iGEM 2017 — Team KUAS_Korea

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

# Team:KUAS Korea/Notebook

[KUAS Korea](https://2017.igem.org/Team%3AKUAS_Korea)

* TEAM
  + [Team](https://2017.igem.org/Team%3AKUAS_Korea/Team)
  + [Attributions★](https://2017.igem.org/Team%3AKUAS_Korea/Attributions)
  + [Collaborations★](https://2017.igem.org/Team%3AKUAS_Korea/Collaborations)
  + [Official Team Profile](https://igem.org/Team.cgi?team_id=2437)* PROJECT
    + [Description](https://2017.igem.org/Team%3AKUAS_Korea/Description)
    + [Design](https://2017.igem.org/Team%3AKUAS_Korea/Design)
    + [Parts](https://2017.igem.org/Team%3AKUAS_Korea/Parts)
    + [Model★](https://2017.igem.org/Team%3AKUAS_Korea/Model)
    + [Acheivement](https://2017.igem.org/Team%3AKUAS_Korea/Acheivement)
  * WET LAB
    + [Experiments](https://2017.igem.org/Team%3AKUAS_Korea/Experiments)
    + [Results](https://2017.igem.org/Team%3AKUAS_Korea/Results)
    + [Contribution★](https://2017.igem.org/Team%3AKUAS_Korea/Contribution)
    + [Safety](https://2017.igem.org/Team%3AKUAS_Korea/Safety)* [NOTEBOOK](https://2017.igem.org/Team%3AKUAS_Korea/Notebook)
    * HUMAN PRACTICES
      + [Overview](https://2017.igem.org/Team%3AKUAS_Korea/HP/Overview)
      + [Human Practices★](https://2017.igem.org/Team%3AKUAS_Korea/HP/Silver)
      + [Integrated Practices★](https://2017.igem.org/Team%3AKUAS_Korea/HP/Gold_Integrated)
      + [Public Engagement](https://2017.igem.org/Team%3AKUAS_Korea/Engagement)

### Notebook

## Notebook

#### Week 1.

1. We researched several gastrointesintal diseases and its causes.
2. Additionally, we researched existing diagnostic methods used in detecting these diseases.

#### Week 2.

1. We looked for gut microbiome distribution in the gut and bacterias that are currently verified and utilized in probiotics.
2. Also, we chose heme, pH, bile salt as our target indicator, which could detect the diseases we previously researched on the first week.

#### Week 3.

We designed a detector device based on analyzation of prior research about sensing heme, pH, and bile salt.

#### Week 4.

1. We received *Lactobacillus plantarum* L67 from professor Se Jong Oh of Chunnam University, and cultured them in MRS media.
2. We collected single colony from them and cultured it.
3. We then cultured these bacteria in culture medium that contain ampicillin, chloramphenicol, tetracycline, gentamycin, rifampicin, streptomycin, kanamycin, and tested antibiotic resistance.

#### Week 5.

1. Our Professor gave us a feedback on the experiment conducted in week 4. Based on the feedback, we revised the problematic part and conducted the experiment again.
2. We compared the test result and verified antibiotic resistance data of *L. plantarum*, and checked if the right bacteria was cultured.
3. We researched on transformation protocol of Lactobacillus transformation and referenced protocol of Eppendorf.

#### Week 6.

1. We selected the gram positive-E. coli shuttle vector for the transformation of the *Lactobacillus Plantarum* L67
2. After taking the shuttle vector out from stock, heat-shock transformation to E. coli BW25113 was processed to amplify the vector. vector was extracted by mini-prep method.
3. We measured the concentration and the checked the size of the outcome using gel electrophoresis.

#### Week 7.

1. *Lactobacillus Plantarum* L67 Electroporation competent cell was produced. (Eppendorf protocol was referred)
2. We tried to transform previously amplified vector to the competent cell at various voltages and amounts of vector.
3. Result was checked using the selective culture.

#### Week 8.

After the failure of transformation on the 7th week, we re-constructed competent cell and carried out another experiment.

#### Week 9.

1. After the re-failure of transformation on the 8th week, we found out another protocol and constructed a new competent cell and performed an experiment once again.
2. Our professor revised the previously designed detecting device.
3. We ordered forward and reverse primer for PCR of hssR and hssS from the IDT.
4. We also ordered sequences of hrtR promoter and HrtR protein attached with iGEM prefix and suffix.

#### Week 10.

We requested for *Bacillus cereus* ATCC from Korean Collection for Type Culture(KCTC) 14579.

#### Week 11.

1. We cultured freeze-drying *B. cereus* from KCTC on Nutrient Agar broth KCTC and got a single colony.
2. We ordered psH71 replicon of Lactic Acid Bacteria from IDT.

#### Week 12.

1. gBlock and primer which we ordered from IDT at week 9 have arrived.
2. gDNA of *B. cereus* was extracted.
3. we processed the PCR, using gDNA and primer but the band corresponding to HssRS was not detected on gel electrophoresis.

#### Week 13.

1. Retried PCR but failed.
2. We requested BBa\_K1033282 to the iGEM Headquarter.

#### Week 14.

1. We selected three preexisting part from the iGEM distribuition kit for
   Characterization.
2. BBa\_K1033282 arrived from iGEM HQ.
3. We cultured *E. coli* Dh5alpha from our cell stock, and made heat-shock competent cell.
4. We cloned hrtR gBlock in the pSB1C3 vector.

#### Week 15.

1. We transformed four existing part in the DH5 alpha and confirmed that two parts, BBa\_K1033282, BBa\_K1696009 had succeeded transforming.
2. The cloning of gBlock also succeeded.

#### Week 16.

1. Seed culture of the DH5alpha which has BBa\_K1033282(amilCP), was done.
2. OD588 was measured at 20 min interval on preparatory experiments.
3. Although pSB1C3 was inserted, additional TA cloning was done since it was hard to sort colonies not having the gBlock.

#### Week 17.

1. We cultured BBa\_K1033282 inserted DH5alpha and measured absorbance of OD588(maximum absorbance) and OD800(cell concentration) in 30 minute interval.
2. We drew graphs showing the change of OD588 in accordance to time, the change of OD588/OD800 ratio to time, and change of OD588 in accordance to OD800 value.
3. We uploaded these graphs and photos on the wiki main page of BBa\_K1033282.
4. We acquired pSB1C3 with HrtR gblock inserted as a result of TA cloning.
5. We digested this by restriction enzyme again, and confirmed the size of the digested DNA fragment.
6. We submitted pSB1C3-hrtR to the iGEM HQ.

[![](https://static.igem.org/mediawiki/2017/2/2a/IDT_Logo_finish.png)](https://www.idt.com/)

[![](https://static.igem.org/mediawiki/2013/1/13/Spon2.png)](https://compbio.korea.ac.kr)

[![](https://static.igem.org/mediawiki/2016/f/f4/Korea_U_Seoul_Sponsor_ku.gif)](https://korea.ac.kr/)

[![](https://static.igem.org/mediawiki/2013/1/1c/Spon3.png)](https://ctl.korea.ac.kr/)

[![](https://static.igem.org/mediawiki/2016/3/34/Korea_U_Seoul_sponsor_kusso.jpg)](https://kusso.korea.ac.kr/)

[![](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:KUAS Korea/Experiments

[KUAS Korea](https://2017.igem.org/Team%3AKUAS_Korea)

* TEAM
  + [Team](https://2017.igem.org/Team%3AKUAS_Korea/Team)
  + [Attributions★](https://2017.igem.org/Team%3AKUAS_Korea/Attributions)
  + [Collaborations★](https://2017.igem.org/Team%3AKUAS_Korea/Collaborations)
  + [Official Team Profile](https://igem.org/Team.cgi?team_id=2437)* PROJECT
    + [Description](https://2017.igem.org/Team%3AKUAS_Korea/Description)
    + [Design](https://2017.igem.org/Team%3AKUAS_Korea/Design)
    + [Parts](https://2017.igem.org/Team%3AKUAS_Korea/Parts)
    + [Model★](https://2017.igem.org/Team%3AKUAS_Korea/Model)
    + [Acheivement](https://2017.igem.org/Team%3AKUAS_Korea/Acheivement)
  * WET LAB
    + [Experiments](https://2017.igem.org/Team%3AKUAS_Korea/Experiments)
    + [Results](https://2017.igem.org/Team%3AKUAS_Korea/Results)
    + [Contribution★](https://2017.igem.org/Team%3AKUAS_Korea/Contribution)
    + [Safety](https://2017.igem.org/Team%3AKUAS_Korea/Safety)* [NOTEBOOK](https://2017.igem.org/Team%3AKUAS_Korea/Notebook)
    * HUMAN PRACTICES
      + [Overview](https://2017.igem.org/Team%3AKUAS_Korea/HP/Overview)
      + [Human Practices★](https://2017.igem.org/Team%3AKUAS_Korea/HP/Silver)
      + [Integrated Practices★](https://2017.igem.org/Team%3AKUAS_Korea/HP/Gold_Integrated)
      + [Public Engagement](https://2017.igem.org/Team%3AKUAS_Korea/Engagement)

### Experiments

##

![](https://static.igem.org/mediawiki/2017/a/a9/T--KUAS_Korea--HrtR.png)

#### 1. Designing the Heme sensing device

We have looked for the precedent researches on heme resistance of gut microbiome, we have decided to select the external detector system using HssS & HssR protein and the internal dectector utilizing HrtR protein. We designed a complete set comprised of promoter, sensing protein, and marker by inserting genetic marker instead of the efflux pump in the conventional mechanism.

#### 2. Constructing the Heme sensing device

➀ In the case of HssS, HssR, we could find the matching sequence in Bacillus cereus ATCC 14579 by BLASTing the sequence that was mentioned in the former research. We received this bacteria from Korean Collection of Type Culture(KCTC), cultured them and collected a single colony. We extracted the gDNA of B.cereus, and added igem prefix, suffix respectively to the front and end of these HssR, HssS sequences. We also conducted PCR of gDNA template using this primer we designed.

➁ We modified HrtR and its promoter by adding iGEM prefix at the front, and suffix at the end. Moreover, we ordered from the IDT as a gBlock. We inserted the arrived gBlock utilizing the 3A assemble and TA cloning. We also screened the bacteria with ones that do not show the expression of RFP. We then cleaved the cloning vector using restriction enzymes and processed gel electrophoresis to check if the part was correct inserted.

#### 3. Transformation of the *Lactobacillus plantarum* L67

We received *L. plantarum* L67 from the lab of Professor Se Jong Oh, Chonnam University. Then, we cultured the bacteria on MRS media and collected single colony. Then we tested antibiotic resistance to prove that we cultured the appropriate bacteria. For the selection of transformation vector of *Lactobacillus*, we have chosen the gram-positive shuttle vector that our lab already owned. We made electron competent cell of *lactobacillus* and attempted transformation in various conditions with different DNA amount and voltage. However, the transformation was unfortunately not successful, and we concluded that the reason was the malfunction of replicon in the *Lactobacillus*. So we additionally ordered the Lactic Acid Bacteria (LAB) replicon for BBa\_K1033206.

#### 4. Construction of the Screenable marker

When defining the presence of occult blood in our feces, we thought it would be difficult for users to recognize if we used general GFP, due to the low fluorescence. Therefore, we planned to use chromoprotein rather than fluorescent proteins like GFP. We found out that 2013 Uppsala team previously used *Lactobacillus* and chromoprotein in their project. So we received the part this team submitted from the iGEM HQ and tested expression.

[![](https://static.igem.org/mediawiki/2017/2/2a/IDT_Logo_finish.png)](https://www.idt.com/)

[![](https://static.igem.org/mediawiki/2013/1/13/Spon2.png)](https://compbio.korea.ac.kr)

[![](https://static.igem.org/mediawiki/2016/f/f4/Korea_U_Seoul_Sponsor_ku.gif)](https://korea.ac.kr/)

[![](https://static.igem.org/mediawiki/2013/1/1c/Spon3.png)](https://ctl.korea.ac.kr/)

[![](https://static.igem.org/mediawiki/2016/3/34/Korea_U_Seoul_sponsor_kusso.jpg)](https://kusso.korea.ac.kr/)

[![](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:KUAS Korea/Results

[KUAS Korea](https://2017.igem.org/Team%3AKUAS_Korea)

* TEAM
  + [Team](https://2017.igem.org/Team%3AKUAS_Korea/Team)
  + [Attributions★](https://2017.igem.org/Team%3AKUAS_Korea/Attributions)
  + [Collaborations★](https://2017.igem.org/Team%3AKUAS_Korea/Collaborations)
  + [Official Team Profile](https://igem.org/Team.cgi?team_id=2437)* PROJECT
    + [Description](https://2017.igem.org/Team%3AKUAS_Korea/Description)
    + [Design](https://2017.igem.org/Team%3AKUAS_Korea/Design)
    + [Parts](https://2017.igem.org/Team%3AKUAS_Korea/Parts)
    + [Model★](https://2017.igem.org/Team%3AKUAS_Korea/Model)
    + [Acheivement](https://2017.igem.org/Team%3AKUAS_Korea/Acheivement)
  * WET LAB
    + [Experiments](https://2017.igem.org/Team%3AKUAS_Korea/Experiments)
    + [Results](https://2017.igem.org/Team%3AKUAS_Korea/Results)
    + [Contribution★](https://2017.igem.org/Team%3AKUAS_Korea/Contribution)
    + [Safety](https://2017.igem.org/Team%3AKUAS_Korea/Safety)* [NOTEBOOK](https://2017.igem.org/Team%3AKUAS_Korea/Notebook)
    * HUMAN PRACTICES
      + [Overview](https://2017.igem.org/Team%3AKUAS_Korea/HP/Overview)
      + [Human Practices★](https://2017.igem.org/Team%3AKUAS_Korea/HP/Silver)
      + [Integrated Practices★](https://2017.igem.org/Team%3AKUAS_Korea/HP/Gold_Integrated)
      + [Public Engagement](https://2017.igem.org/Team%3AKUAS_Korea/Engagement)

### Results

## Results

Our project is to make circuits that express chromoproteins when heme is present. To do this, we used a mechanism in which bacteria respond to heme to make an efflux pump. In this system, HssS and HssR were used to recognize from the outside of the cell and HrtR to recognize from the inside.

#### 1. HrtR System

HrtR is a self-regulator that binds directly to the promoter’s palindrome sequence(ATGACACAGTGTCAT) in front of its CDS. When it binds to the promoter, it acts as an inhibitor that prevents the transcription of itself.

![](https://static.igem.org/mediawiki/2017/9/98/T--KUAS_Korea--Results1.jpg)

We ordered the hrtR sequence of the gram-positive *Lactococcus lactis* in IDT and cloned it into DH5alpha using pSB1C3 and TA cloning. In the picture above, the white colonies were inserted with the HrtR and the RFP was deleted. The red one is that the RFP still exists because HrtR is not inserted normally.

#### 2. Chromoprotein

Commonly used GFPs must be exposed to ultraviolet light in order to see strong green color. So we planned to use chromoprotein instead of fluorescent protein because we thought that fluorescent proteins would be difficult to use in common life. The 2013 Uppsala team's project was using *Lactobacillus* and chromoprotein, and we decided to use these parts. This photograph shows the expression of their amilCP in *E. coli* DH5alpha. AmilCP will be more noticeable because it has a blue color that is complementary to the color of the feces.

![](https://static.igem.org/mediawiki/2017/d/d1/T--KUAS_Korea--Contribution1.jpeg)

Our final design using above system is as follows. HrtR is expressed in cells at a constant concentration by negative feedback. When HtrR binds to heme, its inhibitor function is lost and transcription occurs. Therefore, the concentration of chromoprotein increases and the color of the bacteria in the feces becomes darker.

![](https://static.igem.org/mediawiki/2017/4/4a/T--KUAS_Korea--Results3.jpg)

[![](https://static.igem.org/mediawiki/2017/2/2a/IDT_Logo_finish.png)](https://www.idt.com/)

[![](https://static.igem.org/mediawiki/2013/1/13/Spon2.png)](https://compbio.korea.ac.kr)

[![](https://static.igem.org/mediawiki/2016/f/f4/Korea_U_Seoul_Sponsor_ku.gif)](https://korea.ac.kr/)

[![](https://static.igem.org/mediawiki/2013/1/1c/Spon3.png)](https://ctl.korea.ac.kr/)

[![](https://static.igem.org/mediawiki/2016/3/34/Korea_U_Seoul_sponsor_kusso.jpg)](https://kusso.korea.ac.kr/)

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