iGEM 2017 — Team SIAT-SCIE

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# Team:SIAT-SCIE/Notebook

* [Home](https://2017.igem.org/Team%3ASIAT-SCIE/Home "Home")
* Project

  [Description](https://2017.igem.org/Team%3ASIAT-SCIE/Project_Description)
  [Results](https://2017.igem.org/Team%3ASIAT-SCIE/Results)
  [Mechanism](https://2017.igem.org/Team%3ASIAT-SCIE/Mechanism)
  [Demonstrate](https://2017.igem.org/Team%3ASIAT-SCIE/Demonstrate)
* Notebook

  [iGEM Diary](https://2017.igem.org/Team%3ASIAT-SCIE/IGEM_Diary)
  [Protocol](https://2017.igem.org/Team%3ASIAT-SCIE/Protocol)
* Parts

  [Parts Overview](https://2017.igem.org/Team%3ASIAT-SCIE/Parts_Overview)
  [Basic Parts](https://2017.igem.org/Team%3ASIAT-SCIE/Basic_Parts)
  [Composite Parts](https://2017.igem.org/Team%3ASIAT-SCIE/Composite_Parts)
* [Award](https://2017.igem.org/Team%3ASIAT-SCIE/Award)
* [Modeling](https://2017.igem.org/Team%3ASIAT-SCIE/Model "Modeling")
* Team

  [About us](https://2017.igem.org/Team%3ASIAT-SCIE/About_us)
  [Attribution](https://2017.igem.org/Team%3ASIAT-SCIE/Attributions)
* Human Practice

  [Gold Intergrated](https://2017.igem.org/Team%3ASIAT-SCIE/HP/Gold_Integrated)
  [Silver Intergrated](https://2017.igem.org/Team%3ASIAT-SCIE/HP/Silver)
  [Collaborations](https://2017.igem.org/Team%3ASIAT-SCIE/Collaborations)
* [InterLab](https://2017.igem.org/Team%3ASIAT-SCIE/InterLab "InterLab")

# Notebook

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

##### What should this page have?

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

##### Inspiration

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

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

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


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

# Team:SIAT-SCIE/Experiments

* [Home](https://2017.igem.org/Team%3ASIAT-SCIE/Home "Home")
* Project

  [Description](https://2017.igem.org/Team%3ASIAT-SCIE/Project_Description)
  [Results](https://2017.igem.org/Team%3ASIAT-SCIE/Results)
  [Mechanism](https://2017.igem.org/Team%3ASIAT-SCIE/Mechanism)
  [Demonstrate](https://2017.igem.org/Team%3ASIAT-SCIE/Demonstrate)
* Notebook

  [iGEM Diary](https://2017.igem.org/Team%3ASIAT-SCIE/IGEM_Diary)
  [Protocol](https://2017.igem.org/Team%3ASIAT-SCIE/Protocol)
* Parts

  [Parts Overview](https://2017.igem.org/Team%3ASIAT-SCIE/Parts_Overview)
  [Basic Parts](https://2017.igem.org/Team%3ASIAT-SCIE/Basic_Parts)
  [Composite Parts](https://2017.igem.org/Team%3ASIAT-SCIE/Composite_Parts)
* [Award](https://2017.igem.org/Team%3ASIAT-SCIE/Award)
* [Modeling](https://2017.igem.org/Team%3ASIAT-SCIE/Model "Modeling")
* Team

  [About us](https://2017.igem.org/Team%3ASIAT-SCIE/About_us)
  [Attribution](https://2017.igem.org/Team%3ASIAT-SCIE/Attributions)
* Human Practice

  [Gold Intergrated](https://2017.igem.org/Team%3ASIAT-SCIE/HP/Gold_Integrated)
  [Silver Intergrated](https://2017.igem.org/Team%3ASIAT-SCIE/HP/Silver)
  [Collaborations](https://2017.igem.org/Team%3ASIAT-SCIE/Collaborations)
* [InterLab](https://2017.igem.org/Team%3ASIAT-SCIE/InterLab "InterLab")

# Experiments

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

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

##### What should this page contain?

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

##### Inspiration

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

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

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

# Team:SIAT-SCIE/Results

* [Home](https://2017.igem.org/Team%3ASIAT-SCIE/Home "Home")
* Project

  [Description](https://2017.igem.org/Team%3ASIAT-SCIE/Project_Description)
  [Results](https://2017.igem.org/Team%3ASIAT-SCIE/Results)
  [Mechanism](https://2017.igem.org/Team%3ASIAT-SCIE/Mechanism)
  [Demonstrate](https://2017.igem.org/Team%3ASIAT-SCIE/Demonstrate)
* Notebook

  [iGEM Diary](https://2017.igem.org/Team%3ASIAT-SCIE/IGEM_Diary)
  [Protocol](https://2017.igem.org/Team%3ASIAT-SCIE/Protocol)
* Parts

  [Parts Overview](https://2017.igem.org/Team%3ASIAT-SCIE/Parts_Overview)
  [Basic Parts](https://2017.igem.org/Team%3ASIAT-SCIE/Basic_Parts)
  [Composite Parts](https://2017.igem.org/Team%3ASIAT-SCIE/Composite_Parts)
* [Award](https://2017.igem.org/Team%3ASIAT-SCIE/Award)
* [Modeling](https://2017.igem.org/Team%3ASIAT-SCIE/Model "Modeling")
* Team

  [About us](https://2017.igem.org/Team%3ASIAT-SCIE/About_us)
  [Attribution](https://2017.igem.org/Team%3ASIAT-SCIE/Attributions)
* Human Practice

  [Gold Intergrated](https://2017.igem.org/Team%3ASIAT-SCIE/HP/Gold_Integrated)
  [Silver Intergrated](https://2017.igem.org/Team%3ASIAT-SCIE/HP/Silver)
  [Collaborations](https://2017.igem.org/Team%3ASIAT-SCIE/Collaborations)
* [InterLab](https://2017.igem.org/Team%3ASIAT-SCIE/InterLab "InterLab")

![comic](https://static.igem.org/mediawiki/2017/c/cf/SIAT-SCIE_result.png)

## Vector Construction

#### -Overview:

We are using pMD-19 plasmid backbone for CAHS and p15A plasmid backbone of DSUP.

At the beginning, we tried to use pET28a for protein extraction, but due to the unknown region on the sequencing, we gave it up.

Here, we will show the construction of pMD-19-pTet-tetR-CAHS plasmids, since p15A-pTac-sfGFP-Dsup was constructed by Bluepha.

The assembly method we choose was similar to Gibson assembly, it requires overlaps on both ends, and use exonuclease to cut out the ends for ligation.

#### -PCR:

We used PCR to add the overlapping fragments on our DNA fragment.

![](https://static.igem.org/mediawiki/2017/d/d4/SIAT-SCIE_result1.png "Result1")
![](https://static.igem.org/mediawiki/2017/3/32/SIAT-SCIE_result2.png "Result2")

The result of it is shown by gel electrophoresis.

![](https://static.igem.org/mediawiki/2017/9/98/SIAT-SCIE_result3.png "Result3")

#### -Ligation

After gel-extraction, we added 0.06 pmol of DNA fragments and 0.03 pmol of backbone, incubated for 30 minutes under 37°C in PCR machine, the product was cooled in ice for three minutes, and immediately transform it into the DH5α Chemical Component cell.

#### -Clone PCR

After leaving the DH5α containing previous products in 37°C overnight, we carried out the Clone PCR to verify the ligation

The results are shown by gel electrophoresis.

![](https://static.igem.org/mediawiki/2017/3/3b/SIAT-SCIE_result4.png "Result4")

According to this, we found that Dsup was not successfully inserted into the backbone. After several times, we decided to send it to Bluepha, for the construction of Dsup.

#### -Sequencing

Samples were sent to BGI for sequencing, the results are complementary to our design.

![](https://static.igem.org/mediawiki/2017/a/aa/SIAT-SCIE_result5.png "Result5")

## Protein Expression

#### -Inducer Adding

![](https://static.igem.org/mediawiki/2017/7/71/SIAT-SCIE_result6.jpg "Result6")

#### SDS Page

![](https://static.igem.org/mediawiki/2017/0/03/SIAT-SCIE_result7.png "Result7")
![](https://static.igem.org/mediawiki/2017/e/ed/SIAT-SCIE_result7-1.jpg "Result7-1")

Unfortunately, the SDS-PAGE experiments were done badly, there were always mistakes, including sample contamination, failure in protein expression, faulty in stain and decolor, so we did not get a very reliable result on this.

BUT WE MADE IT!

DURING OUR LAST TRIAL ON OCTOBER 27TH

![Result16]( https://static.igem.org/mediawiki/2017/2/2f/SIAT-SCIE_result16.jpg)

## Desiccation

#### Dilution

In order to find out the survival rate, we had to find out the change of CFU, the number of bacteria is too large in protein expressed broth, since they are in the stationary phase, so we had to do gradient dilution.

![](https://static.igem.org/mediawiki/2017/9/9c/SIAT-SCIE_result8.jpg "Result8")
![](https://static.igem.org/mediawiki/2017/1/15/SIAT-SCIE_result9.jpg "Result9")
![](https://static.igem.org/mediawiki/2017/3/3c/SIAT-SCIE_result9-1.jpg "Result9-1")
![](https://static.igem.org/mediawiki/2017/5/51/SIAT-SCIE_result10.jpg "Result10")
![](https://static.igem.org/mediawiki/2017/e/ee/SIAT-SCIE_result11.jpg "Result11")

And then spread the diluted broth to the petri dish, showed that when the broth was diluted to 10^13 times, we could count the cfu.

#### Desiccation

We used centrifuge machine and concentrator to remove the water.

![](https://static.igem.org/mediawiki/2017/c/c4/SIAT-SCIE_result12.jpg "Result12")

## Irradiation

More information, please go to our [demonstration page.](https://2017.igem.org/Team%3ASIAT-SCIE/Demonstrate)

![](https://static.igem.org/mediawiki/2017/7/75/SIAT-SCIE_result13.jpg "Result13")

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