iGEM 2017 — Team FAFU-CHINA

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# Team:FAFU-CHINA/Notebook

* [Home](https://2017.igem.org/Team%3AFAFU-CHINA)
* Project
  + [Design](https://2017.igem.org/Team%3AFAFU-CHINA/Design)
  + [Background](https://2017.igem.org/Team%3AFAFU-CHINA/Applied_Design)
  + [Interlab](https://2017.igem.org/Team%3AFAFU-CHINA/InterLab)
  + [Module 1: Phyto-route](https://2017.igem.org/Team%3AFAFU-CHINA/Plant)
  + [Module 2: Metal-trap](https://2017.igem.org/Team%3AFAFU-CHINA/Experiments#sss2)
  + [Module 3: Safeguard](https://2017.igem.org/Team%3AFAFU-CHINA/Experiments#sss3)
* Achievements
  + [Demonstrate](https://2017.igem.org/Team%3AFAFU-CHINA/Demonstrate)
  + [Judging Form](https://2017.igem.org/Team%3AFAFU-CHINA/Judging_Form)
* Extras
  + [Collaborations](https://2017.igem.org/Team%3AFAFU-CHINA/Collaborations)
  + [Protocols](https://2017.igem.org/Team%3AFAFU-CHINA/Protocols%20)
  + Notebook
* [Parts](https://2017.igem.org/Team%3AFAFU-CHINA/Parts)
* [Model](https://2017.igem.org/Team%3AFAFU-CHINA/Model)
* [Human Practice](https://2017.igem.org/Team%3AFAFU-CHINA/Human_Practices)
* Team
  + [Team members](https://2017.igem.org/Team%3AFAFU-CHINA/Team)
  + [Attributions](https://2017.igem.org/Team%3AFAFU-CHINA/Attributions)

![](https://static.igem.org/mediawiki/2017/9/9f/T--FAFU-CHINA--banner.png)

**2017 Jun**

**2017 Jul**

**2017 Agu**

**2017 Sep**

**2017 Oct**

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# 2017.05.28-2017.06.03

We collected a lot of soil samples from all over the country, hoping to screen out the bacteria with the ability to dissolve phosphorus. Firstly, we put half of the samples into organic phosphorus and inorganic phosphorus medium for culture.

![](https://static.igem.org/mediawiki/2017/6/64/T--FAFU-CHINA--week1.jpg)

# 2017.06.04-2017.06.10

During the screening of bacteria, total four soil samples, respectively, 37th, 38th, Beishan area, Chinese garden soil and soil samples. Phosphorus enrichment in organic and inorganic found even filtered process 37, 38th samples did not see obvious phenomena of dissolved phosphorus, observed only yellow mucus-like plaque. Stored too long, speculation, and dry, less solubilizing bacteria, strong colony survival and stress resistance (yellow mucus-like plaque), a type of bacteria in less dissolved phosphorus under low phosphorus environment to survive. 37, 38th, soil samples failed. Found in the Beishan screening of soil samples, the only obvious solubilizing, labeled a, its propagation and flat dash found solubilizing effect, as one of candidate species for further experiments. Therefore, crossed the plate picked up three colonies, numbered A1, A2 and A3, respectively, on the inclined plane of bacteria and propagation in preparation for the molybdenum blue Colorimetry. Screening of Chinese garden soil samples found in phosphate, then pick the better of the two colonies (numbered b, c) a flat line, but the result is not ideal c, pass. B the results still can, collect a bacteria retention and propagation in preparation for molybdenum blue Colorimetry. Direct pick the bacteria found in the process of picking fungi in repeated experiments, pick the number of bacteria will not, lead to very different results.

![](https://static.igem.org/mediawiki/2017/c/c4/T--FAFU-CHINA--week2.jpg)

# 2017.06.11-2017.06.17

Then we did the plate scribing, and picked out a single colony with phosphorus solution, measuring its 16SrDNA to identify it.

Finally, we found *Bacillus megaterium*.Genomic DNA was extracted from bacterial isolates using a commercial kit according to the manufacturer’s instructions. Then, DNA was stored at −20℃. Two universal oligonucleotide forward and reverse primers were synthesized based on the standard 16S rRNA gene sequence. The primers used to amplify the 16S rDNA samples were

forward primer:

-5’-CCGAATTCGTCGACAACAGAGTTTGATCCTGGCTCAG-3’.

and reverse primer:

-5’-CCCGGATCCAAGCTTACGGCTACCTTGTTACGACTT-3’.

**PCR Amplification.**

10x PCR Buffer 5μL

10x dNTP 0.5μL

Forward Primer 1μL

Reverse Primer 1μL

Template 1μL

Taq DNA polymerase 1μL

Sterile distill water up to 50 μL

**PCR**

initial denaturation at 95℃ for 3minutes which was followed with 35-cycle consisting of denaturation at ℃ for 30 seconds, annealing at 60℃ for 30 seconds, and elongation at 72℃ for 2minutes.The reaction was completed with an extension step at 72℃for 5 minutes. All samples were removed from the thermal cycler and stored at −20℃. The PCR product was determined by electrophoresis with loading 8 l of PCR product on to 1% agarose gel.

![](https://static.igem.org/mediawiki/2017/e/e0/T--FAFU-CHINA--week3.jpg)

# 2017.06.18-2017.06.24

Considering stability and efficiency of subsequent genetic tranformantion, we bought strain from microbial and inoculum company and then cultivated the *Bacillus megaterium* strain from first generation to seventh generation on egg medium.

![](https://static.igem.org/mediawiki/2017/6/61/T--FAFU-CHINA--week4.2.png)
![](https://static.igem.org/mediawiki/2017/5/57/T--FAFU-CHINA--week4.1.jpg)

# 2017.06.25-2017.07.01

We continually cultivated the *Bacillus megaterium* strain from seventh generation to fifth generation on egg medium. However, we still didn’t find any signs of degradation of the ability to dissolve phosphorus. So we gave up the screening.

![](https://static.igem.org/mediawiki/2017/4/40/T--FAFU-CHINA--week5.jpg)

# 2017.07.02-2017.07.08

The growth curve of *Bacillus megaterium*  was measured in beef extract peptone with different lead concentration. gradient

![](https://static.igem.org/mediawiki/2017/9/91/T--FAFU-CHINA--week6.jpg)

# 2017.07.09-2017.07.15

The growth curve of *Bacillus megaterium*  was measured in beef extract peptone with different cadmium concentration gradient.

![](https://static.igem.org/mediawiki/2017/9/9a/T--FAFU-CHINA--week7.jpg)

# 2017.07.16-2017.07.22

ALL of our parts were by Genscript Company, The Beijing Genomics Institute (BGI) Company and Fuzhou Sunya Biotechnology Company.2 weeks later, we got two parts.

![](https://static.igem.org/mediawiki/2017/9/9a/T--FAFU-CHINA--week7.jpg)

# 2017.07.23-2017.07.29

# 2017.07.30-2017.08.05

ALL of our parts were by Genscript Company, The Beijing Genomics Institute (BGI) Company and Fuzhou Sunya Biotechnology Company.4 weeks later, we got parts one after another.

# 2017.08.06-2017.08.12

ALL of our parts were by Genscript Company, The Beijing Genomics Institute (BGI) Company and Fuzhou Sunya Biotechnology Company.4 weeks later, we got parts one after another.

# 2017.08.13-2017.08.19

ALL of our parts were by Genscript Company, The Beijing Genomics Institute (BGI) Company and Fuzhou Sunya Biotechnology Company.4 weeks later, we got parts one after another.

# 2017.08.20-2017.08.26

ALL of our parts were by Genscript Company, The Beijing Genomics Institute (BGI) Company and Fuzhou Sunya Biotechnology Company.4 weeks later, we got parts one after another.

# 2017.08.27-2017.09.02

We held CCiC

We planted wild-type tobacco and the southeast Sedum ,then we prepared the barrel, and began to prepare hydroponics experimental supplies. we Determination of Heavy Metals in Fertilizers.

# 2017.09.03-2017.09.09

We held CCiC

We planted wild-type tobacco and the southeast Sedum ,then we prepared the barrel, and began to prepare hydroponics experimental supplies. we Determination of Heavy Metals in Fertilizers.

# 2017.09.10-2017.09.16

We finished interlab work

![](https://static.igem.org/mediawiki/2017/7/72/T--FAFU-CHINA--rtstu.png)

After getting genes, we began to transform them to the Bacillus megaterium.And standardizing

# 2017.09.17-2017.09.23

We finished interlab work

![](https://static.igem.org/mediawiki/2017/7/72/T--FAFU-CHINA--rtstu.png)

After getting genes, we began to transform them to the Bacillus megaterium.And standardizing

# 2017.09.24-2017.09.30

In the two weeks, we carried on the induced expression and SDS-page.

![](https://static.igem.org/mediawiki/2017/b/b7/T--FAFU-CHINA--DSBA1.png)
![](https://static.igem.org/mediawiki/2017/9/90/T--FAFU-CHINA--LPP-1.png)
![](https://static.igem.org/mediawiki/2017/1/16/T--FAFU-CHINA--1GST.png)
![](https://static.igem.org/mediawiki/2017/c/c4/T--FAFU-CHINA--1MCPB.png)

# 2017.10.01-2017.10.07

After the Lpp-OmpA-MBP (Pb) and DsbA-MBP (Pb) were transferred to Bacillus megaterium, and cultured them in beef extract peptone medium with lead + xylose for 24 hours. Then we measured the growth curve.

![](https://static.igem.org/mediawiki/2017/5/59/T--FAFU-CHINA--0006.jpg)

We started preparing the experimental of Pb into wild-type tobacco

After GST-CRS5 was transferred to Bacillus megaterium, we measured the growth curve.

![](https://static.igem.org/mediawiki/2017/8/87/T--FAFU-CHINA--0007.jpg)

We started preparing the experimental of Cd into Sedum alfredii Hance

# 2017.10.08-2017.10.14

We finished the Formal experimental treatment of Pb into wild-type tobacco

# 2017.10.15-2017.10.21

We finished the Formal experimental treatment of Cd into Sedum alfredii Hance and take sample collection and treatment of plant, then analysis data.

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# Team:FAFU-CHINA/Experiments

* [Home](https://2017.igem.org/Team%3AFAFU-CHINA)
* Project
  + [Design](https://2017.igem.org/Team%3AFAFU-CHINA/Design)
  + [Background](https://2017.igem.org/Team%3AFAFU-CHINA/Applied_Design)
  + [Interlab](https://2017.igem.org/Team%3AFAFU-CHINA/InterLab)
  + [Module 1: Phyto-route](https://2017.igem.org/Team%3AFAFU-CHINA/Plant)
  + [Module 2: Metal-trap](#sss2)
  + [Module 3: Safeguard](#sss3)
* Achievements
  + [Demonstrate](https://2017.igem.org/Team%3AFAFU-CHINA/Demonstrate)
  + [Judging Form](https://2017.igem.org/Team%3AFAFU-CHINA/Judging_Form)
* Extras
  + [Collaborations](https://2017.igem.org/Team%3AFAFU-CHINA/Collaborations)
  + [Protocols](https://2017.igem.org/Team%3AFAFU-CHINA/Protocols%20)
  + [Notebook](https://2017.igem.org/Team%3AFAFU-CHINA/Notebook)
* [Parts](https://2017.igem.org/Team%3AFAFU-CHINA/Parts)
* [Model](https://2017.igem.org/Team%3AFAFU-CHINA/Model)
* [Human Practice](https://2017.igem.org/Team%3AFAFU-CHINA/Human_Practices)
* Team
  + [Team members](https://2017.igem.org/Team%3AFAFU-CHINA/Team)
  + [Attributions](https://2017.igem.org/Team%3AFAFU-CHINA/Attributions)

## Module 2: Metal-trap

## Module 3: Safeguard

---

* [Module 1: Phyto-route](https://2017.igem.org/Team%3AFAFU-CHINA/Plant)
* [Module 2: Metal-trap](#sss2)
* [Module 3: Safeguard](#sss3)

![](https://static.igem.org/mediawiki/2017/5/57/T--FAFU-CHINA--week4.1.jpg)

![](https://static.igem.org/mediawiki/2017/b/bb/T--FAFU-CHINA--010.png)

Fig 1 Toggle switch, a mechanism that is triggered if a human operator becomes incapacitated, allows cells to live as long as an operator compound (anhydrotetracycline, ATc) is present.

---

**Biochtainment systems** that couple enviromental sensing with circuit-based control of cell viability could be used to prevent escape of genetically modified microbes into the enviroment. In order to guarantee our chassis’ safety of the application in reality , we have taken a robust and significant strategy, dubbed ‘Toggle Switch’([), based on a circuit which the LacI and TetR transcription factors are reciprocally repressive.](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2384014)

---

## Overview

      For [Human Practice](https://2017.igem.org/Team%3AFAFU-CHINA/HP/Gold_Integrated), we have come up with a common question to President Shi that “If our project needs to be applied in practice, what will be the specific operation processes? And of what aspects shall we be conscious? ”. It is a essential reminder of his answers tha there is the need for constant care.
We decided to customize a ‘kill switch’ as a safeguard for our project, simultaneously harbors a neutral statet that allows billions of microbes to happily thrive and avoid unexpectedly escaping of GEMs into the enviroment and transferring of non-natural plasmid DNA to existing soil bacteria.
‘Toggle Switch’, based on a circuit which the LacI and TetR transcription factors are reciprocally repressive, but in which the expression off TetR is favoured owing to modifications in the strength of the ribosommal binding sites of the two transcription factors. Inhibition of TetR expression by anhydrotetracycline(ATc), a compound that is not normally found in nature, is necessary of expression of LacI. LacI directly inhibits expression of a lethal toxin or indirectly prevents inhibition of the expression of our target genes. Removal of ATc from the enviroment activates the expression of TetR, which leads to cell death.

---

**Reference**

Haynes, K. A. (2016). Synthetic biology: Building genetic containment. Nature Publishing Group, 12(2), 55–56. https://doi.org/10.1038/nchembio.2004

Osório, J. (2015). Genetic kill switches — a matter of life or death. Nature Publishing Group, 1979(December), 2015. https://doi.org/10.1038/nrg.2015.29

Chan, C. T. Y., Lee, J. W., Cameron, D. E., Bashor, C. J., & Collins, J. J. (2015). for bacterial containment. Nature Chemical Biology, (December), 1–7. https://doi.org/10.1038/nchembio.1979

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# Team:FAFU-CHINA/Results

# Results

Here you can describe the results of your project and your future plans.

##### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

##### You should also describe what your results mean:

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember all measurement and characterization data must be on part pages in the Registry.
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

##### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

##### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)

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