iGEM 2018 — Team XMU-China

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:XMU-China/Notebook

Team:XMU-China/Notebook - 2018.igem.org

* Project
  + [Description](https://2018.igem.org/Team%3AXMU-China/Description)
  + [Design](https://2018.igem.org/Team%3AXMU-China/Design)
  + [Results](https://2018.igem.org/Team%3AXMU-China/Results)
  + [Demonstrate](https://2018.igem.org/Team%3AXMU-China/Demonstrate)
  + [Parts](https://2018.igem.org/Team%3AXMU-China/Parts)

* Hardware
  + [Overview](https://2018.igem.org/Team%3AXMU-China/Hardware)
  + [Microfluidic Chips](https://2018.igem.org/Team%3AXMU-China/Hardware#Microfluidic_Chips)
  + [Fluorescence Detection](https://2018.igem.org/Team%3AXMU-China/Hardware#Fluorescence_Detection)
  + [Raspberry Pi](https://2018.igem.org/Team%3AXMU-China/Hardware#Raspberry_Pi)
  + [Application](https://2018.igem.org/Team%3AXMU-China/Hardware#Application)
  + [Software](https://2018.igem.org/Team%3AXMU-China/Software)
  + [Product Design](https://2018.igem.org/Team%3AXMU-China/Applied_Design)
* Model
  + [Summary](https://2018.igem.org/Team%3AXMU-China/Model#Summary)
  + [Thermodynamic Model](https://2018.igem.org/Team%3AXMU-China/Model#Thermodynamic_model)
  + [Fluid dynamics Model](https://2018.igem.org/Team%3AXMU-China/Model#Fluid_dynamics_model)
  + [Molecular Docking Model](https://2018.igem.org/Team%3AXMU-China/Model#Molecular_docking_model)
  + [Derivation of Rate Equation](https://2018.igem.org/Team%3AXMU-China/Model#The_dynamic_model)
* Social Works
  + [Human Practice](https://2018.igem.org/Team%3AXMU-China/Human_Practices)
  + [Engagement](https://2018.igem.org/Team%3AXMU-China/Public_Engagement)
  + [Collaborations](https://2018.igem.org/Team%3AXMU-China/Collaborations)
  + [Entrepreneurship](https://2018.igem.org/Team%3AXMU-China/Entrepreneurship)
* Other Works
  + [InterLab](https://2018.igem.org/Team%3AXMU-China/InterLab)
  + [Improve](https://2018.igem.org/Team%3AXMU-China/Improve)
  + [Safety](https://2018.igem.org/Team%3AXMU-China/Safety)
  + [Measurement](https://2018.igem.org/Team%3AXMU-China/Measurement)
* Notebook
  + [Notebook](https://2018.igem.org/Team%3AXMU-China/Notebook)
  + [Experiments](https://2018.igem.org/Team%3AXMU-China/Experiments)
  + [Engineering](https://2018.igem.org/Team%3AXMU-China/Engineering)
* Team
  + [Members](https://2018.igem.org/Team%3AXMU-China/Team)
  + [Attributions](https://2018.igem.org/Team%3AXMU-China/Attributions)
  + [Judging](https://2018.igem.org/Team%3AXMU-China/Judging)
  + [After iGEM](https://2018.igem.org/Team%3AXMU-China/After_iGEM)

![](https://static.igem.org/mediawiki/2018/b/b5/T--XMU-China--singlelogo.png)
![](https://static.igem.org/mediawiki/2018/3/35/T--XMU-China--iGEM_logo.png)

Team

* [Members](https://2018.igem.org/Team%3AXMU-China/Team)
* [Attributions](https://2018.igem.org/Team%3AXMU-China/Attributions)
* [Judging](https://2018.igem.org/Team%3AXMU-China/Judging)
* [After iGEM](https://2018.igem.org/Team%3AXMU-China/After_iGEM)

Notebook

* [Notebook](https://2018.igem.org/Team%3AXMU-China/Notebook)
* [Experiments](https://2018.igem.org/Team%3AXMU-China/Experiments)
* [Engineering](https://2018.igem.org/Team%3AXMU-China/Engineering)

Other Works

* [InterLab](https://2018.igem.org/Team%3AXMU-China/InterLab)
* [Improve](https://2018.igem.org/Team%3AXMU-China/Improve)
* [Safety](https://2018.igem.org/Team%3AXMU-China/Safety)
* [Measurement](https://2018.igem.org/Team%3AXMU-China/Measurement)

Social Works

* [Human Practice](https://2018.igem.org/Team%3AXMU-China/Human_Practices)
* [Engagement](https://2018.igem.org/Team%3AXMU-China/Public_Engagement)
* [Collaborations](https://2018.igem.org/Team%3AXMU-China/Collaborations)
* [Entrepreneurship](https://2018.igem.org/Team%3AXMU-China/Entrepreneurship)

Model

* [Summary](https://2018.igem.org/Team%3AXMU-China/Model)
* [Thermodynamic Model](https://2018.igem.org/Team%3AXMU-China/Model#Thermodynamic_model)
* [Fluid dynamics Model](https://2018.igem.org/Team%3AXMU-China/Model#Fluid_dynamics_model)
* [Molecular Docking Model](https://2018.igem.org/Team%3AXMU-China/Model#Molecular_docking_model)
* [Derivation of Rate Equation](https://2018.igem.org/Team%3AXMU-China/Model#The_dynamic_model)

Hardware

* [Overview](https://2018.igem.org/Team%3AXMU-China/Hardware)
* [Microfluidic Chips](https://2018.igem.org/Team%3AXMU-China/Hardware#Microfluidic_Chips)
* [Fluorescence Detection](https://2018.igem.org/Team%3AXMU-China/Hardware#Fluorescence_Detection)
* [Raspberry Pi](https://2018.igem.org/Team%3AXMU-China/Hardware#Raspberry_Pi)
* [Application](https://2018.igem.org/Team%3AXMU-China/Hardware#Application)
* [Software](https://2018.igem.org/Team%3AXMU-China/Software)
* [Product Design](https://2018.igem.org/Team%3AXMU-China/Applied_Design)

Project

* [Description](https://2018.igem.org/Team%3AXMU-China/Description)
* [Design](https://2018.igem.org/Team%3AXMU-China/Design)
* [Results](https://2018.igem.org/Team%3AXMU-China/Results)
* [Demonstrate](https://2018.igem.org/Team%3AXMU-China/Demonstrate)
* [Parts](https://2018.igem.org/Team%3AXMU-China/Parts)

[![](https://static.igem.org/mediawiki/2018/6/6f/T--XMU-China--jiannanlogo.png)](https://2018.igem.org/Team%3AXMU-China)

Notebook

* Mar
* Apr
* May
* Jun Wk 1-3
* Jun Wk 4
* Jul Wk 3
* Jul Wk 4
* Aug Wk 1
* Aug Wk 2
* Aug Wk 3
* Aug Wk 4
* Sep Wk 1
* Sep Wk 2
* Sep Wk 3
* Sep Wk 4
* Oct Wk 1
* Oct Wk 2
* Oct Wk 3

![](https://static.igem.org/mediawiki/2018/5/59/T--XMU-China--timeline_left.png)
![](https://static.igem.org/mediawiki/2018/0/01/T--XMU-China--timeline_right.png)

# Mar

- Our team was formally founded.
- All student members learned the fundamental knowledge of molecular cloning and iGEM.
- All student members looked through the previous excellent projects and learned the basic notion of synthetic biology and iGEM.

# Apr

- We were divided into four groups and started to brainstorm separately (and we reported each’s progress every weekend).
- Our team was formally accepted by iGEM.

# May

- Our formal project was confirmed.
- The pre-experiment plan was confirmed.
- The division of labor was confirmed.

# Jun Wk1-3

-All student members learned laboratory fundamental qualities and received molecular cloning operation training.

# Jun Wk 4

- 2018 DNA Distribution Kit Plates were received.
- We transformed the biobricks which we needed.
- [BBa\_J04450](http://parts.igem.org/Part%3ABBa_J04450) constructed to get the backbone.

![](https://static.igem.org/mediawiki/2018/e/e8/T--XMU-China--BBa_J04450.jpeg)

# Jul Wk 3

– The genetic circuit of the TDP began to be built.

# Jul Wk 4

– Interlab experiments.

– Verified the activity of EpCAM.

![](https://static.igem.org/mediawiki/2018/2/25/T--XMU-China--notebook-abcd-1.jpg)

# Aug Wk 1

– The genetic circuit of the Kai began to be built.

# Aug Wk 2

– [BBa\_K2623015](http://parts.igem.org/Part%3ABBa_K2623015) constructed.

![](https://static.igem.org/mediawiki/2018/7/72/T--XMU-China--BBa_K2623015.png)
![](https://static.igem.org/mediawiki/2018/a/a9/T--XMU-China--BBa_K2623015_1.jpeg)

- [BBa\_K2623009](http://parts.igem.org/Part%3ABBa_K2623009)constructed.

![](https://static.igem.org/mediawiki/2018/1/17/T--XMU-China--BBa_K2623009.png)

– [BBa\_K2623016](http://parts.igem.org/Part%3ABBa_K2623016)constructed.

![](https://static.igem.org/mediawiki/2018/d/da/T--XMU-China--BBa_K2623016.png)
![](https://static.igem.org/mediawiki/2018/9/98/T--XMU-China--BBa_K2623016_1.jpeg)

- The genetic circuit of SAHS was completed and protein validation began.

# Aug Wk 3

- [BBa\_K2623001](http://parts.igem.org/Part%3ABBa_K2623001) constructed.

![](https://static.igem.org/mediawiki/2018/8/8a/T--XMU-China--BBa_K2623001.png)

- BBa\_K2623022 and [BBa\_K2623024](http://parts.igem.org/Part%3ABBa_K2623024) constructed.

![](https://static.igem.org/mediawiki/2018/c/c7/T--XMU-China--BBa_K2623022_4.jpeg)

- [BBa\_I13401](http://parts.igem.org/Part%3ABBa_I13401) constructed to get the GFP reporter.

![](https://static.igem.org/mediawiki/2018/a/a7/T--XMU-China--BBa_I13401.jpeg)

# Aug Wk 4

- BBa\_K2623021 and [BBa\_K2623023](http://parts.igem.org/Part%3ABBa_K2623023) constructed.

![](https://static.igem.org/mediawiki/2018/9/9a/T--XMU-China--BBa_K2623021_3.jpeg)

- [BBa\_K2623026](http://parts.igem.org/Part%3ABBa_K2623026) constructed.

![](https://static.igem.org/mediawiki/2018/7/7c/T--XMU-China--BBa_K2623026.jpeg)

- [BBa\_K2623002](http://parts.igem.org/Part%3ABBa_K2623002) constructed.

![](https://static.igem.org/mediawiki/2018/d/d7/T--XMU-China--BBa_K2623002.png)

- [BBa\_K2623003](http://parts.igem.org/Part%3ABBa_K2623003) constructed.

![](https://static.igem.org/mediawiki/2018/1/16/T--XMU-China--BBa_K2623003.png)

– Verified that SYL3C aptamer could combine with 10nt complementary chain successfully.

![](https://static.igem.org/mediawiki/2018/5/59/T--XMU-China--notebook-abcd-2.jpg)

# Sep Wk 1

- [BBa\_K2623014](http://parts.igem.org/Part%3ABBa_K2623014) constructed.

![](https://static.igem.org/mediawiki/2018/9/93/T--XMU-China--BBa_K2623014.png)

# Sep Wk 2

- GFP on BBa\_K2623021 and [BBa\_K2623023](http://parts.igem.org/Part%3ABBa_K2623023) successfully expressed.

![](https://static.igem.org/mediawiki/2018/e/e0/T--XMU-China--BBa_K2623021.jpeg)
![](https://static.igem.org/mediawiki/2018/f/f6/T--XMU-China--BBa_K2623023.jpeg)

- [BBa\_K2623004](http://parts.igem.org/Part%3ABBa_K2623004) constructed.

![](https://static.igem.org/mediawiki/2018/c/cd/T--XMU-China--BBa_K2623004.png)

- [BBa\_K2623005](http://parts.igem.org/Part%3ABBa_K2623005) constructed.

![](https://static.igem.org/mediawiki/2018/d/dd/T--XMU-China--BBa_K2623005.png)

- [BBa\_K2623006](http://parts.igem.org/Part%3ABBa_K2623006) constructed.

![](https://static.igem.org/mediawiki/2018/e/ef/T--XMU-China--BBa_K2623006.png)

- First time successful purification of high-purity SAHS protein.

![](https://static.igem.org/mediawiki/2018/f/f0/T--XMU-China--SAHS_protein.png)

- Coating experiment on microfluidic chips succeeded.
- Started bacterial freeze-drying experiment.

# Sep Wk 3

-  [BBa\_E1010](http://parts.igem.org/Part%3ABBa_E1010) constructed to get the mRFP reporter.

![](https://static.igem.org/mediawiki/2018/f/fb/T--XMU-China--BBa_E1010.jpeg)

-  [BBa\_K2623011](http://parts.igem.org/Part%3ABBa_K2623011) constructed.

![](https://static.igem.org/mediawiki/2018/8/8b/T--XMU-China--BBa_K2623011.png)

-  [BBa\_K2623007](http://parts.igem.org/Part%3ABBa_K2623007) constructed.

![](https://static.igem.org/mediawiki/2018/9/92/T--XMU-China--BBa_K2623007.png)

- Competition experiment succeeded.
- Started protein freeze-drying experiment.

– Verified the feasibility of “competition method” successfully.

![](https://static.igem.org/mediawiki/2018/b/bd/T--XMU-China--notebook-abcd-3.jpg)

# Sep Wk 4

-  [BBa\_K2623025](http://parts.igem.org/Part%3ABBa_K2623025) constructed.

![](https://static.igem.org/mediawiki/2018/7/77/T--XMU-China--BBa_K2623025.jpeg)

-  [BBa\_K2623008](http://parts.igem.org/Part%3ABBa_K2623008) constructed.

![](https://static.igem.org/mediawiki/2018/6/60/T--XMU-China--BBa_K2623008.png)

- Started try genetic recombination experiment.

# Oct Wk 1

-  [BBa\_K2623027](http://parts.igem.org/Part%3ABBa_K2623027) constructed.

![](https://static.igem.org/mediawiki/2018/4/48/T--XMU-China--BBa_K2623027.jpeg)

-  [BBa\_K2623013](http://parts.igem.org/Part%3ABBa_K2623013) constructed.

![](https://static.igem.org/mediawiki/2018/c/cb/T--XMU-China--BBa_K2623013.png)

- NG2+5, NG2+6, NG4+5, NG4+6 constructed.

– Verified that As Cas12a could be activated.

![](https://static.igem.org/mediawiki/2018/5/58/T--XMU-China--notebook-abcd-4.jpg)

# Oct Wk 2

-
[BBa\_K2623028](http://parts.igem.org/Part%3ABBa_K2623028),
[BBa\_K2623029](http://parts.igem.org/Part%3A-BBa_K2623029),
[BBa\_K2623030](http://parts.igem.org/Part%3A-BBa_K2623030),
[BBa\_K2623031](http://parts.igem.org/Part%3A-BBa_K2623031) successfully constructed and the fluorescence intensity of OMVs isolated from E.coli containing these four parts were detected by [flow cytometer](http://xmyan.xmu.edu.cn/)

![](https://static.igem.org/mediawiki/2018/8/89/T--XMU-China--Oct_wk2.png)

– Verified the successful combination between SYL3C aptamer and EpCAM.

![](https://static.igem.org/mediawiki/2018/f/f5/T--XMU-China--notebook-abcd-5.jpg)

– Transferred molecular signal to fluorescence signal with DNase Alert.

![](https://static.igem.org/mediawiki/2018/4/43/T--XMU-China--notebook-abcd-6.jpg)

# Oct Wk 3

– Verified the transfer from protein signal to the fluorescent one successfully

![](https://static.igem.org/mediawiki/2018/5/5e/T--XMU-China--notebook-abcd-7.png)

![](https://static.igem.org/mediawiki/2018/3/3d/T--XMU-China--xmu_is_different.png)

* [Home](https://2018.igem.org/Team%3AXMU-China)
* [Model](https://2018.igem.org/Team%3AXMU-China/Model)
* [Parts](https://2018.igem.org/Team%3AXMU-China/Parts)
* [Entrepreneurship](https://2018.igem.org/Team%3AXMU-China/Entrepreneurship)
* [Attributions](https://2018.igem.org/Team%3AXMU-China/Attributions)

* [Design](https://2018.igem.org/Team%3AXMU-China/Design)
* [Hardware](https://2018.igem.org/Team%3AXMU-China/Hardware)
* [Human Practices](https://2018.igem.org/Team%3AXMU-China/Human_Practices)
* [Notebook](https://2018.igem.org/Team%3AXMU-China/Notebook)
* [Judging](https://2018.igem.org/Team%3AXMU-China/Judging)

* [Results](https://2018.igem.org/Team%3AXMU-China/Results)
* [Software](https://2018.igem.org/Team%3AXMU-China/Software)
* [Collaborations](https://2018.igem.org/Team%3AXMU-China/Collaborations)
* [Members](https://2018.igem.org/Team%3AXMU-China/Members)
* [After iGEM](https://2018.igem.org/Team%3AXMU-China/After_iGEM)

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:XMU-China/Engineering

Team:XMU-China/Engineering - 2018.igem.org

* Project
  + [Description](https://2018.igem.org/Team%3AXMU-China/Description)
  + [Design](https://2018.igem.org/Team%3AXMU-China/Design)
  + [Results](https://2018.igem.org/Team%3AXMU-China/Results)
  + [Demonstrate](https://2018.igem.org/Team%3AXMU-China/Demonstrate)
  + [Parts](https://2018.igem.org/Team%3AXMU-China/Parts)

* Hardware
  + [Overview](https://2018.igem.org/Team%3AXMU-China/Hardware)
  + [Microfluidic Chips](https://2018.igem.org/Team%3AXMU-China/Hardware#Microfluidic_Chips)
  + [Fluorescence Detection](https://2018.igem.org/Team%3AXMU-China/Hardware#Fluorescence_Detection)
  + [Raspberry Pi](https://2018.igem.org/Team%3AXMU-China/Hardware#Raspberry_Pi)
  + [Application](https://2018.igem.org/Team%3AXMU-China/Hardware#Application)
  + [Software](https://2018.igem.org/Team%3AXMU-China/Software)
  + [Product Design](https://2018.igem.org/Team%3AXMU-China/Applied_Design)
* Model
  + [Summary](https://2018.igem.org/Team%3AXMU-China/Model#Summary)
  + [Thermodynamic Model](https://2018.igem.org/Team%3AXMU-China/Model#Thermodynamic_model)
  + [Fluid dynamics Model](https://2018.igem.org/Team%3AXMU-China/Model#Fluid_dynamics_model)
  + [Molecular Docking Model](https://2018.igem.org/Team%3AXMU-China/Model#Molecular_docking_model)
  + [Derivation of Rate Equation](https://2018.igem.org/Team%3AXMU-China/Model#The_dynamic_model)
* Social Works
  + [Human Practice](https://2018.igem.org/Team%3AXMU-China/Human_Practices)
  + [Engagement](https://2018.igem.org/Team%3AXMU-China/Public_Engagement)
  + [Collaborations](https://2018.igem.org/Team%3AXMU-China/Collaborations)
  + [Entrepreneurship](https://2018.igem.org/Team%3AXMU-China/Entrepreneurship)
* Other Works
  + [InterLab](https://2018.igem.org/Team%3AXMU-China/InterLab)
  + [Improve](https://2018.igem.org/Team%3AXMU-China/Improve)
  + [Safety](https://2018.igem.org/Team%3AXMU-China/Safety)
  + [Measurement](https://2018.igem.org/Team%3AXMU-China/Measurement)
* Notebook
  + [Notebook](https://2018.igem.org/Team%3AXMU-China/Notebook)
  + [Experiments](https://2018.igem.org/Team%3AXMU-China/Experiments)
  + [Engineering](https://2018.igem.org/Team%3AXMU-China/Engineering)
* Team
  + [Members](https://2018.igem.org/Team%3AXMU-China/Team)
  + [Attributions](https://2018.igem.org/Team%3AXMU-China/Attributions)
  + [Judging](https://2018.igem.org/Team%3AXMU-China/Judging)
  + [After iGEM](https://2018.igem.org/Team%3AXMU-China/After_iGEM)

![](https://static.igem.org/mediawiki/2018/b/b5/T--XMU-China--singlelogo.png)
![](https://static.igem.org/mediawiki/2018/3/35/T--XMU-China--iGEM_logo.png)

Team

* [Members](https://2018.igem.org/Team%3AXMU-China/Team)
* [Attributions](https://2018.igem.org/Team%3AXMU-China/Attributions)
* [Judging](https://2018.igem.org/Team%3AXMU-China/Judging)
* [After iGEM](https://2018.igem.org/Team%3AXMU-China/After_iGEM)

Notebook

* [Notebook](https://2018.igem.org/Team%3AXMU-China/Notebook)
* [Experiments](https://2018.igem.org/Team%3AXMU-China/Experiments)
* [Engineering](https://2018.igem.org/Team%3AXMU-China/Engineering)

Other Works

* [InterLab](https://2018.igem.org/Team%3AXMU-China/InterLab)
* [Improve](https://2018.igem.org/Team%3AXMU-China/Improve)
* [Safety](https://2018.igem.org/Team%3AXMU-China/Safety)
* [Measurement](https://2018.igem.org/Team%3AXMU-China/Measurement)

Social Works

* [Human Practice](https://2018.igem.org/Team%3AXMU-China/Human_Practices)
* [Engagement](https://2018.igem.org/Team%3AXMU-China/Public_Engagement)
* [Collaborations](https://2018.igem.org/Team%3AXMU-China/Collaborations)
* [Entrepreneurship](https://2018.igem.org/Team%3AXMU-China/Entrepreneurship)

Model

* [Summary](https://2018.igem.org/Team%3AXMU-China/Model)
* [Thermodynamic Model](https://2018.igem.org/Team%3AXMU-China/Model#Thermodynamic_model)
* [Fluid dynamics Model](https://2018.igem.org/Team%3AXMU-China/Model#Fluid_dynamics_model)
* [Molecular Docking Model](https://2018.igem.org/Team%3AXMU-China/Model#Molecular_docking_model)
* [Derivation of Rate Equation](https://2018.igem.org/Team%3AXMU-China/Model#The_dynamic_model)

Hardware

* [Overview](https://2018.igem.org/Team%3AXMU-China/Hardware)
* [Microfluidic Chips](https://2018.igem.org/Team%3AXMU-China/Hardware#Microfluidic_Chips)
* [Fluorescence Detection](https://2018.igem.org/Team%3AXMU-China/Hardware#Fluorescence_Detection)
* [Raspberry Pi](https://2018.igem.org/Team%3AXMU-China/Hardware#Raspberry_Pi)
* [Application](https://2018.igem.org/Team%3AXMU-China/Hardware#Application)
* [Software](https://2018.igem.org/Team%3AXMU-China/Software)
* [Product Design](https://2018.igem.org/Team%3AXMU-China/Applied_Design)

Project

* [Description](https://2018.igem.org/Team%3AXMU-China/Description)
* [Design](https://2018.igem.org/Team%3AXMU-China/Design)
* [Results](https://2018.igem.org/Team%3AXMU-China/Results)
* [Demonstrate](https://2018.igem.org/Team%3AXMU-China/Demonstrate)
* [Parts](https://2018.igem.org/Team%3AXMU-China/Parts)

[![](https://static.igem.org/mediawiki/2018/6/6f/T--XMU-China--jiannanlogo.png)](https://2018.igem.org/Team%3AXMU-China)

Engineering

* Jun 17th
* Jun 30th
* Jul 15th
* Jul 22th
* Jul 29th
* Aug 12th
* Aug 19th
* Aug 26th
* Sep 9th
* Sep 16th
* Sep 23th
* Sep 30th
* Oct 2nd

![](https://static.igem.org/mediawiki/2018/5/59/T--XMU-China--timeline_left.png)
![](https://static.igem.org/mediawiki/2018/0/01/T--XMU-China--timeline_right.png)

# Jun 17th

## 1st draft of cell-free detection device

![](https://static.igem.org/mediawiki/2018/d/d7/T--XMU-China--detection_device1.png)

# Jun 30th

## Set up *Fang* project

![](https://static.igem.org/mediawiki/2018/9/96/T--XMU-China--Agamotto_project.png)

# Jul 15th

## 1st generation of circuit

![](https://static.igem.org/mediawiki/2018/a/a9/T--XMU-China--circuit1.png)

# Jul 22th

## 1st generation of microfluidic chips

![](https://static.igem.org/mediawiki/2018/9/9d/T--XMU-China--microfluidic_chips1.png)

# Jul 29th

## 2nd generation of circuit test

![](https://static.igem.org/mediawiki/2018/9/9e/T--XMU-China--circuit2.png)

# Aug 12th

## Control motor speed by app

![](https://static.igem.org/mediawiki/2018/0/06/T--XMU-China--app.png)

# Aug 19th

## 2nd generation of microfluidic chips

![](https://static.igem.org/mediawiki/2018/b/b4/T--XMU-China--microfluidic_chips2.png)

# Aug 26th

## 3rd generation of microfluidic chips

![](https://static.igem.org/mediawiki/2018/6/6e/T--XMU-China--microfluidic_chips3.png)

# Sep 9th

## 1st generation of 3D printing shell

![](https://static.igem.org/mediawiki/2018/b/b2/T--XMU-China--3D_shell1_1.png)
![](https://static.igem.org/mediawiki/2018/4/49/T--XMU-China--3D_shell1_2.png)

# Sep 16th

## Coating experiments

![](https://static.igem.org/mediawiki/2018/9/92/T--XMU-China--Coating_pinciple.jpeg)
![](https://static.igem.org/mediawiki/2018/6/6d/T--XMU-China--Coating_results.jpeg)

## Forming Double Strands Experiments

![](https://static.igem.org/mediawiki/2018/b/bc/T--XMU-China--binding.png)

# Sep 23th

## Testing for “deep learning”

![](https://static.igem.org/mediawiki/2018/6/61/T--XMU-China--deep_learning.png)

# Sep 30th

## 2nd generation of 3D printing shell

![](https://static.igem.org/mediawiki/2018/7/73/T--XMU-China--3D_shell2.png)

# Oct 2nd

## Whole electronic circuit testing

![](https://static.igem.org/mediawiki/2018/2/23/T--XMU-China--electronic_circui_testing1.png)
![](https://static.igem.org/mediawiki/2018/1/16/T--XMU-China--electronic_circui_testing2.png)

![](https://static.igem.org/mediawiki/2018/3/3d/T--XMU-China--xmu_is_different.png)

* [Home](https://2018.igem.org/Team%3AXMU-China)
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* [Entrepreneurship](https://2018.igem.org/Team%3AXMU-China/Entrepreneurship)
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* [Design](https://2018.igem.org/Team%3AXMU-China/Design)
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* [Notebook](https://2018.igem.org/Team%3AXMU-China/Notebook)
* [Judging](https://2018.igem.org/Team%3AXMU-China/Judging)

* [Results](https://2018.igem.org/Team%3AXMU-China/Results)
* [Software](https://2018.igem.org/Team%3AXMU-China/Software)
* [Collaborations](https://2018.igem.org/Team%3AXMU-China/Collaborations)
* [Members](https://2018.igem.org/Team%3AXMU-China/Members)
* [After iGEM](https://2018.igem.org/Team%3AXMU-China/After_iGEM)

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# Team:XMU-China/Experiments

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Editing Team:XMU-China/Experiments

Team:XMU-China/Experiments - 2018.igem.org

* Project
  + [Description](https://2018.igem.org/Team%3AXMU-China/Description)
  + [Design](https://2018.igem.org/Team%3AXMU-China/Design)
  + [Results](https://2018.igem.org/Team%3AXMU-China/Results)
  + [Demonstrate](https://2018.igem.org/Team%3AXMU-China/Demonstrate)
  + [Parts](https://2018.igem.org/Team%3AXMU-China/Parts)

* Hardware
  + [Overview](https://2018.igem.org/Team%3AXMU-China/Hardware)
  + [Microfluidic Chips](https://2018.igem.org/Team%3AXMU-China/Hardware#Microfluidic_Chips)
  + [Fluorescence Detection](https://2018.igem.org/Team%3AXMU-China/Hardware#Fluorescence_Detection)
  + [Raspberry Pi](https://2018.igem.org/Team%3AXMU-China/Hardware#Raspberry_Pi)
  + [Application](https://2018.igem.org/Team%3AXMU-China/Hardware#Application)
  + [Software](https://2018.igem.org/Team%3AXMU-China/Software)
  + [Product Design](https://2018.igem.org/Team%3AXMU-China/Applied_Design)
* Model
  + [Summary](https://2018.igem.org/Team%3AXMU-China/Model#Summary)
  + [Thermodynamic Model](https://2018.igem.org/Team%3AXMU-China/Model#Thermodynamic_model)
  + [Fluid dynamics Model](https://2018.igem.org/Team%3AXMU-China/Model#Fluid_dynamics_model)
  + [Molecular Docking Model](https://2018.igem.org/Team%3AXMU-China/Model#Molecular_docking_model)
  + [Derivation of Rate Equation](https://2018.igem.org/Team%3AXMU-China/Model#The_dynamic_model)
* Social Works
  + [Human Practice](https://2018.igem.org/Team%3AXMU-China/Human_Practices)
  + [Engagement](https://2018.igem.org/Team%3AXMU-China/Public_Engagement)
  + [Collaborations](https://2018.igem.org/Team%3AXMU-China/Collaborations)
  + [Entrepreneurship](https://2018.igem.org/Team%3AXMU-China/Entrepreneurship)
* Other Works
  + [InterLab](https://2018.igem.org/Team%3AXMU-China/InterLab)
  + [Improve](https://2018.igem.org/Team%3AXMU-China/Improve)
  + [Safety](https://2018.igem.org/Team%3AXMU-China/Safety)
  + [Measurement](https://2018.igem.org/Team%3AXMU-China/Measurement)
* Notebook
  + [Notebook](https://2018.igem.org/Team%3AXMU-China/Notebook)
  + [Experiments](https://2018.igem.org/Team%3AXMU-China/Experiments)
  + [Engineering](https://2018.igem.org/Team%3AXMU-China/Engineering)
* Team
  + [Members](https://2018.igem.org/Team%3AXMU-China/Team)
  + [Attributions](https://2018.igem.org/Team%3AXMU-China/Attributions)
  + [Judging](https://2018.igem.org/Team%3AXMU-China/Judging)
  + [After iGEM](https://2018.igem.org/Team%3AXMU-China/After_iGEM)

![](https://static.igem.org/mediawiki/2018/b/b5/T--XMU-China--singlelogo.png)
![](https://static.igem.org/mediawiki/2018/3/35/T--XMU-China--iGEM_logo.png)

Team

* [Members](https://2018.igem.org/Team%3AXMU-China/Team)
* [Attributions](https://2018.igem.org/Team%3AXMU-China/Attributions)
* [Judging](https://2018.igem.org/Team%3AXMU-China/Judging)
* [After iGEM](https://2018.igem.org/Team%3AXMU-China/After_iGEM)

Notebook

* [Notebook](https://2018.igem.org/Team%3AXMU-China/Notebook)
* [Experiments](https://2018.igem.org/Team%3AXMU-China/Experiments)
* [Engineering](https://2018.igem.org/Team%3AXMU-China/Engineering)

Other Works

* [InterLab](https://2018.igem.org/Team%3AXMU-China/InterLab)
* [Improve](https://2018.igem.org/Team%3AXMU-China/Improve)
* [Safety](https://2018.igem.org/Team%3AXMU-China/Safety)
* [Measurement](https://2018.igem.org/Team%3AXMU-China/Measurement)

Social Works

* [Human Practice](https://2018.igem.org/Team%3AXMU-China/Human_Practices)
* [Engagement](https://2018.igem.org/Team%3AXMU-China/Public_Engagement)
* [Collaborations](https://2018.igem.org/Team%3AXMU-China/Collaborations)
* [Entrepreneurship](https://2018.igem.org/Team%3AXMU-China/Entrepreneurship)

Model

* [Summary](https://2018.igem.org/Team%3AXMU-China/Model)
* [Thermodynamic Model](https://2018.igem.org/Team%3AXMU-China/Model#Thermodynamic_model)
* [Fluid dynamics Model](https://2018.igem.org/Team%3AXMU-China/Model#Fluid_dynamics_model)
* [Molecular Docking Model](https://2018.igem.org/Team%3AXMU-China/Model#Molecular_docking_model)
* [Derivation of Rate Equation](https://2018.igem.org/Team%3AXMU-China/Model#The_dynamic_model)

Hardware

* [Overview](https://2018.igem.org/Team%3AXMU-China/Hardware)
* [Microfluidic Chips](https://2018.igem.org/Team%3AXMU-China/Hardware#Microfluidic_Chips)
* [Fluorescence Detection](https://2018.igem.org/Team%3AXMU-China/Hardware#Fluorescence_Detection)
* [Raspberry Pi](https://2018.igem.org/Team%3AXMU-China/Hardware#Raspberry_Pi)
* [Application](https://2018.igem.org/Team%3AXMU-China/Hardware#Application)
* [Software](https://2018.igem.org/Team%3AXMU-China/Software)
* [Product Design](https://2018.igem.org/Team%3AXMU-China/Applied_Design)

Project

* [Description](https://2018.igem.org/Team%3AXMU-China/Description)
* [Design](https://2018.igem.org/Team%3AXMU-China/Design)
* [Results](https://2018.igem.org/Team%3AXMU-China/Results)
* [Demonstrate](https://2018.igem.org/Team%3AXMU-China/Demonstrate)
* [Parts](https://2018.igem.org/Team%3AXMU-China/Parts)

[![](https://static.igem.org/mediawiki/2018/6/6f/T--XMU-China--jiannanlogo.png)](https://2018.igem.org/Team%3AXMU-China)

Experiments

![](https://static.igem.org/mediawiki/2018/e/e6/T--XMU-China--right50.png)
[![](https://static.igem.org/mediawiki/2018/5/50/T--XMU-China--right51.png)
[Molecular Cloning Handbook](#Molecular_Cloning_Handbook)](#Molecular_Cloning_Handbook)
[![](https://static.igem.org/mediawiki/2018/0/04/T--XMU-China--right52.png)
[Competition experiment](#Competition_experiment)](#Competition_experiment)
[![](https://static.igem.org/mediawiki/2018/d/d0/T--XMU-China--right53.png)
[OMVs experiment](#OMVs_experiment)](#OMVs_experiment)
[![](https://static.igem.org/mediawiki/2018/1/11/T--XMU-China--right54.png)
[Hardware experiment](#Hardware_experiment)](#Hardware_experiment)
[![](https://static.igem.org/mediawiki/2018/b/ba/T--XMU-China--right55.png)
[Supporting project experiment](#Supporting_project_experiment)](#Supporting_project_experiment)

Molecular Cloning Handbook

#### [Molecular Cloning Handbook 4.0 BETA of XMU-China](#collapse0)

[Molecular\_Cloning\_Handbook.pdf](https://static.igem.org/mediawiki/2018/b/b5/T--XMU-China--Molecular_Cloning_Handbook.pdf)

Competition experiment

Verifying the EpCAM's activity

#### [I. Materials & Apparatus](#collapse11)

● Ni-bead with EpCAM coated
● 5% PBS-BSA Buffer (see Appendices)
● Antibody of EpCAM (PE labeled)
● Flow Cytometer (BD FACSVerseTM)

#### [II. Before experiment](#collapse114_1_1)

Prepare 5% PBS-BSA: Weigh 0.5 g BSA, then add 1×PBS to the volume being 10 mL, then mix it evenly to make sure the solid dissolved.

#### [III. Steps](#collapse115_1_1)

**1. Make distributions:**Add reagent to 3 1.5 mL tubes respectively according to the following instructions. Add PBS-BSA first then the antibody.
1) Sample Group: 5 μL EpCAM-Ni bead + 185 μL PBS-BSA + 10 μL antibody of EpCAM.
2) Positive Control: 5 μL EpCAM-Ni bead + 185 μL PBS-BSA + 10 μL antibody of EpCAM.
3) Negative Control: 5 μL EpCAM-Ni bead + 195 μL PBS-BSA.
**2. Wash the free antibody:**Take out the filter of a 10 μL filter tip and insert it into a 200 μL tip to make a 200 μL filter tip.
The above three tubes are respectively subjected to the following operations:
1) Transfer the 200 μL of solution to the 200 μL filter tip, then pipette it up and down to mix evenly.
2) Use a syringe to squeeze out the solution in the 200 μL filter tip.
3) Remove the filter tip and place it in the previous tube.
4) Rinse the tip with 450 μL of 1×PBS Buffer.
5) Take out the tip, then transfer 150μL of Buffer from 4) to the filter tip and pipette up and down to mix evenly.
6) Use a syringe to squeeze out the solution in the 200 μL filter tip.
7) Repeat 5) to 6) three times, at which time 450 μL of the solution from 4) was filtered.
8) Use a pair of scissors to cut the thin end of the filter's head and loosen the filter.
9) Transfer 200 μL of 1×PBS Buffer to a new 1.5 ml tube.
10) Use a pipette to insert the cut filter tip into the tube from 9) and pipette it up and down for several times.
**3. Flow Cytometer test:**Precautions:
1) Turn on the regulator first, then turn on the computer, and finally turn on the instrument 30 minutes before measuring.
2) Check if the sheath tank has a solution, if the waste container is full, and if the dehumidifier is turned on.
3) Remember to perform a two-step **self-test**.
4) Please use **daily clean** after measuring.
5) Shutdown sequence: Click **shut down** on the software, turn off the instrument, then turn off the computer, and finally turn off the regulator.

SYL3C binding EpCAM

#### [I. Materials & Apparatus](#collapse113_1)

● SYL3C-FAM (Sangon Biotech®, see Appendices)
● EpCAM (ACROBiosystems®)
● Binding Buffer (see Appendices)
● Fluorescence Spectrometer (Shimadzu® RF-6000)
● Polarizer (from a pair of 3D glasses)

#### [II. Steps](#collapse12)

**1. Make distributions in 1.5 mL centrifuge tubes:**1) Tube 1 (Aptamer Group): 1 μL 100 μM (100 pmol) SYL3C-FAM + 179 μL binding Buffer.
2) Tube 2 (Aptamer-EpCAM Group): 1 μL 100 μM (100 pmol) SYL3C-FAM + 179 μL binding Buffer.
**2. Sample processing:**The above 2 tubes are simultaneously subjected to the following steps:
1) Denature at 95℃ for 10 minutes.
2) Remove the tubes, then put them at room temperature for about 20 minutes.
3) Add 20 μL of 100 μg/mL EpCAM protein to Tube 2.
4) Add 20 μL of binding Buffer to Tube 1.
5) Incubate all tubes in a shaker for 40 minutes (37℃, 200 rpm).
Note: Avoiding light in whole process is necessary.
**3. Measure fluorescence intensity from vertical/parallel direction:**1) Turn on the instrument for 30 minutes before measuring.
2) Choose "Emission Mode". The excitation wave length is 495 nm, and the range of emission wave length is from 500 nm to 600 nm.
3) Before measuring, dilute the solution from step 2 to about 3 mL with binding Buffer in the fluorescence cuvette.
4) Attach a polarizer to the exit of the excitation light, and attach another polarizer to the receiving inlet of the emission light to make sure the polarization directions of the two polarizers are vertical/parallel.
5) Start measuring to obtain I∥, max (Aptamer), I⊥, max (Aptamer), I∥, max (Aptamer + EpCAM), I⊥, max (Aptamer + EpCAM).

#### [III. Appendices](#collapse13)

● Sequence of SYL3C-FAM:
5'-CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTG-FAM-3'
● Binding Buffer (pH 7.3~7.5) contains:
1×PBS Buffer with 5 mM MgCl2

Forming double strands

#### [I. Materials & Apparatus](#collapse14)

● SYL3C-TEG-biotin (BoruiTM, see Appendices)
● C3-FITC (BoruiTM, see Appendices)
● C4-FITC (BoruiTM, see Appendices)
● Binding Buffer (see Appendices)
● 50×TAE concentrate solution (Solarbio®)
● Agarose (Biowest®)
● DNA dye (TransGen® GelStain)
● Distilled water
● Microwave oven
● 10×Loading Buffer (Takara®)
● Electrophoresis instrument

#### [II. Before Experiment](#collapse15_1)

Dilute 50×TAE concentrate solution to 1×TAE Buffer with distilled water.

#### [III. Steps](#collapse16)

**1. Make distributions in 1.5 mL centrifuge tubes according to the following:**
1) Tube 1 (Aptamer Group): 1 μL 100 μM (100 pmol) SYL3C-TEG-biotin + 19 μL binding Buffer.
2) Tube 2 (C4 Group): 1 μL 100 μM (100 pmol) C4-FITC + 19 μL binding Buffer.
3) Tube 3 (C3 Group): 1 μL 100 μM (100 pmol) C3-FITC + 19 μL binding Buffer.
4) Tube 4 (C4&Aptamer Group): 1 μL 100 μM (100 pmol) SYL3C-TEG-biotin +1 μL 100 μM (100 pmol) C4-FITC + 18 μL binding Buffer.
5) Tube 5 (C3&Aptamer Group): 1 μL 100 μM (100 pmol) SYL3C-TEG-biotin +1 μL 100 μM (100 pmol) C3-FITC + 18 μL binding Buffer.
Note: The Tube 2, 3, 4 and 5 should be coated by tin foil properly after distributing.
**2. Sample processing:**
The above 5 tubes are simultaneously subjected to the following steps:
1) Denature at 95℃ for 10 minutes.
2) Anneal to room temperature for 20 minutes.
**3. Prepare 3% agarose gel:**
1) Weigh 0.9 g of agarose in a 100 mL Erlenmeyer flask.
2) Add 30 mL of 1×TAE Buffer into the flask from 1).
3) Make agarose melt by microwave oven (medium-high heat, about 3 minutes).
4) Add 3 μL of GelStain, mix by shocking.
5) Assemble gel pouring apparatus by inserting gate into slots.
6) Pour agarose gel into the gel tray.
7) Cool for 40 minutes to solidify the DNA agarose gel.
8) Remove the pouring apparatus, put the gel into an electrophoresis instrument.
**4. Electrophoresis:** 1) Add 2 μL of 10×Loading Buffer to each tube, then pipette up and down to evenly mix.
2) Pipette all samples which have been mixed with loading Buffer into the slots.
3) Turn on the electrophoresis instrument, set the working electric current at 150 mA and the working electric voltage at 110 V.
4) Electrophoresis for 50 minutes.
**5. Gel imaging:** Turn off the instrument, take the gel into the gel formatter to take and save photos.

#### [IV. Appendices](#collapse15_1_1)

● Sequence of SYL3C-TEG-biotin:
5'-CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTG-TEG-Biotin-3'
● Sequence of C3-FITC:
5'-CTCTGTAGTGTTTTTTTTTTTTTT-FITC-3'
● Sequence of C4-FITC:
5'-TCTGTAGTGTTTTTTTTTTTTTTT-FITC-3'
● Binding Buffer (pH 7.3~7.5)contains:
1×PBS Buffer with 5 mM MgCl2

Competition

#### [I. Materials & Apparatus](#collapse17)

● SYL3C-TEG-biotin (BoruiTM, see Appendices)
● C3-FITC (BoruiTM, see Appendices)
● EpCAM (ACROBiosystems®)
● Magnetic Beads (InvitrogenTM)
● Binding & Washing Buffer (see Appendices)
● Incubation Buffer (see Appendices)
● Vortex (IKA® lab dancer)
● Fluorescence Spectrometer (Shimadzu® RF-6000)

#### [II. Before experiment](#collapse17_1)

1.8 μL of 100 μM SYL3C-TEG-biotin is diluted by 1×B&W Buffer up to 75 μL, and 1.2 μL of C3-FITC is also diluted by 1×B&W up to 50 μL.
The tube of 100 μg/mL C3-FITC should be coated properly by tin foil in order to avoid being quenched.

#### [III. Steps](#collapse18)

**1. Make distributions in 1.5 mL centrifuge tubes according to the following:**
Tube 1 (Blank Control): 100 μL incubation Buffer.
Tube 2 (Negative Control): 25 μL diluted SYL3C-TEG-biotin + 25 μL 1×B&W Buffer.
Tube 3 (Competition Group): 25 μL diluted SYL3C-TEG-biotin + 25 μL diluted C3-FITC.
Tube 4 (Competition Group without EpCAM): 25 μL diluted SYL3C-TEG-biotin + 25 μL diluted C3-FITC.
Tube 5 (Positive Control): 0.6 μL 100 μM C3-FITC + 100 μL incubation Buffer.
Note: The Tube 3, 4 and 5 should be coated by tin foil properly after distributing.
**2. Denature, anneal and renature:**The Tube 2, 3, and 4 are denatured at 95°C for 5 minutes, then anneal to room temperature.
**3. Wash magnetic beads:**
1) Resuspend the beads and vortex for 10 seconds to make them even.
2) Transfer 75 μL of 10 mg / mL beads to 3 200 μL centrifuge tubes (ie 25 μL per tube).
3) Add 25 μL of 2×B&W Buffer to each tube.
4) Vortex for 5 seconds to resuspend.
5) Place the tubes in a magnetic field for 1 minute, then aspirate the supernatant.
6) Remove the tubes from the magnetic field and add 50 μL of 1×B&W Buffer to each tube.
7) Repeat steps 4) to 5).
8) Add 50 μL of 1×B&W Buffer to each tube.
**4. Bind to magnetic beads:**1) Add 50 μL of solution of Tube 2,3 and 4 from step 2 to 50 μL of washed beads, respectively.
2) Vortex for 5 seconds to resuspend.
3) Incubate at room temperature for 30 minutes in the dark.
4) Vortex for 5 seconds to resuspend.
5) Place the tubes in a magnetic field for 2 minute, then aspirate the supernatant.
6) Remove the tubes from the magnetic field and add 100 μL of 1×B&W Buffer to each tube.
7) Repeat steps 4) to 5).
8) Add 100 μL of incubation Buffer to Tube2, 3 and 4, respectively.
**5. Compete:**1) Add 17 μL of diluted EpCAM to Tube 2 and 3. And add 17 μL of incubation Buffer to the last each tube.
2) Incubate at room temperature for 40 minutes in the dark.
3) Place the tubes in a magnetic field for 2~3 minutes, then pipette 100 μL of the supernatant into a new 200 μL centrifuge tube and mark it.
4) Measure the fluorescence intensity of Tube 1, 5 and the supernatant from 3).
Note: Before measuring, the solution of each tube will be further diluted by adding 3, 000 μL of incubation Buffer into it. The excitation wave length is 495 nm, and the range of emission wave length is from 500 nm to 600 nm.

#### [IV. Appendices](#collapse19)

● Sequence of SYL3C-TEG-biotin:
5'-CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTG-TEG-Biotin-3'
● Sequence of C3-FITC:
5'-CTCTGTAGTGTTTTTTTTTTTTTT-FITC-3'
● 2×Binding & Washing Buffer (pH 7.5) contains:
10 mM Tris 1 mM EDTA 2 M NaCl
● Incubation Buffer (pH 7.5) contains:
20 mM Tris 140 mM NaCl 5 mM KCl 1 mM MgCl2

Trans-cleavage activity of Cas12a

#### [I. Materials & Apparatus](#collapse113)

● Cas12a (Alt-R® CRISPR-Cas12a (Cpf1) System)
● crRNA (IDT®)
● TE Buffer (Sangon Biotech®)
● [DNaseAlertTM Substrate Nuclease Detection System[1]](https://static.igem.org/mediawiki/2018/4/4c/T--XMU-China--dnasealert-detection-manual.pdf)(IDT®, see Appendices)
● 21nt ssDNA (BoruiTM, see Appendices)
● Black 96-well plate (Corning®)
● Microplate reader (Tecan Infinite® M200 Pro)

#### [II. Before experiment](#collapse114_1)

● Add 26.7 μL of filtered TE Buffer into the tube of crRNA to make a 75 μM stock.
● Add 88.6 μL of filtered TE Buffer into the tube of ssDNA to make a 100 μM stock.

#### [III. Steps](#collapse114)

**1. Form the RNP complex:**1) Prepare 3 200 μL tubes. Add 1 μL of Cas12a and 1 μL of diluted crRNA into each nuclease-free 200 μL tube.
2) Incubate Cas12a with crRNA at 37℃ for 30 minutes to form the RNP complex.
 **2. Pretreatment of substrate:**1) Prepare 3 DNaseAlertTM Substrate single-use tubes. Add 5 μL of Nuclease-Free Water to each tube.
2) Add 5 μL of 10×DNaseAlertTM Buffer to each tube.
**3. Make distributions:**
 1) Cas12a Group: DNaseAlertTM Substrate (from step 2) + 2 μL RNP complex (from step 1) + 1 μL diluted ssDNA + 37 μL filtered TE Buffer + 40 μL Nuclease-Free Water.
2) Negative Control: DNaseAlertTM Substrate (from step 2) + 40 μL Nuclease-Free Water + 40 μL Nuclease-Free Water.
3) Positive Control: DNaseAlertTM Substrate (from step 2) + 1 μL DNase I + 39 μL filtered TE Buffer + 40 μL Nuclease-Free Water.
**4. Measure fluorescence intensity by microplate reader:**1) Incubate the reaction system at 37℃ for 30 minutes, then transform the reaction system into the black 96-well plate.
2) Before measure, add 12 μL of TE Buffer into each tube.
3) Set temperature: 37℃, choose plate type: Black. Set excitation wavelength: 536 nm and emission wavelength: 556 nm. Choose mode: Optimal.
4) Measure fluorescence intensity at 30 min, 40 min, 50 min, 60 min, 70 min, 90 min, 110 min (Timing is from the beginning of incubation).

#### [IV. Appendices](#collapse115)

● DNaseAlertTM Substrate Nuclease Detection System contains:
1) DNaseAlertTM Substrate (25 single-use tubes (50 pmol per tube) or 2 tubes bulk substrate (2 nmol per tube))
2) DNaseAlertTM Buffer (250 μL)
3) Nuclease-Free Water (2 mL)
4) DNase I Enzyme (25 μL)
5) Nuclease Decontamination Solution (50 mL)
● Sequence of 21nt ssDNA:
5'-CAGGCCAACCCCCCATGACAA-3'

#### [V. Reference](#collapse115-1)

[1] <https://static.igem.org/mediawiki/2018/4/4c/T--XMU-China--dnasealert-detection-manual.pdf>

Signal transformation

#### [I. Materials & Apparatus](#collapse113_1_1)

● TE Buffer (Sangon Biotech®)
● [DNaseAlertTM Substrate Nuclease Detection System[1]](https://static.igem.org/mediawiki/2018/4/4c/T--XMU-China--dnasealert-detection-manual.pdf) (IDT®, see Appendices)
● Fluorescence spectrometer (Shimadzu® RF-6000)

#### [II. Steps](#collapse114_2_2)

**1. Make distributions:**Add reagent into 2 DNaseAlertTM Substrate single-use tubes according to following instruction. Avoiding light is necessary during the whole process.
1) Negative Control: 5 μL Nuclease-Free Water + 5 μL 10×DNaseAlert Buffer + 40 μL Nuclease-Free Water + 40 μL Nuclease-Free Water.
2) Enzyme Group: 5 μL Nuclease-Free Water + 5 μL 10×DNaseAlert Buffer + 39 μL Nuclease-Free Water + 40 μL Nuclease-Free Water.
Place the two tubes at room temperature for 30 minutes to enough dissolve fluorescent substance.
**2. Make distributions further according to the time gradient:**1) Prepare 6 200μL-PCR tubes in advance, then divide into 2 groups (3 tubes per group). Mark "W" and "I" on the tubes of two groups, respectively. Further mark the tubes according to the time gradient of 3 min, 15 min and 30 min (e.g. "W 3", "I 15").
2) Add 1 μL of DNase I into the DNaseAlertTM Substrate single-use tube of Enzyme Group.
3) Mix immediately, then transfer 30 μL of reaction liquid from each single-use tube to each PCR tube marked before. Place all PCR tubes in 37℃ water bath. Then start timing.
4) Each tube: React for corresponding time, then measure florescence intensity immediately.
**3. Measure fluorescence intensity:**1) Turn on the instrument for 30 minutes before measuring.
2) Choose "Emission Mode". The excitation wave length is 536 nm, and the range of emission wave length is from 540 nm to 580 nm.
3) Before measuring, dilute the reaction liquid to about 3 mL with TE Buffer in the fluorescence cuvette.

#### [III. Appendices](#collapse115_2)

● DNaseAlertTM Substrate Nuclease Detection System contains:
1) DNaseAlertTM Substrate (25 single-use tubes (50 pmol per tube) or 2 tubes bulk substrate (2 nmol per tube))
2) DNaseAlertTM Buffer (250 μL)
3) Nuclease-Free Water (2 mL)
4) DNase I Enzyme (25 μL)
5) Nuclease Decontamination Solution (50 mL)

#### [V. Reference](#collapse115-2)

[1] <https://static.igem.org/mediawiki/2018/4/4c/T--XMU-China--dnasealert-detection-manual.pdf>

Integrated experiment

#### [I. Materials & apparatus](#collapse38-1)

● SYL3C-TEG-biotin (BoruiTM, see Appendices)
● C3 (BoruiTM, see Appendices)
● EpCAM (ACROBiosystems®)
● Magnetic Beads (InvitrogenTM)
● Binding & Washing Buffer (see Appendices)
● Incubation Buffer (see Appendices)
● Cas12a (Alt-R® CRISPR-Cas12a (Cpf1) System)
● crRNA (BoruiTM, see Appendices)
● TE Buffer (Sangon Biotech®)
● [DNaseAlertTM Substrate Nuclease Detection System[1]](https://static.igem.org/mediawiki/2018/4/4c/T--XMU-China--dnasealert-detection-manual.pdf) (IDT®, see Appendices)
● Vortex (IKA® lab dancer)
● Fluorescence Spectrometer (Shimadzu® RF-6000)

#### [II. Before experiment](#collapse39)

0.6 μL of 100 μM SYL3C-TEG-biotin is diluted by 1×B&W buffer up to 25 μL, and 0.6 μL of C3 is also diluted by 1×B&W up to 25 μL.

#### [III. Steps](#collapse310-1)

**1. Make distributions in a 1.5 mL centrifuge tube according to the following:**25 μL diluted SYL3C-TEG-biotin + 25 μL diluted C3.
**2. Denature, anneal and renature:**The tube are denatured at 95°C for 10 minutes, then anneal to room temperature.
**3. Wash magnetic beads:**1) Resuspend the beads and vortex for 10 seconds to make them even.
2) Transfer 25 μL of 10 mg / mL beads to a 200 μL centrifuge tube.
3) Add 25 μL of 2×B&W buffer to the tube.
4) Vortex for 5 seconds to resuspend.
5) Place the tube in a magnetic field for 1 minute, then aspirate the supernatant.
6) Remove the tube from the magnetic field and add 50 μL of 1×B&W buffer to the tube.
7) Repeat steps 4) to 5).
8) Add 50 μL of 1×B&W buffer to the tube.
**4. Bind to magnetic beads:**1) Add 50 μL of solution of the tube from step 2 to 50 μL of washed beads, respectively.
2) Vortex for 5 seconds to resuspend.
3) Incubate at room temperature for 30 minutes.
4) Vortex for 5 seconds to resuspend.
5) Place the tube in a magnetic field for 2 minute, then aspirate the supernatant.
6) Remove the tube from the magnetic field and add 100 μL of 1×B&W buffer to the tube.
7) Repeat steps 4) to 5).
8) Add 20 μL of incubation buffer to the tube.
**5. Compete:**1) Add 30 μL of 100 μg/mL EpCAM to the tube.
2) Incubate at room temperature for 40 minutes.
3) Place the tube in a magnetic field for 2~3 minutes, then pipette 38 μL of the supernatant into a new 200 μL centrifuge tube and mark it.
**6. Form the RNP complex:**1) Prepare a 200 μL tube. Add 1 μL of 63 μM Cas12a and 1 μL of diluted crRNA (75 μM) into the tube.
2) Incubate Cas12a with crRNA at room temperature for 30 minutes to form the RNP complex.
**7. Detect using DNaseAlertTM:**1) Add reagent into a DNaseAlertTM Substrate single-use tube according to the following instruction. Avoiding light is necessary during the whole process.
5 μL Nuclease-Free Water + 5 μL 10×DNaseAlert Buffer + 40 μL **supernatant （from the tube in step 5 & the RNP complex）** + 40 μL Nuclease-Free Water.
2) Incubate the reaction system at 37℃ for 30 minutes in the dark.
3) Measure fluorescence intensity:
① Turn on the instrument for 30 minutes before measuring.
② Choose “Emission Mode”. The excitation wave length is 536 nm, and the range of emission wave length is from 540 nm to 580 nm.
③ Before measuring, dilute the reaction liquid to about 3 mL with TE buffer in the fluorescence cuvette.

#### [IV. Appendices](#collapse311)

● Sequence of SYL3C-TEG-biotin:
5'-CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTG-TEG-Biotin-3'
● Sequence of C3:
5'-CTCTGTAGTGTTTTTTTTTTTTTT-3'
● Sequence of crRNA:
5'-UAAUUUCUACUCUUGUAGAUAAAAAAAAAAACACUACAGAG-3’
● 2×Binding & Washing Buffer (pH 7.5) contains:
10 mM Tris 1 mM EDTA 2 M NaCl
● Incubation Buffer (pH 7.5) contains:
20 mM Tris 140 mM NaCl 5 mM KCl 1 mM MgCl2

#### [V. Reference](#collapse115-3)

[1] <https://static.igem.org/mediawiki/2018/4/4c/T--XMU-China--dnasealert-detection-manual.pdf>

OMVs experiment

Preparation of OMVs-free culture medium

#### [I. Requirements](#collapse31)

● 0.45 μm syringe filters
● LB broth culture
● OptimaTM XE-90 Ultracentrifuge (Beckman Coulter)
● SW41 Ti Swinging-Bucket Rotor

#### [II. Protocols](#collapse32)

1) Prepare 100 mL 10×LB broth medium.
2) Pass the culture through a 0.45 μm syringe filter.
3) Ultracentrifuge each tube for 18 hours at 100,000 g in a Swinging-Bucket rotor to precipitate the biomass.
4) Recover the supernatant of each tube and dilute them to 1×LB broth culture.
5) Sterilize the 1×LB broth culture.

OMVs Extraction for HSFCM[1] analysis and electron microscope analysis

#### [I. Requirements](#collapse33)

● PBS
● 0.45 μm syringe filters
● OMVs-free LB broth culture
● OptimaTM MAX-XP Ultracentrifuge (Beckman Coulter)
● TLA120.2 Rotor

#### [II. Protocols](#collapse34)

**1. Bacteria Growth:**
1) Inoculate each strain for analysis to 10 mL OMVs-free culture with appropriate antibiotic, and place them on a shaking incubator at 37°C, 200 rpm.
2) After about 5 hours of incubation (when the OD600 of each culture is 0.6-0.8), add any necessary inducing agents (IPTG at 0.5 mM or Arabinose at a 0.2% final concentration) into the cultures, and then leave them on the shaking incubator at 37°C, 200 rpm overnight.
**2. Cell Extraction:**
1) Measure the OD600 of each culture before OMVs harvest.
2) Centrifuge each culture for 10 min at 1,000 g and then 10 min at 2,000 g in a fixed angle rotor to pellet the biomass.
3) Recover the supernatants and then pass them through a 0.45 μm syringe filter.
**3. OMVs Purification:**
1) Load 10 mL syringe-filtered supernatant into ultracentrifugal tubes, and then ultracentrifuge them at 100,000 g in a TLA120.2 rotor for 17 min at 4°C。
2) Discard the supernatant, add enough PBS Buffer and repeat the step 1 above, and then discard the supernatant again.
3) Add 100 μL PBS into the tubes and then pipet up and down for about 200 times.
4) OMVs are stored at 4°C until analysis.

#### [III. Reference](#collapse35)

[[1] https://pubs.acs.org/doi/10.1021/nn505162u](https://pubs.acs.org/doi/10.1021/nn505162u)

OMVs Extraction for SDS-PAGE analysis

#### [I. Requirements](#collapse36)

● PBS
● 0.45 μm syringe filters
● OMVs-free LB broth culture
● OptimaTM XE-90 Ultracentrifuge (Beckman Coulter)
● SW41 Ti Swinging-Bucket Rotor

#### [II. Protocols](#collapse37)

**1. Bacterial Growth:**
1) Inoculate each strain for analysis into 100 mL OMVs-free culture with appropriate antibiotic, and then place them on a shaking incubator at 37°C, 200 rpm.
2) After about 5 hours of incubation (when the OD600 of each culture is 0.6-0.8), add any necessary inducing agents (IPTG at 0.5 mM or Arabinose at a 0.2% final concentration) into the cultures, and leave them on the shaking incubator at 37°C overnight.
**2. Cell Extraction:**
1) Measure the OD600 of each culture before OMVs harvest.
2) Centrifuge each culture for 10 min at 1,000 g and then 10 min at 2,000 g in a fixed angle rotor to pellet the biomass.
3) Recover the supernatants and then pass them through a 0.45 μm syringe filter.
**3. OMVs Purification:**
1) Load 100 mL syringe-filtered supernatant into ultracentrifugal tubes, and then ultracentrifuge them at 100,000 g in a Swinging-Bucket rotor for 2 hours at 4°C。
2) Recover the supernatant, repeat the step 1 above, and then discard the supernatant.
3) Add 100 μL PBS into the tube and then pipet up and down for about 200 times.
4) OMVs are stored at 4°C until analysis.
**4. SDS-PAGE:**
1) Prepare the 1×Tris-Glycine electrophoresis Buffer (2 L)
2) Add 5×Protein loading Buffer (V/V) to the OMVs pellet, and then pipet up and down to mix the sample.
3) Put the tube in 100°C water bath for 10 min.
4) Make the gel for SDS-PAGE according to the manufacture's instructions.
5) Add 10 μL protein marker to the first well and add about 25 μL samples to other wells.
Note: You must keep enough sample loaded in every well. You can add 25 μL firstly and after the sample pass through the stacking gel, stop the instrument immediately and add 25 μL sample again.
6) Start the electrophoresis apparatus, the parameters are showed below:
7) Turn off the instrument, take the gel into a clean plate with deionized water, ish it gently and then discard the water.
8) Add the Coomassie blue stain into the plate and then heat it to about 50°C.
9) Incubate for 30 min on a shaking bed.
10) Discard the stain and add the destaining solution (acetic acid: ethyl alcohol: deionized water=10: 50: 40, V/V/V), then heat it to about 50°C.
11) Incubate for 30 min on a shaking bed.
12) Repeat step10-11 for 3 times.
13) Take the gel into the gel formatter to take and save photos.

Evaluate the efficiency of the C/Dbox

#### [I. Requirements](#collapse38)

● PBS
● 0.45 μm syringe filters
● LB broth culture
● OptimaTM MAX-XP Ultracentrifuge (Beckman Coulter)
● TLA120.2 Rotor

#### [II. Protocols](#collapse39-1)

**1. Bacterial Growth:**
1) Inoculate each strain for analysis into 100 mL OMVs-free culture with appropriate antibiotic, and then place them on a shaking incubator at 37°C, 200 rpm.
2) After about 5 hours of incubation (when the OD600 of each culture is 0.6-0.8), add arabinose at a 0.2% final concentration to initiate the production of siRNA-C/Dbox.
After an additional 3 h incubation period, add IPTG to a final concentration of 0.5 mM to initiate the production of L7Ae-SpyCatcher. Incubate the culture at 37°C and 200 rpm for an additional 18 h.
**2. Cell Extraction:**
1) Measure the OD600 of each culture before OMVs harvest.
2) Centrifuge each culture for 10 min at 1,000 g and then 10 min at 2,000 g in a fixed angle rotor to pellet the biomass.
3) Recover the supernatants and then pass them through a 0.45 μm syringe filter.
**3. OMVs Purification:**
1) Load 100 mL syringe-filtered supernatant into ultracentrifugal tubes, and then ultracentrifuge them at 100,000 g in a Swinging-Bucket rotor for 2 hours at 4°C.
2) Discard the supernatant, add enough PBS Buffer and repeat the step one above, then discard the supernatant again.
3) Add 100 μL PBS into the tube and then pipet up and down for about 200 times.
4) OMVs are stored at 4°C until analysis.
**4. RNASelect dye:**
The experiment below is designed according to the manufacture' s instruction.[2]
1) Prepare a 1 mM DMSO stock solution of the SYTOTM RNASelectTM.
2) Add 1 μL of the dye stock solution to the 100 μL OMVs sample and mix them to obtain a final dye concentration of 10 μM. Incubate it at 37°C for 20 min (protect from light).
3) Analyze the efficiency of OMVs labeling using HSFCM.

#### [III. Reference](#collapse310)

[[2] https://www.thermofisher.com/order/catalog/product/S32703](https://www.thermofisher.com/order/catalog/product/S32703)

Hardware experimrnt

Motor speed testing

#### [I. Requirements](#collapse21)

● PBS solution with protein EpCAM (imitative plasma)
● Microfluidic chips

#### [II. Protocols](#collapse22)

1) Pipette appropriate imitative plasma solution into the sample chamber.
2) Gradually increase the motor speed by software and find the critical speed for the second chamber in which the competition reaction happens.
3) Find a second critical speed for the third chamber-incubation chamber.

Combine the aptamer with ssDNA

#### [I. Requirements](#collapse23)

● ssDNA (10 nt)
● Biotinylated aptamers
● PBS contained Mg2+ (5.0 mmol)
● 100× SYBR Green Ⅰ (exited by 488 nm)
● Microplate Reader(Tecan Infinite® M200 Pro)

#### [II. Protocols](#collapse24)

1) Mix equivalent aptamer and ssDNA(dissolved in the PBS with Mg2+).
2) Keep the solution away from light at room temperature/ 37℃ water bath / PCR 95 ℃ for 10 min.
3) Mix 100×SYBR Green Ⅰ with the mixed solution for 20 min at room temperature.
4) Use Microplate Reader to detect the fluorescence of the solution(using the solution contained only aptamer or ssDNA as blank control).

Ternary affinity coating (TERACOAT) method

#### [I. Requirements](#collapse25)

● Biotinylated BSA
● NeutrAvidin
● Combined aptamer-ssDNA(with the fluorophore)
● PBS-0.1% Tween 20

#### [II. Protocols [1]](#collapse26)

1) Pour and coat Biotinylated BSA on our device for 10 min.
2) ish the remaining Biotinylated BSA with PBS-Tween for 5 min.
3) Pour and coat Neutravidin on our device for 10 min.
4) ish the remaining Neutravidin with PBS-Tween for 5 min.
5) Pour and coat Combined aptamers-ssDNA on our device for 10 min.
6) ish the remaining Neutravidin with PBS-Tween for 5 min.
7) Detect the fluorescence of ishing solution from step 6 to find whether ssDNA is tightly bonded to aptamer.

#### [III. Reference](#collapse27)

[1] Piraino, Francescoet, *et al. ACS nano*. **2016**, 10: 1699-1710.

Fluorescence detection for EpCAM

#### [I. Requirements](#collapse28)

● Coated microfuidic chips
● EpCAM
● PBS-0.1% Tween 20
● Cas12a & crDNA systems

#### [II. Protocols](#collapse29)

1) Pipette appropriate imitative plasma solution into the sample chamber.
2) Control the motor speed and make the solution flow into the competitive chamber, and then remain for 20 min at room temperature.
3) Increase the motor speed and make the solution flow into the detection chamber, and then remain for 10 min at room temperature.
4) Collect the solution from the last chamber and detect whether there is any fluorescence.
5) Use the camera of the hardware to find out whether there is any fluorescence and compare the data.

Supporting project experiment

#### [I. Protocol（SDS-PAGE）](#collapse41)

1) Prepare polyacrylamide gel according to standard protocol. The parameters are showed below:
![](https://static.igem.org/mediawiki/2018/4/41/T--XMU-China--exp_supporting_table1.png)

2) At this point, the gel can either be transferred to a membrane or stained with Coomassie blue (see below).
3) Place gel in a plastic container, cover over with isopropanol fixing solution and shake at room temperature (RT). For 0.75 mm-thick gels, shake for 10 to 15 min; for 1.5 mm thick gels, shake for 30 to 60 min.
4) Pour off fixing solution, cover over with Coomassie blue staining solution and shake at RT for 2 h.
5) Pour off staining solution, ish the gel with 10% acetic acid to destain and shaking at RT overnight.

#### [II. Protein purification protocol](#collapse42)

**1. Transformation:**
1) Transform E. coli BL21star (DE3) with pET28a + SAHS plasmids.
2) Inoculate each single colon with 10 mL of LB broth culture with kanamycin at 60 mg/mL.
3) Shake each culture at 200 rpm, 37 ˚C overnight.
**2. Induced expression:**
1) After about 3 hours of incubation (when the OD600 reaches to 0.5), add IPTG at a final concentration for 1 mM to induce expression.
2) Incubate the cultures for 8 h and pellet the cells at 7,000g, 25 ˚C for 10 min.
**3. Centrifugation and lysis:**
1) Store the cells pellet at -20 ˚C until use.
2) Resuspend the pellets in 12.5 mL PBS.
3) Lyse the cells by Ultrasonic Cell Disruptor.
4) Cool the lysates at 4 ˚C for 30 min.
5) Remove insoluble components by centrifugation at 25,000g, 4 ˚C for 20 min.
6) Harvest the supernatants to purify the SAHS protein.
**4. Chromatography:**
1) Equilibrate the Ni-NTA Agarose with PBS and then adjust the A280 value to the neutral line.
2) Load appropriate amount of supernatant into the Ni-NTA Agarose column and ish with lysis Buffer until the A280 value is below 0.01.
3) Clean the column with PBS until the A280 value is below 0.01.
4) Elute the SAHS protein with Elution Buffer and collect all eluates according to its A280.
5) Heat treatment: Collect all equates and place them in water bath at 85 ˚C for 15min. Remove insoluble components by centrifugation at 13,000g for 20 min.

#### [III. Making Electrocompetent Cells Protocol](#collapse43)

1) Preferably, select single colony of E. coli from fresh LB plate for inoculating a 10 mL 2XYT overnight (O/N) starter culture. Alternatively, streak out frozen glycerol stock of bacterial cells onto LB plate, grow plate O/N, and then select single colony for starter culture. Grow 10 mL starter culture O/N in 37°C shaker (250 rpm).
2) Inoculate 1L of 2XYT media and place culture in 37°C shaker. Grow cells and measure OD600 every 45 min. When the OD600 equals 0.6-0.9 (log phase growth), remove the cells from the shaker and place on ice.
Note: It is very important to keep the cells at 4°C (or on ice) for the remainder of the procedure.
3) Split the 1 L culture into four equal parts by pouring ~250 mL of culture into each chilled 250mL Corning pointed bottle.
4) Spin (#1) in GPR centrifuge at 4000 rpm, 25 min at 4°C.
(if you chose to use the J6/ JS-4.2 rotor (E. Davidson Lab), use 1L bottles , fill half full, spin 4000rpm, 20min, at 4°C.)
5) Place bottles on ice. Remove supernatant immediately as cell pellet begins to lift off quickly. Gently resuspend each pellet in 200 mL ice-cold dH20.
6) Spin (#2) in GPR centrifuge at 4000 rpm, 25 min at 4°C.
7) Place bottles on ice. Remove supernate. Gently resuspend each pellet in 100ml of ice-cold dH20.
8) Spin (#3) in GPR centrifuge at 4000rpm, 25min at 4°C.
9) Place bottles on ice. Remove supernatant. Gently resuspend each pellet in 20mL ice-cold 10% glycerol. For each pair of 250 mL Corning bottles, transfer both 20 mL cell suspension into one chilled 50 mL conical tube- therefore you should end up with two 50 mL conical tubes on ice where each tube contains ~40 mL of cells in 10% glycerol.
10) Spin (#4) in GPR centrifuge at 4000 rpm, 10 min at 4°C.
11) Place tubes on ice. Remove supernatant. Gently resuspend each cell pellet in 1 mL of ice-cold 10% glycerol. Final OD600 of resuspended cells » 200-250.
12) With cell suspensions on ice, prepared 70l aliquots of cells in pre-chilled 1.5 mL eppendorf tubes. Snap freeze tubes containing cells in liquid N2. Store frozen cells at -80°C.
Note: liquid N2 very hazardous- use carefully and don't contact N2 directly!

#### [IV. Electrocompetent Cells Transformation Protocol](#collapse44)

1) Thaw electrocompetent cells on ice.
2) Transfer 50 mL of electrocompetent cells to a pre-chilled electroporation cuvette with 1 mm gap.
3) Add 1 mL of the assembly product to electrocompetent cells.
4) Mix gently by pipetting up and down.
5) Once DNA is added to the cells, electroporation can be carried out immediately. It is not necessary to incubate DNA with cells.
6) Add 950 mL of room-temperature SOC media to the cuvette immediately after electroporation.
7) Place the tube at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
8) Warm selection plates to 37°C.
9) Spread 100 mL of the cells onto the plates.
10) Incubate overnight at 37°C.

#### [V. Homologous recombination](#collapse45)

1) Transfer the plasmid pKD46 into **E.coli** DH5α, and Incubate at 30° for 12h.
2) Make electro-competent cell with *E.coli* DH5α contains pKD46.
3) PCR amplification of CAHS fragments, and add two segments homologous arm.
4) DNA gel electrophoresis and gel extraction and purification.
5) Transfer the fragments into the electro-competent cell.
6) Add 100 mM L-Arabinose, then induced for 90min at 37°.
7) Activate for 2h in 37° shaking incubator.
8) Add 1 M IPTG to make the final concentration to be 1 mM.
9) Add 250 μL of the bacteria solution to plate.
10) Incubate at 37°C.
11) That the colony’s color turning to be red is regarded as success.

#### [VI. Gibson Assembly® Protocol](#collapse46)

1) Set up the following reaction on ice:
![](https://static.igem.org/mediawiki/2018/d/da/T--XMU-China--exp_supporting_table2.jpeg)
\* Optimized cloning efficiency is 50–100 ng of vectors with 2–3 fold of excess inserts. Use 5 times more of inserts if size is less than 200 bps. Total volume of unpurified PCR fragments in Gibson Assembly reaction should not exceed 20%.
\*\* Control reagents are provided for 5 experiments.
\*\*\* If greater numbers of fragments are assembled, additional Gibson Assembly Master Mix may be required.
2) Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples on ice or at –20°C for subsequent transformation.
Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases (for further details see FAQ section).
3) Transform NEB 5-alpha Competent E. coli cells (provided with the kit) with 2 μL of the assembly reaction, following the transformation protocol.

#### [VII. Freeze-dry protein protocol](#collapse47)

1) Add a total of 100 μL of protein sample into centrifuge tubes.
2) Pre-freezing: put the centrifuge tubes into the refrigerator to freeze at -80 ℃ for 12h.
3) Operate the lyophilizer: start the lyophilizer to allow the chamber temperature to drop to -50℃.
4) Freeze-drying: after pre-freezing of the sample, put it into the lyophilizer. Then sublimation for 24 hours under vacuum condition which called Primary drying; Lastly, increase shelf temperature to about 30 ℃ to desorbe the residual water under vacuum condition.
5) Store the lyophilized sample under low temperature and dry conditions.

![](https://static.igem.org/mediawiki/2018/3/3d/T--XMU-China--xmu_is_different.png)

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# Team:XMU-China/Results

Team:XMU-China/Results - 2018.igem.org

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  + [Attributions](https://2018.igem.org/Team%3AXMU-China/Attributions)
  + [Judging](https://2018.igem.org/Team%3AXMU-China/Judging)
  + [After iGEM](https://2018.igem.org/Team%3AXMU-China/After_iGEM)

![](https://static.igem.org/mediawiki/2018/b/b5/T--XMU-China--singlelogo.png)
![](https://static.igem.org/mediawiki/2018/3/35/T--XMU-China--iGEM_logo.png)

Team

* [Members](https://2018.igem.org/Team%3AXMU-China/Team)
* [Attributions](https://2018.igem.org/Team%3AXMU-China/Attributions)
* [Judging](https://2018.igem.org/Team%3AXMU-China/Judging)
* [After iGEM](https://2018.igem.org/Team%3AXMU-China/After_iGEM)

Notebook

* [Notebook](https://2018.igem.org/Team%3AXMU-China/Notebook)
* [Experiments](https://2018.igem.org/Team%3AXMU-China/Experiments)
* [Engineering](https://2018.igem.org/Team%3AXMU-China/Engineering)

Other Works

* [InterLab](https://2018.igem.org/Team%3AXMU-China/InterLab)
* [Improve](https://2018.igem.org/Team%3AXMU-China/Improve)
* [Safety](https://2018.igem.org/Team%3AXMU-China/Safety)
* [Measurement](https://2018.igem.org/Team%3AXMU-China/Measurement)

Social Works

* [Human Practice](https://2018.igem.org/Team%3AXMU-China/Human_Practices)
* [Engagement](https://2018.igem.org/Team%3AXMU-China/Public_Engagement)
* [Collaborations](https://2018.igem.org/Team%3AXMU-China/Collaborations)
* [Entrepreneurship](https://2018.igem.org/Team%3AXMU-China/Entrepreneurship)

Model

* [Summary](https://2018.igem.org/Team%3AXMU-China/Model)
* [Thermodynamic Model](https://2018.igem.org/Team%3AXMU-China/Model#Thermodynamic_model)
* [Fluid dynamics Model](https://2018.igem.org/Team%3AXMU-China/Model#Fluid_dynamics_model)
* [Molecular Docking Model](https://2018.igem.org/Team%3AXMU-China/Model#Molecular_docking_model)
* [Derivation of Rate Equation](https://2018.igem.org/Team%3AXMU-China/Model#The_dynamic_model)

Hardware

* [Overview](https://2018.igem.org/Team%3AXMU-China/Hardware)
* [Microfluidic Chips](https://2018.igem.org/Team%3AXMU-China/Hardware#Microfluidic_Chips)
* [Fluorescence Detection](https://2018.igem.org/Team%3AXMU-China/Hardware#Fluorescence_Detection)
* [Raspberry Pi](https://2018.igem.org/Team%3AXMU-China/Hardware#Raspberry_Pi)
* [Application](https://2018.igem.org/Team%3AXMU-China/Hardware#Application)
* [Software](https://2018.igem.org/Team%3AXMU-China/Software)
* [Product Design](https://2018.igem.org/Team%3AXMU-China/Applied_Design)

Project

* [Description](https://2018.igem.org/Team%3AXMU-China/Description)
* [Design](https://2018.igem.org/Team%3AXMU-China/Design)
* [Results](https://2018.igem.org/Team%3AXMU-China/Results)
* [Demonstrate](https://2018.igem.org/Team%3AXMU-China/Demonstrate)
* [Parts](https://2018.igem.org/Team%3AXMU-China/Parts)

[![](https://static.igem.org/mediawiki/2018/6/6f/T--XMU-China--jiannanlogo.png)](https://2018.igem.org/Team%3AXMU-China)

Results

![](https://static.igem.org/mediawiki/2018/f/f5/T--XMU-China--right0.png)
[![](https://static.igem.org/mediawiki/2018/d/db/T--XMU-China--right1.png)
[ABCD system](#ABCDsystem)](#ABCDsystem)
[![](https://static.igem.org/mediawiki/2018/c/cd/T--XMU-China--right2.png)
[OMVs](#OMVs)](#OMVs)
[![](https://static.igem.org/mediawiki/2018/1/1c/T--XMU-China--right3.png)
[Supporting](#Supporting)](#Supporting)

The result of ABCD system

# What work we've done:

Verified the concept of our design for three modules respectively.

Verified the activity of EpCAM.

Verified the successful combination between SYL3C aptamer and EpCAM.

Verified that SYL3C aptamer could combine with 10nt complementary chain successfully.

Verified the feasibility of "competition method" successfully.

Verified that As Cas12a could be activated.

Verified the trans-cleavage activity.

Transferred molecular signal to fluorescence signal with DNaseAlert.

Verified the transfer from protein signal to the fluorescent one successfully.

# The work hasn’t finished yet:

Quantitative analysis of the effect of "competition method" and "As Cas12a system" respectively.

Verified the detection limit of "competition method", "As Cas12a system", and then our whole system.

Other designs to transfer signals for reporting devices.

# Activity of EpCAM (Flow Cytometry)

We got EpCAM directly from ACROBiosystems®. In order to prove that the protein was still bio-active, we chose antibody of EpCAM with fluorescence and flow cytometry to measure it. Furnished by the research group laboratory, the fluorescent antibody was proved effective through previous experiments.

We bought EpCAM with His-tag. It's widely known that His-tag could chelate with nickel ions, so we incubated EpCAM protein with nickel beads to bind them together. Adding the antibody and then incubating, we measured them with flow cytometry. At the same time, we started two other control group experiments, with the result shown below. Get more information of our experiment, [click here.](https://2018.igem.org/Team%3AXMU-China/Experiments)

![]( https://static.igem.org/mediawiki/2018/6/66/T--XMU-China--measurement-1017_7.png)

| Name | Events | % Parent | % Grandparent | % Total | PE-A Mean | PE-A Geo Mean |
| --- | --- | --- | --- | --- | --- | --- |
| ni-beads:P1 | 5,087 | 94.52 | \*\*\* | 94.52 | 108 | 82 |
| ni-beads+antiepcam:P1 | 5,208 | 91.45 | \*\*\* | 91.45 | 2,880 | 2,626 |
| EpCAM+Anti-EpCAM:P1 | 4,238 | 93.53 | \*\*\* | 93.53 | 14,816 | 14,211 |

**Figure 1.** The result of detecting the binding of EpCAM with fluorescent antibody, with flow cytometry.
a) Black line: pure nickel beads;
b) Blue line: nickel beads + fluorescent antibody (measure the non-specific adsorption between beads and antibody);
c) Red line: nickel beads-EpCAM complex + fluorescent antibody.

From the graph and data above, we could find out that the non-specific adsorption between nickel beads and fluorescent antibody does exist, but in the presence of EpCAM, the fluorescence intensity increased seven times compared with the original one, so we could prove that our bio-active EpCAM protein successfully combined with fluorescent antibody.

# Combination between SYL3C and EpCAM (Fluorescence Polarization)

Although EpCAM and aptamer SYL3C are directly quoted in the literature [1], we believed that we still needed to verify this step and that we could ensure the validity of our material.

We struggled with this step for a long period, and we suspected that our failure was caused by impure materials. We used 30 KDa ultrafiltration tube--through which unbound fluorescent aptamer could pass, but the aptamer combined with protein could not. We also used flow cytometry to directly detect whether there were binding fluorescent aptamer on EpCAM, without satisfying results in the end.

Fortunately, we successfully validated this step with the fluorescence polarization method after replacing materials, with the proposal from Prof. Zhi Zhu. We incubated two groups, namely pure fluorescent aptamer as well as fluorescent aptamer with EpCAM without light at the same time, then adding binding buffer to the total volume of 200 μL. After incubation, we added 3 mL binding buffer for fluorescence polarization. Get more information of our experiment, [click here.](https://2018.igem.org/Team%3AXMU-China/Experiments)

![](https://static.igem.org/mediawiki/2018/e/e2/T--XMU-China--result1-2.png)

**Figure 2.1** Polarizing fluorescence intensity of fluorescent aptamer.

![](https://static.igem.org/mediawiki/2018/b/b9/T--XMU-China--result1-3.png)

**Figure 2.2** Polarizing fluorescence intensity of fluorescent aptamer with EpCAM, after incubation.

According to the principle of fluorescence polarization, the FP value would depend directly on the effective molecular size of the fluorescent group at fixed temperature and viscosity of solution. For small molecules (fluorescent aptamer) with fast Brownian rotation in solution, the FP value was lower; while for larger molecules (fluorescent aptamer combined with EpCAM), the FP value was higher. [2]

The FP value could be calculated by the following formula:

![](https://static.igem.org/mediawiki/2018/a/a2/T--XMU-China--result1-4.png)

Below are four maximum fluorescence values for calculating FP values:

![](https://static.igem.org/mediawiki/2018/3/3b/T--XMU-China--result1-5.png)

![](https://static.igem.org/mediawiki/2018/f/fb/T--XMU-China--result1-6.png)

Later, we could calculate whether the FP value of fluorescent aptamer increased with EpCAM incubation according to the principle. If the FP value increased, it showed that fluorescent aptamer did bind to EpCAM. We calculated the difference herein:

![](https://static.igem.org/mediawiki/2018/1/11/T--XMU-China--result1-7.png)

With an increase of 0.142, the difference was rather obvious, which demonstrated that the fluorescent aptamer did combine with EpCAM.

# Combination between SYL3C and the "complementary strand" (DNA Agarose Gel Electrophoresis)

We've been struggling for some time due to one problem about how to prove that our aptamer and the complementary chains were combined, which would be paramount for our following experiments.

We tried SYBR Green I, a fluorescent dye that could be embedded in a double helix of DNA and thus significantly enhance fluorescence intensity, since it could distinguish ssDNA from dsDNA obviously. However, there's no significant difference according to our results. We speculated that the sequence might be not long enough, coupled with the formation of secondary structure of aptamers, leading to no difference in fluorescence intensity.

Finally, we exploited the conventional gel electrophoresis method to prove this block, and then gel electrophoresis was performed immediately after grouping and denaturing annealing. Because the sequence was too short, we did not have the appropriate DNA marker to use. Get more information of our experiment, [click here.](https://2018.igem.org/Team%3AXMU-China/Experiments)

![](https://static.igem.org/mediawiki/2018/6/62/T--XMU-China--result1-8.png)

**Figure 3.** Gel image of aptamer combined with complementary chain.
a) W1: aptamer;
 b) W2: 24nt complementary chain with a 9nt complementary part;
c) W3: 24nt complementary chain with a 10nt complementary part;
 d) W4: Aptamer + 24nt complementary chain with a 9nt complementary part;
e) W5: Aptamer + 24nt complementary chain with a 10nt complementary part.

We could find that there were three distinct bands between the aptamer + complementary strands with a 10nt complementary part (W5), while 9nt did not. The lane 1 and 2 acted as markers, so we could clearly identify the positions of the aptamers and complementary strands in the three bands. The last band, obviously, was the result of an increase in molecular weight, which meant that the aptamer and the complementary strand combined together successfully.

Therefore, we have proved that the aptamer could combine with its complementary strand successfully.

# The competition (Fluorescence Spectrometer)

We used the competition method to transfer molecular signal, because we considered that the competition method was best operated automatically later, so that users did not need to have a deep understanding toward technical basis. Therefore, it's quite important for us to verify that EpCAM could compete successfully, from a theoretical prospective to support our hardware and the activation of Cas12a as well.

Below are the exact procedures of our experimental group. First, we successfully hatched the streptavidin beads, biotin-modified aptamers and fluorescent complementary strands to form the complexes, then fixed the beads with magnets and discarded the supernatant, which would still contain fluorescent complementary strands that did not form complexes. After that, we incubated the complexes with EpCAM to achieve the process of competition.

Additionally, considering the non-specific adsorption, we set up several control groups, and the variables amongst them were replaced by buffer. After competition, we fixed the magnetic beads with magnet, then extracted the supernatant and added 3 mL incubation buffer in tandem. Below is the plot about the fluorescence intensity detected in different groups. Get more information of our experiment, [click here.](https://2018.igem.org/Team%3AXMU-China/Experiments)

![](https://static.igem.org/mediawiki/2018/3/37/T--XMU-China--result1-9.png)

**Figure 4.** Fluorescence intensity images of competition results.
a) Black line: incubation buffer;
b) Red line: magnetic beads-aptamer + incubation buffer;
c) Blue line: magnetic beads-aptamer-complementary strand complex + incubation buffer;
d) Green line: magnetic beads-aptamer-complementary strand complex + EpCAM + incubation buffer.

Comparing the four groups, we can conclude that the non-specific adsorption of complementary strands on the magnetic beads does exist, and the magnetic beads-aptamer complex, like the buffer solution, had almost no fluorescence intensity. Significantly, the fluorescence intensity of the competition group with EpCAM was about 2.5 times larger than that of the non-specific adsorption group, which demonstrated that the fluorescence complementary strand of the competition group with EpCAM was successful, and so was our competition method.

# Trans-cleavage activity of Cas12a (Microplate Reader)

It's reported that Cas12a could be activated by ssDNA and has trans-cleavage activity this year, so we hope to use this function in our system to amplify the signal. [3]

We operated the experiment according to the instructions from IDT. We incubated the Cas12a protein with crRNA to form a complex and then added our ssDNA to be recognized. Then, we used the commercial DNaseAlertTM Kit to explore its trans-cleavage activity. We drawed the fluorescence curve over time using a microplate reader, and measured the fluorescence intensity from 30 minutes to 110 minutes.

In order to control variables, we also set up a negative (DNaseAlert) and a positive (DNase I + DNaseAlert) control group. Get more information of our experiment, [click here.](https://2018.igem.org/Team%3AXMU-China/Experiments)

![](https://static.igem.org/mediawiki/2018/d/df/T--XMU-China--result1-10.png)

**Figure 5.** Fluorescence time curve image of activation effect of Cas12a.
a) Black line: negative group, DNaseAlert only;
b) Red line: positive group, DNase I + DNaseAlert;
c) Blue line: experimental group, activated Cas12a + DNaseAlert;

We fitted three sets of data points with Gaussian fitting curve in Figure 5. From the fitting results, we could see that the negative group and the positive group were declining, while the Cas12a protein group increased at first and then decreased. This is in line with our expectations, and the reason for the decline was, we suspected, the quenching caused by continuous measurements and the weakening of the fluorescence itself as the experiment proceeds. However, it's enough to prove that Cas12a could meet the expected result of amplifying signals.

# Signal transformation by DNaseAlertTM Kit (Fluorescence Spectrometer)

Although we have used DNaseAlert to detect Cas12a, as the last of the three modules we designed, we needed to prove that DNaseAlert was indeed cut and would emit fluorescence to our expectations, so that it could be used to transfer our signals.

Here, we chose DNase I to verify directly. In the presence or absence of DNase I, we measured fluorescence intensity in the DNase alert tube. Considering the experiences we got previously that long-term measurement in the microplate reader would lead to reduction in fluorescence intensity, resulting in experimental error, we chose fluorescence spectrometer instead.

We set up the experimental group (with DNase I) and the control group (without DNase I), with the time gradient of 3, 15, 30 minutes. All groups started at the same time. We didn't operate continuous measurement, to avoid excitation that may cause partial fluorescence quenching. After incubation, 3 mL TE buffer was added, and then detect the fluorescence intensity. Get more information of our experiment, [click here.](https://2018.igem.org/Team%3AXMU-China/Experiments)

![](https://static.igem.org/mediawiki/2018/d/dc/T--XMU-China--result1-11.png)

**Figure 6.** Fluorescence intensity curve in 3rd minutes, with or without DNase I.

![](https://static.igem.org/mediawiki/2018/b/b8/T--XMU-China--result1-12.png)

**Figure 7.** Fluorescence intensity curve in 15th minutes, with or without DNase I.

![](https://static.igem.org/mediawiki/2018/3/3d/T--XMU-China--result1-13.png)

**Figure 8.** Fluorescence intensity curve in 30th minutes, with or without DNase I.

From the Figure 6-8, we could find that cutting efficiency of DNase I was very high, almost all of the DNaseAlert substrate has been cut in 3 minutes, fluorescence intensity in 15 and 30 minutes and 3 minutes almost kept the same level. As we expected, DNaseAlert could indeed be cleaved and then re-released as a fluorescent group. In other words, DNaseAlert could transfer the molecular signal to the fluorescent signal actually.

# Integrated experiment

After making each part work effectively, we accomplished the integrated experiment finally, transferring the protein signal to fluorescent one.

Similarly, following procedures mentioned in the "competition method" part, we successfully hatched the streptavidin beads, biotin-modified aptamers and fluorescent complementary strands to form the complexes, then fixed the beads with magnets and discarded the supernatant, which would still contain fluorescent complementary strands that did not form complexes. After that, we incubated the complex with EpCAM to achieve the process of competition. After competition, we fixed the magnetic beads with magnet, adding the supernatant extracted into Cas12a-crRNA complex incubated previously to activate Cas12a protein. Finally, we added it into DNaseAlert, supplementing 3 mL TE buffer after 30 minutes to measure the fluorescent intensity. Below is the corresponding plot we've obtained, indicating that we made the integrated experiment successfully.

![](https://static.igem.org/mediawiki/2018/5/5e/T--XMU-China--notebook-abcd-7.png)

**Figure 9.** Integrated experiment.

# References

[1] Yanling Song, Zhi Zhu, Yuan An, Weiting Zhang, Huimin Zhang, Dan Liu, Chundong Yu, Wei Duan, Chaoyong James Yang. Selection of DNA Aptamers against Epithelial Cell Adhesion Molecule for Cancer Cell Imaging and Circulating Tumor Cell Capture. *Anal. Chem*. **2013**, 85: 4141-4149.
[2] DS Smith, SA Eremin, Fluorescence polarization immunoassays and related methods for simple, high-throughput screening of small molecules. *Analytical and bioanalytical chemistry*. **2008**, 391(5): 1499.
[3] Chen JS, Ma E, Harrington LB, Da Costa M, Tian X, Palefsky JM, Doudna JA. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. *Science*. **2018**, 360(6387): seaar6245.

The result of OMVs

# The growth curve of IPTG-induced and Arabinose-induced *E. coli* BL21

To ensure when we should add the inducer, we measured the growth curve of BBa\_K2623026 (IPTG-induced) and BBa\_K2623027 (Arabinose-induced). According to figure 1a, we found that after incubation for 5 h, the OD600 of BBa\_K2623026 (IPTG-induced) reached to about 0.6, which is suitable for adding IPTG to a final concentration at 0.5 mM. Figure 1b shows that after incubation for 5 h, the OD600 of BBa\_K2623027 (Arabinose-induced) reached to about 0.6, which is suitable for adding Arabinose to a 0.2% final concentration.

![](https://static.igem.org/mediawiki/2018/5/53/T--XMU-China--results2-1.png)

**Figure 1.** Growth curve of (a) IPTG-induced and (b) Arabinose-induced *E. coli* BL21.

# OmpA-ST could be transported to OMVs

OmpA-SpyTag (OmpA-ST) is transported to bacterial outer membrane and therefore OMVs. However, *E. coli* could produce natural OmpA and then transport it to its outer membrane and OMVs as well. It's necessary to know how much OmpA-ST is transported to OMVs or what the ratio of the OMVs-ST is in total OMVs. To evaluate the efficiency of its transport to outer membrane and OMVs, we fused wtGFP and our OmpA-ST (BBa\_K2623022 and BBa\_K2623024) together in order to construct OmpA-ST-GFP (BBa\_K2623021 and BBa\_K2623023) (Figure 2).

![](https://static.igem.org/mediawiki/2018/b/b8/T--XMU-China--results2-2.png)

**Figure 2.** The spectrogram of wtGFP [1].

We cultured bacteria transfected with these four parts respectively in 10 mL OMVs-free LB broth culture and extracted the OMVs according to our protocols, in which two forms of non-GFP OMVs were set as negative controls. Transmission electron microscopy (TEM) pictures identified what we extracted were exactly OMVs with membrane structure and 100-nm diameter (Figure 3).

![](https://static.igem.org/mediawiki/2018/0/06/T--XMU-China--results2-3.png)

**Figure 3.** TEM figures identify our OMVs.

Building upon Yan's lab-built high-sensitivity flow cytometer [2-3], we successfully detected the OMVs labelled with GFP and evaluated the efficiency of its transport to OMVs (Figure 4). Herein, we'd like to extend our heartfelt thanks toward Yan's lab due to the very kind and selfless help during the experiment.

![](https://static.igem.org/mediawiki/2018/f/f4/T--XMU-China--results2-4.png)

**Figure 4.** HSFCM analysis of OmpA-ST-GFP inside OMVs.

Figure 4 are bivariate dot-plots of GFP green fluorescence versus side scattering (SSC) for our OMVs isolates. *E. coli* transfected with BBa\_K2623021 (Figure 4a) could secret more OMVs labeled with GFP than *E. coli* transfected with BBa\_K2623022 (Figure 4b). Similarly, *E. coli* transfected with BBa\_K2623023 (Figure 4c) would secret more OMVs labeled with GFP than *E. coli* transfected with BBa\_K2623024 (Figure 4d). Moreover, we could see *E. coli* transfected with BBa\_K2623021 (Figure 4a) would transport OmpA-ST to OMVs more efficiently than *E. coli* transfected with BBa\_K2623023 (Figure 4c). The remarkable difference of GFP-OMVs ratio between these two parts might account for the different inserted site of ST. SpyTag (ST) in BBa\_K2623021 (Figure 4a) is inserted to N-termini of OmpA, while ST in BBa\_K2623023 (Figure 4c) is inserted to C-termini of OmpA. Our HSFCM data shows that BBa\_K2623021 is more efficient to transport ST-OmpA-GFP to OMVs and might be a good candidate for the following study.

# References

[1] https://www.semrock.com/
[2] Zhu S, Ma L, Wang S, *et al*. Light-Scattering Detection below the Level of Single Fluorescent Molecules for High-Resolution Characterization of Functional Nanoparticles. *Acs Nano*. **2014**, 8(10): 10998-11006.
[3] Tian Y, Ma L, Gong M, *et al*. Protein Profiling and Sizing of Extracellular Vesicles from Colorectal Cancer Patients via Flow Cytometry[J]. *Acs Nano*. **2018**, 12(1): 671-680.

# Demonstrate the OmpA-ST inside *E. coli* BL21 and OMVs

As our HSFCM data show, OmpA-ST-GFP (BBa\_K2623021 and BBa\_K2623023) transported to OMVs could be detected. In our total circuit, we would use non-GFP part (BBa\_K2623022 and BBa\_K2623024) to establish SpyTag/SpyCatcher (ST/SC) conjugation. For this purpose, we'd like to demonstrate the OmpA-ST (BBa\_K2623022 and BBa\_K2623024) inside both *E. coli* BL21 and OMVs, as well as SDS-PAGE used (Figure 5).

![](https://static.igem.org/mediawiki/2018/a/ad/T--XMU-China--results2-5.png)

**Figure 5.** Lane 1: *E. coli* BL21 transfected with BBa\_K2623022.
Lane 2: OMVs secreted by *E. coli* BL21 transfected with BBa\_K2623022.
Lane 3: *E. coli* BL21 transfected with BBa\_K2623024.
Lane 4: OMVs secreted by *E. coli* BL21 transfected with BBa\_K2623024.

As our SDS-PAGE figure shows, ideal band at about 50 kDa are shown in *E. coli* BL21 transfected with BBa\_K2623022 and BBa\_K2623024, respectively. However, we could see neither bands nor traces in lane 2 and lane 4 loading with OMVs samples. We suppose that our sample loadings are not enough for the LOD (limit of detection) of SDS-PAGE followed by Coomassie blue staining. Because of limited time, we couldn't repeat this experiment. Whatever, our HSFCM data have shown that OmpA-ST could be encapsulated into OMVs.

# Demonstrate the SC-L7Ae inside *E. coli* BL21

Similarly, in our total circuit, we used non-RFP part (BBa\_K2623026) to fulfill L7Ae-C/Dbox linkage. It's demonstrated that the SpyCatcher (SC)-L7Ae could be expressed inside *E. coli* BL21 firstly. Under the induction of IPTG at 0.5 mM, we could detect the expression of SC-L7Ae (Figure 6).

![](https://static.igem.org/mediawiki/2018/9/99/T--XMU-China--results2-6.png)

**Figure 6.** SDS-PAGE figure of the SC-L7Ae.
Lane 1: induced-BL21.
Lane 2: uninduced-BL21. The red arrow points to the band of the SC-L7Ae.

Though no IPTG was added into *E. coli* BL21 as negative control (lane 2), we could still see our band at about 70.4 kDa indicated for SC-L7Ae. However, SDS-PAGE is not an ideal approach for identify our targeted protein. In our future plans, we would use anti-SC mAb to immunoblot our SC-LA7e.

# Demonstrate the ST/SC conjugation inside OMVs

Though we have revealed the OmpA-ST inside OMVs, we are inclined to figure out the ST/SC bioconjugation inside OMVs. Hence, we fused mRFP1 to SC-L7Ae (BBa\_K2623026) to build SC-L7Ae-RFP (BBa\_K2623025) (Figure 7). We linked our two non-GFP OmpA-ST parts (BBa\_K2623022 and BBa\_K2623024) with BBa\_K2623025 and BBa\_K2623026 to form four new parts:

1) BBa\_K2623022 & BBa\_K2623025: BBa\_K2623028 (RFP form)
2) BBa\_K2623022 & BBa\_K2623026: BBa\_K2623029
3) BBa\_K2623024 & BBa\_K2623025: BBa\_K2623030 (RFP form)
4) BBa\_K2623024 & BBa\_K2623026: BBa\_K2623031

Under the induction of IPTG, L7Ae could be expressed with SpyCatcher and RFP modification and then be encapsulated into OMVs through ST/SC conjugation. OMVs labeled with RFP will be detected through HSFCM.

![](https://static.igem.org/mediawiki/2018/7/73/T--XMU-China--results2-7.png)

**Figure 7.** a) Genetic circuit of SC-L7Ae-RFP [1].
b) The spectrogram of mRFP1.

![](https://static.igem.org/mediawiki/2018/a/a1/T--XMU-China--results2-8.png)

**Figure 8.** HSFCM analysis of ST/SC bioconjugation inside OMVs.

Figure 8 contains bivariate dot-plots of RFP red fluorescence versus side scattering (SSC) for our OMVs isolates. *E. coli* BL21 transfected with BBa\_K2623028, BBa\_K2623029, BBa\_K2623030, BBa\_K2623031 were incubated with OMVs-free LB culture for about 5 hours to get a 0.6-0.8 OD600 respectively, in which BBa\_K2623029 and BBa\_K2623031 were set as negative controls. Then IPTG was added to a final concentration at 0.5 mM and nurture the bacteria overnight. OMVs were isolated according to our protocols and then analyzed by HSFCM. It’s interesting to note that *E. coli* transfected with BBa\_K2623030 (Figure 3c) could secret more RFP-OMVs than BBa\_K2623028 (Figure 3a), indicating that ST/SC conjugation inside OMVs is more efficient with ST at the C-termini of OmpA. This result is inconsistent with the result shown in Figure 4, in which ST at the N-termini of OmpA (BBa\_K2623028) has a higher efficiency to be encapsulated inside OMVs. We propose that GFP inserted to OmpA might interfere the transport of OmpA-ST (BBa\_K2623021 and BBa\_K2623023) to OMVs.

# References:

[1] https://www.semrock.com/

# Demonstrate the L7Ae-C/Dbox conjugation inside OMVs

Our OMVs module is designed for siRNA delivery and then for cancer treatment. To demonstrate the conjugation of the L7Ae-C/Dbox inside OMVs, we linked ST/SC part with the box part (BBa\_K2623027, see Figure 9) to form our total circuit (BBa\_K2623032).

![](https://static.igem.org/mediawiki/2018/d/db/T--XMU-China--OMVs26.png)

**Figure 9.** Genetic circuit of the Box part (BBa\_K2623027).

The box part can code siRNA target for Kras in human pancreatic ductal adenocarcinoma (PDAC) with a C/Dbox RNA structure to bind with L7Ae.

Under the induction of arabinose at a 0.2% final concentration, we isolated the OMVs according to our protocols and then stained the OMVs isolates with SYTOTM RNASelectTM (selectively stain for RNA) at 10 μM (Figure 10). The stained isolates were analyzed by HSFCM (Figure 11).

![](https://static.igem.org/mediawiki/2018/b/bf/T--XMU-China--results2-10.png)

**Figure 10.** a) Schematic illustration of our total circuit.
b) The spectrogram of SYTOTM RNASelectTM [1].

![](https://static.igem.org/mediawiki/2018/9/9b/T--XMU-China--results2-11.png)

**Figure 11.** HSFCM analysis of OMVs stained with SYTOTM RNASelectTM.

Figure 11 are bivariate dot-plots of green fluorescence versus side scattering (SSC) for our OMVs isolates. *E. coli* BL21 transfected with BBa\_K2623029 and BBa\_K2623032 respectively were cultured in 100 mL OMVs-free LB culture for about 5 hours to get a OD600 at 0.6-0.8, in which BBa\_K2623029 was set as negative control. Then Arabinose was added to a final concentration at 0.2% and after incubation for another 2 hours, IPTG was added to a final concentration at 0.5 mM and then nurtured the bacteria overnight. OMVs were isolated according to our protocols and stained with SYTOTM RNASelectTM. The stain OMVs were analyzed by HSFCM. As we expected, OMVs isolated from BBa\_K2623032 could secret more OMVs containing with RNA stained positively by SYTOTM RNASelectTM (Figure 11b) than BBa\_K2623029, indicating that our L7Ae and C/Dbox could fulfill their function. It’s a pity that we didn’t have cell culture experiment to test our siOMVs to silence Kras in human pancreatic ductal adenocarcinoma (PDAC). In our future plans, we'll finish our cell experiment and demonstrate the function of our siOMVs.

# References

[1] https://www.semrock.com/

# Future Plan

It is not practical to transfer our ideas to a fully-completed project or even a portable device just after an enriched summer. We should learn more for the sake of improving our project, making it suitable for feasible usage. Therefore, we list our future plans to develop our projects.

**Design-1**. Though we have characterized OmpA-ST through SDS-PAGE, in many cases SDS-PAGE is not an optimal method to characterize specific protein in complex samples, like bacterial lysates. For further characterizing our OmpA-ST, we would be inclined to find monoclonal antibody (mAb) specific for SpyTag and Western Blot analysis alike. Exploiting with anti-SpyTag mAb, we supposed that the band at about 50 kDa would be stained positively.

**Design-2**. Similar to Design-1, we would also use monoclonal antibody (mAb) specific for L7Ae and SC to validate our L7Ae-SC.

**Design-3**. For our siRNA-C/Dbox, due to poor experimental instrument, we could only characterize the promotion of RNA level inside our engineering bacteria. The problem is that we could not ensure the presence of our siRNA target for KrasG12D. To characterize this part better, we'd like to use qPCR, FISH (fluorescence in situ hybridization) or low-throughput miRNA target identification method[1] to demonstrate our siRNA could target KrasG12D. Through the incubation of siOMVs (OMVs containing siRNA) and human pancreatic ductal adenocarcinoma (PDAC) cell line PANC-1, the silence effect of the siOMVs could be told.

**Design-4**. As mentioned above, our chassis is bacterium. Some researchers have demonstrated not only bacteria but also their OMVs could be pathogenetic. Though some governments have approved the usage of OMVs vaccines, the side effects of the OMVs are still ambiguous. Extracellular vesicles (EVs), originated from eukaryocytes, which are often referred to exosomes and mircovesicles, are better choices for nucleic acid delivery for its biocompatibility and homology. We could conjugate L7Ae to the C-terminus of tetraspanin CD63 and insert a C/Dbox into the 3′-untranslated region (3′-UTR) of the gene coding for siRNA[2].

# References

[1] Michael J V, Jgt W, Mao G F, *et al*. Platelet microparticles infiltrating solid tumors transfer miRNAs that suppress tumor growth. *Blood*. **2017**, 130(5): blood-2016-11-751099.
[2] Kojima R, Bojar D, Rizzi G, *et al*. Designer exosomes produced by implanted cells intracerebrally deliver therapeutic cargo for Parkinson’s disease treatment. *Nature Communications*. **2018**, 9(1): 1305.

The result of Supporting

# Verify the expression of SAHS protein

Firstly, we used the [BBa\_E1010](http://parts.igem.org/Part%3ABBa_E1010) (mRFP) as our report gene to make sure the circuit for expressing the SAHS protein was constructed precisely. But we did not observe the distinct color on the plate as it was so weak unless the *E.coli* was cultured in a tube and centrifuged. So we had a new test by using the [BBa\_K592009](http://parts.igem.org/Part%3ABBa_K592009)（blue chromoprotein）as our report gene and got a distinct color on the plate.

![](https://static.igem.org/mediawiki/2018/2/25/T--XMU-China--results3-1.png)

**Figure 1.** These are the pictures our fluorescently characterized plate and bacterial pellets. The report gene on the left is BBa\_K592009, and the one on the right is E1010. Some colonies on the left are blue, which are the DH5α that we successfully transferred to the designed genetic loop. The leftmost EP tube in the right photo is the control, and the other two EP tubes contain DH5α which we had successfully transferred to the designed genetic loop.

And then, we transformed the plasmid to the *E.coli* BL21, which is always used to express proteins with high efficiency to verify the SAHS protein was produced successfully with a small scale. Besides, whether the SAHS protein with a signal peptide could be secreted was determined by SDS-PAGE.

![](https://static.igem.org/mediawiki/2018/0/02/T--XMU-China--measurement-kai1017-4.png)

**Figure 2.** The marker is on the lest, followed by our control group (the BL21 with the empty plasmid), and the third well is the concentrated supernatant.

As shown in the image above, we also explored the appropriate temperature for SAHS protein expression. As we expected, protein expression was be more efficient at 30 °C. However, we did not find the SAHS protein band in the supernatant even when it was concentrated by ultrafiltration. So we speculated that cell wall obstructed the secretion of protein on the basis of 2016 Peking University's working. See more information on [Come form wiki](https://2016.igem.org/Team%3APeking/Secretion).

Therefore, we have to design a new protein purification program. To get enough protein produces for the following experiment, the gene was cloned into the vector pET-28a with high expression lever combined with *E.coli* BL21. Besides, Two HIS-TAGs on the end of N-and C-terminal was produced and allow SAHS protein to bind with Nickel column (like Ni-NTA) for purification. Meanwhile, we purified the sample with heat water bath considering the characterization of heat stability by following the reference.

![](https://static.igem.org/mediawiki/2018/4/47/T--XMU-China--results3-3.png)

**Figure 3.** The picture on the left is a series of temperature gradient processed protein samples. The number represents the temperature (°C). And the picture on the right is the gel map of our final purified high purity SAHS protein.

So, we designed a set of temperature gradients from 70°C to 90°C, to explore the appropriate condition. What's more, we chose the 85°C for 15 min finally for large scale purification. But given that there were many other protein bands, we combined heating with Nickel column for producing high quality protein. Finally, we tested two patterns, heating-Ni-NTA and Ni-NTA-heating, and found that the later one is better.

After that, we obtained a protein sample with high concentration and purity by using this protein purification method（Figure 3）.

# Verify the role of SAHS protein in preserving biological activity

In order to verify the preservation effect of SAHS protein, we used the lyophilization method. First, we tested the concentration of purified SAHS protein, which ranged from 0.3 g/L to 0.6 g/L. Then, we concentrated the purified protein to control the protein concentration to about 1 g/L.

We selected *Taq* enzyme as the protein preserved in our experiment, using SAHS protein as protective agent, and set a series of concentration gradients: 1 g/L, 0.5 g/L, 0.1 g/L, 0.01 g/L and 0 g/L， then stored with lyophilization. In order to verify the preservation effect of SAHS protein, we resuscited the lyophilized sample and performed PCR experiments to verify the activity of *Taq* enzyme. The activity of *Taq* enzyme can be reflected by the product of PCR.

![](https://static.igem.org/mediawiki/2018/0/01/T--XMU-China--results3-4.png)

**Figure 4.** PCR results of *Taq* enzymes preserved at different SAHS protein concentrations.

We selected different PCR systems for verification. We found that some short fragments were easy to succeed, but there was no obvious gradient. However, when we replaced them with some other fragments that are difficult to succeed or very long, there would show a very obvious gradient. We think it must be that a shorter fragments were too easy to succeed, and made the base too large. The two fragments above are pET-28a, which had a relatively obvious gradient and also had a good repeatability.

![](https://static.igem.org/mediawiki/2018/d/da/T--XMU-China--results3-5.png)

**Figure 5.** The result of the comparison of PBS and ultra-pure water. Blank is the group that the *Taq* enzyme has not been lyophilized and without SAHS protein.

In addition, we also made a comparison between PBS and ultra-pure water. We found that regardless of the presence of TDP, the lyophilized system added with PBS could not make the correct band. On the contrary, there was only one large band which was much larger than 8000bp. We think it may be a PCR mismatch. Considering that PBS contained a large amount of salt, it was likely that these salt ions affected the binding of *Taq* enzyme to Mg2+.

![](https://static.igem.org/mediawiki/2018/a/ac/T--XMU-China--results3-6.png)

**Figure 6.** The ruselt of the success rate of PCR with *Taq* enzymes preserved at different SAHS protein concentrations.

Then, we tried some shorter fragments for PCR, and interfered with the PCR system by reducing time extension and reducing the concentration of the template in order to explore the success rate of PCR. As the figure showed, when the concentration of SAHS protein was 0.5 g/L, the success rate of PCR was the highest. We suspected that excessive concentration of SAHS protein might affect the normal function of the *Taq* enzyme and might affect the binding of the enzyme to the substrate. This reminded us of the results of TUDelft in 2017. They also got the result of that 0.5 g/L was better than 1 g/L in some cases. Eventually, they took a concentration of 1 g/L to preserve Cas13a. They discovered that while CAHS 94205 preserved Cas13a's RNase-like activity after drying, CAHS 94205 could not preserve its specificity. This is exactly the same as ours when the concentration is 1 g/L. Therefore, we believe that it is possible that they just did not find the most suitable storage concentration, and if the concentration is properly reduced, the results may be much better.

# Conclusion

According to the experimental results of lyophilization above, we can clearly see the preservation effect of SAHS protein on protein activity. Secondly, when the concentration is too high, SAHS protein may affect the normal function of protein. Therefore, it is necessary to find the most appropriate concentration. The optimal concentration may vary slightly among different proteins, but the preservation of biological activity by SAHS proteins is undoubtable. We believe that SAHS protein is a promising bioactive protective agent for different preservative substances, as long as the appropriate preservation system is set up.

Verify the expression of CAHS protein

![](https://static.igem.org/mediawiki/2018/7/7f/T--XMU-China--results3-7.png)

**Figure 7.** The circuit of BBa\_K2623014

In the circuit, the Lac I was expressed constantly, and Lac O, lac repressor was bound to Lac O. Following the CAHS gene was our reporter gene mRFP (J04650). When we added IPTG in the bacterial solution, the IPTG bound to repressor and inactivated it. So the CAHS gene and mRFP would start to express.

![](https://static.igem.org/mediawiki/2018/d/dc/T--XMU-China--results3-8.png)

**Figure 8.** the *E.coli* DH5а with BBa\_K2623014 and IPTG turns red.(D+ is *E.coli* DH5а with BBa\_K2623014 and IPTG, D- is *E.coli* DH5а with BBa\_K2623014 without IPTG, D0 is *E.coli* DH5а without BBa\_K2623014)

We found that the red fluorescent protein expressed very slowly. After we added IPTG in the 4mL bacterial solution, we put the bacterial solution in 25°C shaker culture. After 12 hours, we saw the centrifugal precipitation color showed red.

We also did experiment with *E.coli* BL21, but the bacteria didn't turn red.

We cultured the *E.coli* BL21 which contained the plasmid of CAHS protein (BBa\_K2623014) in the fluid medium. When the value of OD600 reached about 0.6, we took 4mL bacteria solution in the glass test tubes and added 4 μL IPTG in the tubes. And then put them in 25°C shaker culture for 5 hours. Then, we took 1mL bacterial liquid to centrifugalize. We picked the precipitation and added 100 μL DDW and 20 μL SDS loading buffer. After that, we heated and boiled it for 15 minutes. And then, it was separated by SDS-PAGE and stained with Coomassie brilliant blue for 40 minutes. Ultimately, we decolorized it and observed the results.

We find the CAHS is surely expressed in the *E.coli* BL21 after adding IPTG. Here is the result.

![](https://static.igem.org/mediawiki/2018/c/c0/T--XMU-China--results3-9.png)

**Figure 9.** the CAHS protein express in SDS-PAGE. (1, 2, 3 is all with BBa\_K2623014. 1 is BL21 with IPTG and pre-heat, 2 is BL21 with IPTG, 3 is BL21 without IPTG, 4 is BL21 without the BBa\_K2623014.)

The picture of SDS-PAGE shows the CAHS protein is surely expressed in B+ group (the *E.coli* BL21 with BBa\_K2623014 with IPTG). And CAHS protein isn't expressed in the B- group (the *E.coli* BL21 with BBa\_K2623014 without IPTG) and B0 group (the *E.coli* BL21 without BBa\_K2623014).

We also did experiments with *E.coli* DH5а, but the SDS-PAGE showed there was no CAHS protein. Because the *E.coli* BL21 is a kind of strain which can knock out proteases while the *E.coli* DH5а not. We guess the CAHS protein in *E.coli* DH5а is degraded. So we chose the BL21 to verify the function of CAHS.

# Verify the role of CAHS protein in preserving biological activity

The SDS-PAGE showed the CAHS protein was really expressed in BL21. We cultured the *E.coli* BL21 bacteria which contained the plasmid BBa\_K2623014. We picked the *E.coli* BL21 bacterial liquid for freeze-drying. First of all, we picked 1mL bacterial liquid to centrifugalize, added 100 μL protective agent (LB+5% Sucrose), and put them in -20°C refrigerator for 2 hours. And then, we put them in -80°C for one thorough night, and then freeze-dried them (Advantage ES-53).

After lyophilization, we added 500 μL PBS in each centrifuge tube, and put them in 37°C shaker culture for 15mins. We also added 500 μL DDW in each centrifuge tube. And then, we took 100 μL the dilute bacteria liquid, added in 900 μL ddH《》, repeated the step, and carried out gradient dilution to 104, 105, 106. Then, we picked 200 μL dilute bacteria liquid, and coated the plate with chloramphenicol.

![](https://static.igem.org/mediawiki/2018/3/37/T--XMU-China--results3-10.png)

**Figure 10.** gradient dilution.

The results showed that CAHS protein could improve the preservation effect of freeze-drying bacteria significantly!

The B+ group's CFU is 100 times higher than the B0 group, 30 times better than the B- group. So we think the CAHS is leaking expression in B- group, which has some of the protection for the bacteria in freeze-drying.

After being irradiated by UV for 1min, the B0 group was nearly fully dead, and the CFU of B- group had been decrease to a very low level, while the B+ group was higher. So we think the CAHS is can provide some of certain protection against UV.

![](https://static.igem.org/mediawiki/2018/b/b8/T--XMU-China--results3-11.png)

**Figure 11.** The CFU of BL21. (B0 is *E.coli* BL21 without BBa\_K2623014, B+ is *E.coli* BL21 with BBa\_K2623014 and IPTG, B- is *E.coli* BL21 with BBa\_K2623014, UV means the desiccant bacterial under UV for 1min.)

This result shows that the CAHS can provide production for bacteria in freeze-drying. And the CAHS can also provide CAHS for bacteria suffering from UV. We think the CAHS protein is a very potential protective agent, so we can use the CAHS protein to store plasmid and bacteria. We can even design a kit for the bacteria storage with CAHS protein.

After being irradiated by UV for 1min, the B+ group was still alive. Therefore, we think the CAHS protein can provide protection for bacteria against UV and can protect the nucleic acid in the bacteria. The water bear can survive from UV, and the bacteria can also survive from UV, so it is certain the CAHS protein works.

As for the colony recovery rate, the CAHS also showed great effect. The B+ group colony recovery rate was highest, which reached about 40%. While those of the B- group and B0 group were no more than 2%. After being irradiated by UV for 1min, the B+ group was still the highest in the three group (UB\_B0, UV\_B+ and UV\_B-).

![](https://static.igem.org/mediawiki/2018/b/bb/T--XMU-China--results3-12.png)

**Figure 12.** The colony recovery rate. (B0 is *E.coli* BL21 without BBa\_K2623014, B+ is *E.coli* BL21 with BBa\_K2623014 and IPTG, B- is *E.coli* BL21 with BBa\_K2623014, UV means the desiccant bacterial under UV for 1min.)

Here are the pictures of the culture plate. We used the disposable plastic petri dishes, and appended the sealing film on the aperture. When we started, we used the glass petri dishes, but the effect was not obvious at all. All the petri dishes had chloramphenicol, and the B0 group bacteria had transformed BBa\_S0100.

![](https://static.igem.org/mediawiki/2018/6/61/T--XMU-China--results3-13.png)

**Figure 13.** the plate of colony for counting. (B0 is *E.coli* BL21 without BBa\_K2623014, B+ is *E.coli* BL21 with BBa\_K2623014 and IPTG, B- is *E.coli* BL21 with BBa\_K2623014, UV means the desiccant bacterial under UV for 1min. And the 104 & 105 means Dilution ratio)

# Conclusion

Our experiment shows the great function of CAHS protein in improving the desiccation tolerance and UV tolerance. We think that the CAHS protein is a very potential protective agent for bacteria storage. Except expressing the CAHS protein with plasmid, we can also knock the CAHS protein gene into the genome of bacteria through homologous recombination. And we believe that a new way for storing the bacteria is available.

![](https://static.igem.org/mediawiki/2018/3/3d/T--XMU-China--xmu_is_different.png)

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