iGEM 2017 — Team BIT-China

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# Team:BIT-China/Notebook

BIT-CHINA

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* Project
  + [Description](https://2017.igem.org/Team%3ABIT-China/Project/Description)
  + [Receptor Expression](https://2017.igem.org/Team%3ABIT-China/Project/Sense)
  + [Pathway optimization](https://2017.igem.org/Team%3ABIT-China/Project/Transduction)
  + [Signal Output](https://2017.igem.org/Team%3ABIT-China/Project/Detection)
  + [Result](https://2017.igem.org/Team%3ABIT-China/Project/Results)
* Modeling
  + [Overview](https://2017.igem.org/Team%3ABIT-China/Model)
  + [Docking](https://2017.igem.org/Team%3ABIT-China/Model/Docking)
  + [GPCR pathway](https://2017.igem.org/Team%3ABIT-China/Model/GPCRpathway)
* Laboratory
  + [Material](https://2017.igem.org/Team%3ABIT-China/Material)
  + [Notebook](https://2017.igem.org/Team%3ABIT-China/Notebook)
  + [Protocol](https://2017.igem.org/Team%3ABIT-China/Protocol)
  + [Equipment](https://2017.igem.org/Team%3ABIT-China/Equipment)
  + [Safety](https://2017.igem.org/Team%3ABIT-China/Safety)
* Achievement
  + [Parts](https://2017.igem.org/Team%3ABIT-China/Parts)
  + [Medal](https://2017.igem.org/Team%3ABIT-China/Medal)
* HP
  + [HP-Silver](https://2017.igem.org/Team%3ABIT-China/HP/Silver)
  + [integrated＆Gold](https://2017.igem.org/Team%3ABIT-China/HP/Gold_Integrated)
  + [Engagement](https://2017.igem.org/Team%3ABIT-China/Engagement)
  + [Collaboration](https://2017.igem.org/Team%3ABIT-China/Collaborations)
* Team
  + [Member](https://2017.igem.org/Team%3ABIT-China/Member)
  + [Attribution](https://2017.igem.org/Team%3ABIT-China/Attributions)
  + [Sponsor](https://2017.igem.org/Team%3ABIT-China/Sponsor)

![](https://static.igem.org/mediawiki/2017/e/ec/T--BIT-China--2017laboratory.jpg)

## NOTEBOOK

### Breakthrough

#### 2.14-4.14:team recruitment & brainstorm

#### 4.14-6.20: core part synthesis and heterologous expression

4.14-5.2: core part-T1R2/T1R3 synthesis completely through OE-PCR.

4.30-6.20: heterologous expression of T1R2/T1R3. [Click here to see more details.](https://2017.igem.org/Team%3ABIT-China/Project/Sense)

#### 4.30-5.15：modeling

4.30-5.5: finished several sweeteners'docking as well as T1R2/T1R3 PDB file construction. Different sweeteners docked to the sweet taste receptor T1R2/T1R3.[Click here to see more details.](https://2017.igem.org/Team%3ABIT-China/Model/Docking)

5.5-5.15: MAPK pathway signal modeling. [Click here to see more details.](https://2017.igem.org/Team%3ABIT-China/Model/GPCRpathway)

#### 5.2-8.20: pathway optimization

5.2-7.1: single gene knockout

7.1-8.5: any two of the three genes knockout and three genes knockout[Click here to see more details.](https://2017.igem.org/Team%3ABIT-China/Project/Transduction)

8.5-8.20: characterization of engineered strains

#### 8.21-10.20: Verification of the new pathway

8.21-9.13: establishment of the detection circuit .[Click here to see more details.](https://2017.igem.org/Team%3ABIT-China/Project/Detection)

9.13-9.26: verification of the new pathway

9.27-10.10: sweetness meter verification

10.8-10.20: parts standardization

It appears you don't have a PDF plugin for this browser.
No biggie... you can [click here to
download the PDF file.](https://static.igem.org/mediawiki/2017/7/7c/Bit-china-notebook.pdf)

For more details,please see our [project](https://2017.igem.org/Team%3ABIT-China/Project/Description).

[![](https://static.igem.org/mediawiki/2017/a/a4/T--BIT-China--2017previous.png)](https://2017.igem.org/Team%3ABIT-China/Material)
[![](https://static.igem.org/mediawiki/2017/6/6f/T--BIT-China--2017next.png)](https://2017.igem.org/Team%3ABIT-China/Protocol)

[TOP
![](https://static.igem.org/mediawiki/2017/f/fd/Bit-china-2017backTop.png)](#mytop)

##### Contact

Institute of Biotransformation and synthetic biosystem

School of life science

Beijing Institute of Technology

100081, Beijing

Email: lichun@bit.edu.cn

![](https://static.igem.org/mediawiki/2017/7/71/T--BIT-China--2017sponsor_pic1.png)
![](https://static.igem.org/mediawiki/2017/d/da/T--BIT-China--2017sponsor_pic3.png)
![](https://static.igem.org/mediawiki/2017/2/28/T--BIT-China--2017sponsor_pic5.png)

![](https://static.igem.org/mediawiki/2017/b/b8/T--BIT-China--2017sponsor_pic8.png)
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Copyright © 2017 BIT-China

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# Team:BIT-China/Experiments

BIT-CHINA

[![](https://static.igem.org/mediawiki/2017/f/f5/T--BIT-China--2017nav_logo.gif)](https://2017.igem.org/Team%3ABIT-China)

* Project
  + [Description](https://2017.igem.org/Team%3ABIT-China/Project/Description)
  + [Receptor Expression](https://2017.igem.org/Team%3ABIT-China/Project/Sense)
  + [Pathway optimization](https://2017.igem.org/Team%3ABIT-China/Project/Transduction)
  + [Signal Output](https://2017.igem.org/Team%3ABIT-China/Project/Detection)
  + [Result](https://2017.igem.org/Team%3ABIT-China/Project/Results)
* Modeling
  + [Overview](https://2017.igem.org/Team%3ABIT-China/Model)
  + [Docking](https://2017.igem.org/Team%3ABIT-China/Model/Docking)
  + [GPCR pathway](https://2017.igem.org/Team%3ABIT-China/Model/GPCRpathway)
* Laboratory
  + [Material](https://2017.igem.org/Team%3ABIT-China/Material)
  + [Notebook](https://2017.igem.org/Team%3ABIT-China/Notebook)
  + [Protocol](https://2017.igem.org/Team%3ABIT-China/Protocol)
  + [Equipment](https://2017.igem.org/Team%3ABIT-China/Equipment)
  + [Safety](https://2017.igem.org/Team%3ABIT-China/Safety)
* Achievement
  + [Parts](https://2017.igem.org/Team%3ABIT-China/Parts)
  + [Medal](https://2017.igem.org/Team%3ABIT-China/Medal)
* HP
  + [HP-Silver](https://2017.igem.org/Team%3ABIT-China/HP/Silver)
  + [integrated＆Gold](https://2017.igem.org/Team%3ABIT-China/HP/Gold_Integrated)
  + [Engagement](https://2017.igem.org/Team%3ABIT-China/Engagement)
  + [Collaboration](https://2017.igem.org/Team%3ABIT-China/Collaborations)
* Team
  + [Member](https://2017.igem.org/Team%3ABIT-China/Member)
  + [Attribution](https://2017.igem.org/Team%3ABIT-China/Attributions)
  + [Sponsor](https://2017.igem.org/Team%3ABIT-China/Sponsor)

# 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:BIT-China/Results

BIT-CHINA

[![](https://static.igem.org/mediawiki/2017/f/f5/T--BIT-China--2017nav_logo.gif)](https://2017.igem.org/Team%3ABIT-China)

* Project
  + [Description](https://2017.igem.org/Team%3ABIT-China/Project/Description)
  + [Receptor Expression](https://2017.igem.org/Team%3ABIT-China/Project/Sense)
  + [Pathway optimization](https://2017.igem.org/Team%3ABIT-China/Project/Transduction)
  + [Signal Output](https://2017.igem.org/Team%3ABIT-China/Project/Detection)
  + [Result](https://2017.igem.org/Team%3ABIT-China/Project/Results)
* Modeling
  + [Overview](https://2017.igem.org/Team%3ABIT-China/Model)
  + [Docking](https://2017.igem.org/Team%3ABIT-China/Model/Docking)
  + [GPCR pathway](https://2017.igem.org/Team%3ABIT-China/Model/GPCRpathway)
* Laboratory
  + [Material](https://2017.igem.org/Team%3ABIT-China/Material)
  + [Notebook](https://2017.igem.org/Team%3ABIT-China/Notebook)
  + [Protocol](https://2017.igem.org/Team%3ABIT-China/Protocol)
  + [Equipment](https://2017.igem.org/Team%3ABIT-China/Equipment)
  + [Safety](https://2017.igem.org/Team%3ABIT-China/Safety)
* Achievement
  + [Parts](https://2017.igem.org/Team%3ABIT-China/Parts)
  + [Medal](https://2017.igem.org/Team%3ABIT-China/Medal)
* HP
  + [HP-Silver](https://2017.igem.org/Team%3ABIT-China/HP/Silver)
  + [integrated＆Gold](https://2017.igem.org/Team%3ABIT-China/HP/Gold_Integrated)
  + [Engagement](https://2017.igem.org/Team%3ABIT-China/Engagement)
  + [Collaboration](https://2017.igem.org/Team%3ABIT-China/Collaborations)
* Team
  + [Member](https://2017.igem.org/Team%3ABIT-China/Member)
  + [Attribution](https://2017.igem.org/Team%3ABIT-China/Attributions)
  + [Sponsor](https://2017.igem.org/Team%3ABIT-China/Sponsor)

![](https://static.igem.org/mediawiki/2017/d/d9/Bit-china-2017project.jpg)

## Result

The strategy for the function of engineered CEN.PK2-1C stains lacks △ far1+△sst2, △sst and △far1+△sst2+△ste2 were divided into the following parts;

(1). Testing the inhibitive effects on the growth of engineered CEN.PK2-1C, whose cellular period can be stopped at the G1 stage by α factor.

(2). Expression and identification of Pfus+RFP+cyc1t+PRS42K in engineered CEN.PK2-1C strains △far1+△sst2, △far1+△sst2+△ste2.

### Antibacterial circle

#### The plan and method

The growth of CEN.PK2-1C is inhibited by α factor,We want to test the function of △sst2, △far1+△sst2 and △far1+△sst2+△ste2 by comparing with CEN.PK2-1C .

(1)The influence of the growth of bacteria caused by different amount of α factor in the solid plate.

(2)The observed difference among the function of engineered CEN.PK2-1C strains, △far1+△sst2, △far1+△sst2+△ste2 acted on the growth of Cen.PK2-1C, △sst2, △far1+△sst2, △far1+△sst2+△ste2 impaired by different amount of α factor.

#### The results and discussion

(1)The bacteria strain of CEN.PK2-1C stops growing in G1 due to the effects acted by the α factor and antibacterial circle appears in the panel.

![](https://static.igem.org/mediawiki/2017/4/46/T-BIT-China-2017result-1.png)

(2)△sst2 strain (sst2 is knocked out successfully) and the sensitivity of △sst2 strain to α factor will be increased. Meanwhile the larger antibacterial circle will appear when the amount of α factor is reduced.

![](https://static.igem.org/mediawiki/2017/9/94/T-BIT-China-2017result-2.png)
![](https://static.igem.org/mediawiki/2017/a/a8/T-BIT-China-2017result-3.png)

(3)The far1 of △far1+△sst2, △far1+△sst2+△ste2 is knocked out successfully, and this strain will still keep growing under the effects acted by α factor and antibacterial circle will not be shown either.

![](https://static.igem.org/mediawiki/2017/c/c5/T-BIT-China-2017result-4.png)
![](https://static.igem.org/mediawiki/2017/6/6d/T-BIT-China-2017result-5.png)

Conclusion： Because far1 can make cellular period stop at the G1 stage by α factor and sst2 can reduce the intensity of the transmitted signal. So we want to knock out of far1 and sst2.From the above results we can see that in the α factor concentration range, the strain will be inhibited by α factor. With the increase of the concentration of the alpha factor,the radius of Antibacterial circle increases and the inhibitory effect of the growth of the strain was enhanced. And more obvious. Knocking out of the sst2 gene, making the strain respond to α factor enhanced.

### The growth curve

The impacts acted by the various concentration of α factor to the growth of the bacteria immersed into liquid.

The research of the impacts acted by various concentration of α factor to the growth of Cen.PK2-1C, △sst2 of CEN.PK2-1C.

#### The plan and method

The gowth of engineered CEN.PK2-1C strains were determined by measuring the initial rates of α factor at concentrations of 0-15 µM . We take out 5ml cultivating bacteria liquid of each of them during 30h and correct them by the YPD solution which not inoculates yeasts after shake culture. We obtain the value of OD600 at position of wavelength of 600nm by UV-Vis spectrophotometer.

#### The results and discuss

(1)With the increasing concentration of α factor, the growth curve of CEN.PK2-1C is inhibited by α factor.

![](https://static.igem.org/mediawiki/2017/e/e3/T-BIT-China-2017result-6.png)

(2)the growth curve of △sst2 is bit lower than CEN.PK2-1C. It’s strongly inhibited by α factor.

![](https://static.igem.org/mediawiki/2017/4/45/T-BIT-China-2017result-7.png)

Conclusion：From the above results we can see that in the α factor concentration range, the strain will be inhibited by α factor. With the increase of the concentration of the alpha factor, the inhibitory effect of the growth of the strain was enhanced. And more obvious. Knocking down the sst2 gene, making the strain respond to α factor enhanced.

### Fluorescence

Test the expression of Pfus+mRFP+cyc1t+pRS42K in engineered CEN.PK2-1C strains, △far1+△sst2, △far1+△sst2+△ste2, △sst2.

We research the impacts acted by various concentration of α factor to the amount of expression of fluorescence as well as the the growth of engineered CEN.PK2-1C strains, △far1+△sst2、△far1+△sst2+△ste2，△sst2 containing the examined circuit.

#### The plan and method

We set the concentration of alpha factor as 0umol/L，1 umol/L， 2.5 umol/L， 5 umol/L，10 umol/L，15 umol/L.We take out 5ml cultivating bacteria liquid of each of them after 0、4、8、10、12、14、16、18、20、22、24、26、28、30h respectively and correct them by the YPD solution which not inoculated yeasts after shake culture. Afterwards,the intensity of triggered fluorescence is measured in microplate reader(584/607).We create the coordianate system of the expression of fluorescence of engeneering yeast by setting the value of that intensity of triggered fluorescence/OD600 as Y-axis and time taken as X- axis.

#### The results and discuss

![](https://static.igem.org/mediawiki/2017/0/00/T-BIT-China-2017result-8.png)

![](https://static.igem.org/mediawiki/2017/f/f6/T-BIT-China-2017result-9.png)

![](https://static.igem.org/mediawiki/2017/0/08/T-BIT-China-2017result-10.png)

Conclusion：From the above results we can see that in the α factor concentration range, the expression of mRFP was induced. With the increase of the concentration of the alpha factor, the fluorescence expression was enhanced. And more obvious. Knocking down the sst2 and far1gene, making the strain respond to α factor enhanced.

[TOP
![](https://static.igem.org/mediawiki/2017/f/fd/Bit-china-2017backTop.png)](#mytop)

##### Contact

Institute of Biotransformation and synthetic biosystem

School of life science

Beijing Institute of Technology

100081, Beijing

Email: lichun@bit.edu.cn

![](https://static.igem.org/mediawiki/2017/7/71/T--BIT-China--2017sponsor_pic1.png)
![](https://static.igem.org/mediawiki/2017/d/da/T--BIT-China--2017sponsor_pic3.png)
![](https://static.igem.org/mediawiki/2017/2/28/T--BIT-China--2017sponsor_pic5.png)

![](https://static.igem.org/mediawiki/2017/b/b8/T--BIT-China--2017sponsor_pic8.png)
![](https://static.igem.org/mediawiki/2017/7/7f/T--BIT-China--2017sponsor_pic10.png)
![](https://static.igem.org/mediawiki/2017/3/36/T--BIT-China--2017sponsor_pic9.png)

Copyright © 2017 BIT-China

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