iGEM 2018 — Team HAFS

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

# Team:HAFS/Notebook

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

# Team:HAFS/Experiments

# Experiments

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

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

### What should this page contain?

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

### Inspiration

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

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

# Team:HAFS/Results

﻿

[# iGEM HAFS](https://2018.igem.org/Team%3AHAFS)

* PROJECT ▾
  + [Description](https://2018.igem.org/Team%3AHAFS/Description)
  + [Design](https://2018.igem.org/Team%3AHAFS/Applied_Design)
  + [Model](https://2018.igem.org/Team%3AHAFS/Model)
  + [Results](https://2018.igem.org/Team%3AHAFS/Results)
  + [Parts](https://2018.igem.org/Team%3AHAFS/Improve)
* HUMAN PRACTICES ▾
  + [Human Practices](https://2018.igem.org/Team%3AHAFS/Human_Practices)
  + [Engagement](https://2018.igem.org/Team%3AHAFS/Public_Engagement)* [INTERLAB](https://2018.igem.org/Team%3AHAFS/InterLab)
  * TEAM ▾
    + [Team](https://2018.igem.org/Team%3AHAFS/Team)
    + [Attributions](https://2018.igem.org/Team%3AHAFS/Attributions)
    + [Collaborations](https://2018.igem.org/Team%3AHAFS/Collaborations)

![](https://static.igem.org/mediawiki/2018/1/1e/T--HAFS--Result_logo.png)

Results

## Preparation of Minicell

We obtained *E. coli* ME8077 strain (minA minB thr-1 leuB6 azi lacY1 tsx ara-13 gal-6 malA1 thi-1 xyl-7 rpsL mtl-2 tonA2 supE44) from NBRP (NIG, Japan). Since ME8077 is a knockout of minB locus (which includes minCDE gene cluster), it produces minicells. We incubated ME8077 strain in LB broth overnight, and observed the morphology of cells under confocal microscope to confirm the production of spherical minicell and long rod shaped cell, which are both result of a uneven distribution of genetic material during cell division. Furthermore, we measured and plotted the length of cells from the confocal photomicrography using ImageJ.

![](https://static.igem.org/mediawiki/2018/5/52/T--HAFS--Result_minicell.png)  ![](https://static.igem.org/mediawiki/2018/b/bc/T--HAFS--Result_Mini_2.png)

Figure 1. (a) Minicells observed from *E. coli* ME8077 (b) Rod shaped cells that produce minicells

![](https://static.igem.org/mediawiki/2018/2/2e/T--HAFS--Result_length.png)

Figure 2. Cell length distribution of *E. coli* ME8077 strain

### [▲ top](#top)

## Insulin production

After synthesizing the Insulin production cassette flanked by biobrick prefix and suffix, we first digested the part and pSB1C3 vector with EcoR1 and Pst1 and then ligated the two parts. The resulting plasmid was transformed into E.Coli DH5-α.

![](https://static.igem.org/mediawiki/2018/c/c8/T--HAFS--Ligation.jpeg)

Figure 3. *E. coli* DH5 alpha transformed with ligated plasmid

We runned gel with the miniprepped DNA but by EcoR1, which should be about 2.5k long. We found out that only one of the three plasmids, were correctly transformed, which was the part with constitutive promoter J23100.

![](https://static.igem.org/mediawiki/2018/e/ef/T--HAFS--Result_Gel.png)

Figure 4. 1.2% Agarose Gel electrophoresis result of pSB1C3 cut with EcoR1

In order to detect Insulin production, we conducted SDS-page gel using the DH5 alpha tranformed with the J23100-CPP-Insulin in pSB1c3 grown in LB medium overnight.
![](https://bit.ly/gaattcgcggccgcttctagagttgacggctagctcagtcctaggtacagtgctagcaaagaggagaaa3)

Figure 5. 10% SDS-Page Gel showing expression of protein

In future study, we are planning to transform the ME8077 strain using the prepared DNA.

### [▲ top](#top)

Team: AQA\_Unesp

![](https://static.igem.org/mediawiki/2018/8/80/T--HAFS--Sponsor.png)

Hankuk Academy of Foreign Studies
International studies
Gyeong gi, South Korea
kyuhee0622@gmail.com