iGEM 2017 — Team NEU-China

[![](https://static.igem.org/mediawiki/2017/8/8b/HQ_page_logo.jpg)](https://2017.igem.org)

# Team:NEU-China/Notebook

[![](http://placehold.it/350x150)](https://2017.igem.org/Team%3ANEU-China)

MENU

[HOME](https://2017.igem.org/Team%3ANEU-China)

TEAM

[Team](https://2017.igem.org/Team%3ANEU-China/Team)
[Collaborations](https://2017.igem.org/Team%3ANEU-China/Collaborations)

PROJECT

[Description](https://2017.igem.org/Team%3ANEU-China/Description)
[Design](https://2017.igem.org/Team%3ANEU-China/Design)
[Experiments](https://2017.igem.org/Team%3ANEU-China/Experiments)
[Notebook](https://2017.igem.org/Team%3ANEU-China/Notebook)
[InterLab](https://2017.igem.org/Team%3ANEU-China/InterLab)
[Contribution](https://2017.igem.org/Team%3ANEU-China/Contribution)
[Model](https://2017.igem.org/Team%3ANEU-China/Model)
[Results](https://2017.igem.org/Team%3ANEU-China/Results)
[Demonstrate](https://2017.igem.org/Team%3ANEU-China/Demonstrate)
[Improve](https://2017.igem.org/Team%3ANEU-China/Improve)
[Attributions](https://2017.igem.org/Team%3ANEU-China/Attributions)

PARTS

[Parts](https://2017.igem.org/Team%3ANEU-China/Parts)
[Basic Parts](https://2017.igem.org/Team%3ANEU-China/Basic_Part)
[Composite Parts](https://2017.igem.org/Team%3ANEU-China/Composite_Part)
[Part Collection](https://2017.igem.org/Team%3ANEU-China/Part_Collection)

[SAFETY](https://2017.igem.org/Team%3ANEU-China/Safety)

HUMAN PRACTICES

[Silver HP](https://2017.igem.org/Team%3ANEU-China/HP/Silver)
[Integrated and Gold](https://2017.igem.org/Team%3ANEU-China/HP/Gold_Integrated)
[Public Engagement](https://2017.igem.org/Team%3ANEU-China/Engagement)

AWARDS

[Applied Design](https://2017.igem.org/Team%3ANEU-China/Applied_Design)
[Entrepreneurship](https://2017.igem.org/Team%3ANEU-China/Entrepreneurship)
[Hardware](https://2017.igem.org/Team%3ANEU-China/Hardware)
[Measurement](https://2017.igem.org/Team%3ANEU-China/Measurement)
[Model](https://2017.igem.org/Team%3ANEU-China/Model)
[Plant](https://2017.igem.org/Team%3ANEU-China/Plant)
[Software](https://2017.igem.org/Team%3ANEU-China/Software)

[JUDGING FORM](https://igem.org/2017_Judging_Form?team=NEU-China)

# NEU-China

# 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:

* [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/2017/b/bf/HQ_menu_logo.jpg)](https://2017.igem.org)


[![](https://static.igem.org/mediawiki/2017/8/8b/HQ_page_logo.jpg)](https://2017.igem.org)

# Team:NEU-China/Experiments

Toggle navigation

[# ![logo](https://static.igem.org/mediawiki/2017/4/4d/NEU-China-logo.png)](index.html)

* [Home](https://2017.igem.org/Team%3ANEU-China)
* Project
  + [Background](https://2017.igem.org/Team%3ANEU-China/Background)
  + [Description](https://2017.igem.org/Team%3ANEU-China/Description)
  + [Design](https://2017.igem.org/Team%3ANEU-China/Design)
  + [Experiment & Results](https://2017.igem.org/Team%3ANEU-China/Experiments)
  + [Demostrate](https://2017.igem.org/Team%3ANEU-China/Demonstrate)
  + [Model](https://2017.igem.org/Team%3ANEU-China/Model)
* Notebook
  + [Progress](https://2017.igem.org/Team%3ANEU-China/Progress)
  + [Protocol](https://2017.igem.org/Team%3ANEU-China/Protocol)
* Parts
  + [Our Parts](https://2017.igem.org/Team%3ANEU-China/Parts)
  + [Biobricks For Medal](https://2017.igem.org/Team%3ANEU-China/BiobricksForMedal)
  + [Basic Parts](https://2017.igem.org/Team%3ANEU-China/Basic_Part)
  + [Safety](https://2017.igem.org/Team%3ANEU-China/Safety)
* [InterLab](https://2017.igem.org/Team%3ANEU-China/InterLab)
* [Collaborations](https://2017.igem.org/Team%3ANEU-China/Collaborations)
* Human Practices
  + [Human Practices](https://2017.igem.org/Team%3ANEU-China/Human_Practices)
  + [Silver](https://2017.igem.org/Team%3ANEU-China/HP/Silver)
  + [Gold](https://2017.igem.org/Team%3ANEU-China/HP/Gold_Integrated)
* Lab
  + [Team](https://2017.igem.org/Team%3ANEU-China/Team)
  + [Attributions](https://2017.igem.org/Team%3ANEU-China/Attributions)
  + [Lab Life](https://2017.igem.org/Team%3ANEU-China/LabLife)

# Experiments and Results

## Bacterial Strains and Growth Cultures

### E. coli DH5α was used for all cloning purposes. Cultures for cloning were grown in antibiotic-supplemented Lysogeny Broth (LB) at 37°C. The E. coli expression host cultures were grown in antibiotic-supplemented Lysogeny Broth (LB) at 37°C. Bacterial cultures were grown overnight (16-20 hours) to stationary phase before being subcultured for characterization experiments.

## Plasmid Construction & Gene Cloning

### Cloning Strategy: 1. PCR-amplify OR1A1 sequences from genomic DNA 2. Add Rho-tag sequencing to N-terminal during PCR 3. Ligate the PCR fragment to pcDNA3.1+ plasmid

| Parts | Plasmid Construction | Transformation to Host Strain |
| --- | --- | --- |
| OR1A1 | pcDNA3.1+ | E.coli.DH5α |
| OR1D2 | pcDNA3.1+ | E.coli.DH5α |

![](https://static.igem.org/mediawiki/2017/a/a3/NEU-China-Experiments1.png)

OR1A1 and pcDNA3.1+ fragments gel electrophoresis

![](https://static.igem.org/mediawiki/2017/d/d4/NEU-China-Experiments2.png)

Sanger sequencing of OR1A1

![](https://static.igem.org/mediawiki/2017/b/b5/NEU-China-Experiments3.png)

Sanger sequencing of OR1D2

### Test the sensitivity and specificity of this basic odor biosensors with their cognate odors β-citronellol or bourgeonal.

![](https://static.igem.org/mediawiki/2017/2/26/NEU-China-Experiments4.png)

![](https://static.igem.org/mediawiki/2017/d/db/NEU-China-Experiments5.png)

### In order to further verify the specificity of OR1A1, we also used some different odors to stimulate it.

![](https://static.igem.org/mediawiki/2017/7/75/NEU-China-Experiments6.png)

### Not surprisingly, only when we stimulated with β-citronellol could we detect an enhance in luminescence. Judging from it we can consider that OR1A1 only shows a specific role in β-citronellol recognition.

## CRE-luciferase reporter does tranduce cAMP signaling in HEK293 cells

### As for the cAMP-activated reporter gene system, we choose Forskolin to induce expression of the reporter gene, and expression of the reporter protein luciferase was confirmed by treatment with 10 millimole（mM）forskolin.

![](https://static.igem.org/mediawiki/2017/f/fb/NEU-China-Experiments7.png)

### After a 24 h incubation, cracked the cell and added the substrate reacting with luciferase, then detected it by fluorescent microplate reader. Here is the data we got. Cells expressing luciferase treated only with 0.1%DMSO was used as a negative control. We can see that the luminescence data gets significantly improved, which proves that the reporter gene we choose is available.

## Cloning into lenti sgRNA(MS2)-puro backbone vector for CRISPRa（gRNA）

### Referencing to some papers, we knew that there are three genes which can help the olfactory receptors anchored in the membrane. So we aimed to activate these three key accessory protein to enhance the express of odor receptors.

| Parts | Plasmid Construction | Transformation to Host Strain |
| --- | --- | --- |
| GNAL | Lenti MS2 | E.coli.DH5α |
| RIC8B | Lenti MS2 | E.coli.DH5α |
| RTP1 | Lenti MS2 | E.coli.DH5α |

![](https://static.igem.org/mediawiki/2017/d/d6/NEU-China-Experiments8.png)

### Here shows multiple sgRNAs we designed to target the core genes, GNAL, RIC8B and RTP1 in the signal pathway.

![](https://static.igem.org/mediawiki/2017/5/5b/NEU-China-DemonstrateNewGNAL.png)

![](https://static.igem.org/mediawiki/2017/b/b2/NEU-China-DemonstrateNewRIC8B.png)

![](https://static.igem.org/mediawiki/2017/d/da/NEU-China-DemonstrateNewRTP1.png)

### The Sanger sequencing results shows we’ve got correct sequence and successfully cloned them into the sgRNA-expressing vector.

## Virus infection experiment

### First, we transfected HEK293 with lenti dCas9-VP64-Blast, lenti MS2-P65-HSF1-Hygro and lenti sgRNA to produce virus. Then, we coinfected HEK293 with the virus of dCas9-VP64-Blast and MS2-P65-HSF1-Hygro to construct stable cell lines. Based on these cell lines, we infected different sgRNAs of GNAL,RIC8B and RTP1 respectively. This are the results of real-time PCR.

![](https://static.igem.org/mediawiki/2017/9/9f/NEU-China-Experiments9.png)

![](https://static.igem.org/mediawiki/2017/6/67/NEU-China-Experiments10.png)

![](https://static.igem.org/mediawiki/2017/2/28/NEU-China-Experiments11.png)

### These three graphs show that our sgRNAs has successfully activated the three genes that mentioned before.

## OR1A1 biosensor evaluation with its cognate odor molecule

### Here comes the most essential experiment. We infected the stable cell lines with all virus to construct a super cell line, which has a high expression of the three genes. Then we transfected OR1A1 and OR1D2 respectively in the super cell, and stimulated with β-citronellol and bourgeonal, which can recognized by OR1A1 and OR1D2 respectively.

### This is the luminescence result of OR1A1.

![](https://static.igem.org/mediawiki/2017/c/cb/NEU-China-Experiments12.png)

### **Conclusion：**In general, the iSmeller we constructed using CRISPR / Cas9 Synergistic Activation Mediator successfully amplifies the signal of the core components of the olfactory pathway and enhances its sensitivity to odor response. In addition, by stimulateing with different scent, our iSmeller's specificity of odor recognization has also been validated.

![](https://static.igem.org/mediawiki/2017/9/99/NEU-China-under.png)

![](https://static.igem.org/mediawiki/2017/7/7e/NEU-China-igem.png)

![](https://static.igem.org/mediawiki/2017/f/fc/NEU-China-NEU.png)

![](https://static.igem.org/mediawiki/2017/1/17/NEU-China-CLHS.png)

![](https://static.igem.org/mediawiki/2017/4/45/NEU-China-SC.jpeg)

![](https://static.igem.org/mediawiki/2017/e/e5/NEU-China-BMIE.jpeg)

![](https://static.igem.org/mediawiki/2017/5/5e/NEU-China-addgene.png)

![](https://static.igem.org/mediawiki/2017/d/d9/NEU-China-snapgene.png)

![](https://static.igem.org/mediawiki/2017/d/db/NEU-China-sam.png)

![](https://static.igem.org/mediawiki/2017/5/5c/NEU-China-igv.png)

![](https://static.igem.org/mediawiki/2017/e/e5/NEU-China-NCBI.png)

Copyright © 2017 NEU-CHINA Team. All Rights Reserved.

[![](https://static.igem.org/mediawiki/2017/b/bf/HQ_menu_logo.jpg)](https://2017.igem.org)

[![](https://static.igem.org/mediawiki/2017/8/8b/HQ_page_logo.jpg)](https://2017.igem.org)

# Team:NEU-China/Results

[![](http://placehold.it/350x150)](https://2017.igem.org/Team%3ANEU-China)

MENU

[HOME](https://2017.igem.org/Team%3ANEU-China)

TEAM

[Team](https://2017.igem.org/Team%3ANEU-China/Team)
[Collaborations](https://2017.igem.org/Team%3ANEU-China/Collaborations)

PROJECT

[Description](https://2017.igem.org/Team%3ANEU-China/Description)
[Design](https://2017.igem.org/Team%3ANEU-China/Design)
[Experiments](https://2017.igem.org/Team%3ANEU-China/Experiments)
[Notebook](https://2017.igem.org/Team%3ANEU-China/Notebook)
[InterLab](https://2017.igem.org/Team%3ANEU-China/InterLab)
[Contribution](https://2017.igem.org/Team%3ANEU-China/Contribution)
[Model](https://2017.igem.org/Team%3ANEU-China/Model)
[Results](https://2017.igem.org/Team%3ANEU-China/Results)
[Demonstrate](https://2017.igem.org/Team%3ANEU-China/Demonstrate)
[Improve](https://2017.igem.org/Team%3ANEU-China/Improve)
[Attributions](https://2017.igem.org/Team%3ANEU-China/Attributions)

PARTS

[Parts](https://2017.igem.org/Team%3ANEU-China/Parts)
[Basic Parts](https://2017.igem.org/Team%3ANEU-China/Basic_Part)
[Composite Parts](https://2017.igem.org/Team%3ANEU-China/Composite_Part)
[Part Collection](https://2017.igem.org/Team%3ANEU-China/Part_Collection)

[SAFETY](https://2017.igem.org/Team%3ANEU-China/Safety)

HUMAN PRACTICES

[Silver HP](https://2017.igem.org/Team%3ANEU-China/HP/Silver)
[Integrated and Gold](https://2017.igem.org/Team%3ANEU-China/HP/Gold_Integrated)
[Public Engagement](https://2017.igem.org/Team%3ANEU-China/Engagement)

AWARDS

[Applied Design](https://2017.igem.org/Team%3ANEU-China/Applied_Design)
[Entrepreneurship](https://2017.igem.org/Team%3ANEU-China/Entrepreneurship)
[Hardware](https://2017.igem.org/Team%3ANEU-China/Hardware)
[Measurement](https://2017.igem.org/Team%3ANEU-China/Measurement)
[Model](https://2017.igem.org/Team%3ANEU-China/Model)
[Plant](https://2017.igem.org/Team%3ANEU-China/Plant)
[Software](https://2017.igem.org/Team%3ANEU-China/Software)

[JUDGING FORM](https://igem.org/2017_Judging_Form?team=NEU-China)

# NEU-China

# Results

Here you can describe the results of your project and your future plans.

##### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

##### You should also describe what your results mean:

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember all measurement and characterization data must be on part pages in the Registry.
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

##### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

##### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)

[![](https://static.igem.org/mediawiki/2017/b/bf/HQ_menu_logo.jpg)](https://2017.igem.org)