINA/Experiments

Template:2018\_NAU-CHINA

# 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.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

### 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/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:NAU-CHINA/Results

Template:2018\_NAU-CHINA

header

[![](https://static.igem.org/mediawiki/2018/4/4d/T--NAU-CHINA--MOSFET_LOGO.png)](https://2018.igem.org/Team%3ANAU-CHINA)

* TEAM
  + [Team members](https://2018.igem.org/Team%3ANAU-CHINA/Team_members)
  + [Collaborations](https://2018.igem.org/Team%3ANAU-CHINA/Collaborations)
  + [Attributions](https://2018.igem.org/Team%3ANAU-CHINA/Attributions)
* PROJECT
  + [Overview](https://2018.igem.org/Team%3ANAU-CHINA/Overview)
  + [Design](https://2018.igem.org/Team%3ANAU-CHINA/Design)
  + [InterLab](https://2018.igem.org/Team%3ANAU-CHINA/InterLab)
  + [Protocols](https://2018.igem.org/Team%3ANAU-CHINA/Protocols)
  + [Application Prospects](https://2018.igem.org/Team%3ANAU-CHINA/Application_prospects)
* RESULTS
  + [Demonstrate](https://2018.igem.org/Team%3ANAU-CHINA/Demonstrate)
  + [Basic Part](https://2018.igem.org/Team%3ANAU-CHINA/Basic_Part)
  + [Composite Part](https://2018.igem.org/Team%3ANAU-CHINA/Composite_Part)
  + [Improved Parts](https://2018.igem.org/Team%3ANAU-CHINA/Improve)
  + [Part Collection](https://2018.igem.org/Team%3ANAU-CHINA/Part_Collection)
* MODEL
  + [Overview](https://2018.igem.org/Team%3ANAU-CHINA/Model)
  + [Model Illustration](https://2018.igem.org/Team%3ANAU-CHINA/Model%20Details)
  + [Auxiliary Understanding](https://2018.igem.org/Team%3ANAU-CHINA/Auxiliary%20Understanding)
* [iGEMCloud](https://2018.igem.org/Team%3ANAU-CHINA/Software)
* HUMAN PRACTICES
  + [Overview](https://2018.igem.org/Team%3ANAU-CHINA/Human_Practices)
  + [Integrated HP](https://2018.igem.org/Team%3ANAU-CHINA/IntegratedHP)
  + [Public Engagement](https://2018.igem.org/Team%3ANAU-CHINA/Public_Engagement)
* [SAFETY](https://2018.igem.org/Team%3ANAU-CHINA/Safety)
* [NOTEBOOK](https://2018.igem.org/Team%3ANAU-CHINA/Notebook)

![](#)

# results

Effects of cell surface-expressed GFP on synNotch-TEV.

![](https://static.igem.org/mediawiki/2018/thumb/5/59/T--NAU-China--results1.jpg/1200px-T--NAU-China--results1.jpg.png)

A. 1.Fluorescence microscope observation of HEK 293 T transfected with plasmid containing synNotch. The cells were cross-pnked with GFP. The results showed that synNotch can be located to the membrane. 2.Blank control (without transfection).
![](https://static.igem.org/mediawiki/2018/thumb/6/6e/T--NAU-China--result3.jpg/930px-T--NAU-China--result3.jpg.png)
![](https://static.igem.org/mediawiki/2018/thumb/a/a8/T--NAU-China--T--NAU-China--results4.jpg/1059px-T--NAU-China--T--NAU-China--results4.jpg.png)
B. Assay of the synNotch-TEV and FLAG-tagged TEV concentration affected by cell surface-expressed GFP. C,D. Relative level of the synNotch-TEV and FLAG-tagged TEV affected by cell surface-expressed GFP. synNotch-TEV affected by surface-expressed GFP can be resolved into FLAG-TEV and V5-mNotch. Data are mean ±S.E. (n=3). \*\*, p < 0.01; N.S., no significance.

Effects of tetR on GFP expression with tetO sequence.

![](https://static.igem.org/mediawiki/2018/thumb/5/56/T--NAU-China--result5.jpg/908px-T--NAU-China--result5.jpg.png)

Stably transferred cell pne containing tetO-miniCMV-EGFP sequence.
1. Expression of GFP 6 h after nucleofection with plasmid containing tetR.
2.Expression of GFP 72 h after nucleofection.

Pronuclear verification of recombinase

Two plasmids with different origins of reppcation were used for functional verification of the recombinase, which have different resistances. One of them is a reporter gene plasmid, which uses the constitutive promoter J23119. The recombination site is located on both sides of the promoter, one side of the promoter is sfGFP, and the other side is mRFP. The other is a recombinase expression plasmid using PBAD, an inducible promoter, which is induced by arabinose. When the two plasmids were co-transfected into E. cop, the reporter plasmid expressed sfGFP, a green fluorescent protein, when the inducer arabinose was added, the recombinant enzyme was expressed, the promoter was inverted, and the mRFP red fluorescent protein was expressed.
Due to the use of two different resistant plasmids, kana and chloramphenicol, we used a plate containing two resistances of kana and chloramphenicol for screening, and grew many colonies, randomly selected 9 singles. After the colonies, we made colony PCR (Fig. 1) and the results showed that both plasmids were transferred.
![](https://static.igem.org/mediawiki/2018/4/40/T--NAU-China--protocal3.jpg)
Figure 1: A total of 9 single colonies were verified in the order of: single colony 1-9 recombinase expression plasmid vapdation, 2000 marker, single colony 1-9 reporter gene expression plasmid vapdation.
The verified E. cop was placed in M9 minimal medium supplemented with glycerol and arabinose, and the culture was divided into two at OD 600 = 0.1, designated time 0, and 0.2% glucose was added to each pair. In the pair. Samples were obtained at 0 and 30 minutes, 60 minutes and 120 minutes before the culture was isolated. (Refer to the description of BBa\_I0500)