iGEM 2018 — Team Peking

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# Team:Peking/Notebook

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* [Home](https://2018.igem.org/Team%3APeking)
* Project
  + [Description](https://2018.igem.org/Team%3APeking/Project)
  + [Design](https://2018.igem.org/Team%3APeking/Design)
  + [Demonstration](https://2018.igem.org/Team%3APeking/Demonstrate)
  + [Perspective](https://2018.igem.org/Team%3APeking/Perspective)
* [Modeling](https://2018.igem.org/Team%3APeking/Model)
* [Software](https://2018.igem.org/Team%3APeking/Software)
* [Human Practices](https://2018.igem.org/Team%3APeking/Human_Practices)
* Achievement
  + [Judging Form](https://2018.igem.org/Team%3APeking/Judging_Form)
  + [Parts](https://2018.igem.org/Team%3APeking/Parts)
  + [Improvement](https://2018.igem.org/Team%3APeking/Improve)
  + [InterLab](https://2018.igem.org/Team%3APeking/InterLab)
* Lab
  + [Team Members](https://2018.igem.org/Team%3APeking/Team_Members)
  + [Attributions](https://2018.igem.org/Team%3APeking/Attributions)
  + [Notebook](https://2018.igem.org/Team%3APeking/Notebook)
  + [Collaborations](https://2018.igem.org/Team%3APeking/Collaborations)
  + [Safety](https://2018.igem.org/Team%3APeking/Safety)
  + [Acknowledgement](https://2018.igem.org/Team%3APeking/Acknowledgement)

# Notebook

#### [TEAM MEMBERS](https://2018.igem.org/Team%3APeking/Team_Members)

#### [ATTRIBUTIONS](https://2018.igem.org/Team%3APeking/Attributions)

#### [NOTEBOOK](https://2018.igem.org/Team%3APeking/Notebook)

#### [COLLABORATIONS](https://2018.igem.org/Team%3APeking/Collaborations)

#### [SAFETY](https://2018.igem.org/Team%3APeking/Safety)

#### [ACKNOWLEDGEMENT](https://2018.igem.org/Team%3APeking/Acknowledgement)

Protocol

---

[Carotene Extraction Protocol.pdf](https://static.igem.org/mediawiki/2018/5/57/T--Peking--Carotene_Extraction_Protocol.pdf)
[Carotene Identification Protocol.pdf](https://static.igem.org/mediawiki/2018/7/7e/T--Peking--Carotene_Identify_Protocol.pdf)
[Carotene Production Protocol.pdf](https://static.igem.org/mediawiki/2018/2/24/T--Peking--Carotene_Production_Protocol.pdf)
[Molecular Cloning Plasmid extraction/DNA electrophoresis/DNA clean.pdf](https://static.igem.org/mediawiki/2018/c/cd/T--Peking--Molecular_Cloning_%28Plasmid_extraction_DNA_electrophoresis_DNA_clean%29.pdf)
[Molecular Cloning Protocol DNA recombination.pdf](https://static.igem.org/mediawiki/2018/4/49/T--Peking--Molecular_Cloning_Protocol_%28DNA_recombanation%29.pdf)
[Cloning Protocol E.coli transformation.pdf](https://static.igem.org/mediawiki/2018/9/9d/T--Peking--Molecular_Cloning_Protocol_%28E_coli_transformation%29.pdf)
[Molecular Cloning Protocol PCR.pdf](https://static.igem.org/mediawiki/2018/1/11/T--Peking--Molecular_Cloning_Protocol_%28PCR%29.pdf)
[Yeast Transformation Protocol.pdf](https://static.igem.org/mediawiki/2018/c/cd/T--Peking--Yeast_Transformation_Protocol.pdf)
[Yeast Dry Weight and OD Measurement Protocol.pdf](https://static.igem.org/mediawiki/2018/9/96/T--Peking--yeastweighprotocol.pdf)

Diary

---

0

### 2018.5.13

Confirmed project plan.

Prepared experiment equipment and reagents.

1

### 2018.5.14-2018.5.20

Molecular cloning test.
Yeast transformation test.

2

### 2018.5.21-2018.5.27

Modeling beginning.

3

### 2018.5.28-2018.6.3

Plasmid backbone propagation：
*E. coli* expression: pET-28a
yeast expression: pNH603, pNH604, pNH605, pNH606
Required gene segment synthesis beginning.

4

### 2018.6.4-2018.6.10

Gibson assembly, golden gate assembly, and other seamless cloning test.
IPTG induced expression test.

5

### 2018.6.11-2018.6.17

Restriction recombination test.
Began to construct SUMO-SIM system.

6

### 2018.6.18-2018.6.24

Construction *E. coli* expression plasmid：
pET-28a-FKBP-yEGFP-HOTag3
pET-28a-Frb-yEGFP-HOTag6

7

### 2018.6.25-2018.7.1

Fluorescence observation test.

8

### 2018.7.2-2018.7.8

Joined iGEM conference at Nankai University held by NKU\_CHINA.

9

### 2018.7.9-2018.7.15

Construction yeast transformation plasmid of carotene synthesis:
pNH603-crtE/crtI/crtYB
pNH604-crtE/crtI/crtYB
pNH605-crtE/crtI/crtYB
pNH606-crtE/crtI/crtYB

10

### 2018.7.16-2018.7.22

Meeting up with Fudan-CHINA.
Insert 4 kinds of promoters PDH3, TEF1, Ura3, Tet07 into 4 kinds of yeast transformation plasmids pNH603, pNH604, pNH605, pNH606.

11

### 2018.7.23-2018.7.29

Construction yeast transformation plasmids of SPOT:
pNH604-PDH3/TEF1/Ura3/Tet07-FKBP-yEGFP-HOTag3
pNH605-PDH3/TEF1/Ura3/Tet07-Frb-yEGFP-HOTag6
pNH604-PDH3/TEF1/Ura3/Tet07-SUMO-yEGFP-HOTag3
pNH605-PDH3/TEF1/Ura3/Tet07-SIM-yEGFP-HOTag6

12

### 2018.7.30-2018.8.5

Transferred FKBP-Frb SPOT system and SUMO-SIM SPOT system into yeast.
Nucleic localization test.
Fluorescence microscope observation and flow cytometry measurement of transformed yeast cell lines.

13

### 2018.8.6-2018.8.12

Rapamycin induction concentration test of FKBP-Frb SPOT system.

Construction yeast transformation plasmids of carotene production:
pNH603-PDH3/TEF1/Ura3/Tet07-Frb-crtI-HOTag6
pNH605-PDH3/TEF1/Ura3/Tet07-Frb-crtE-HOTag6
pNH606-PDH3/TEF1/Ura3/Tet07-Frb-crtYB-HOTag6

Construction yeast transformation plasmids of light induced SPOT system:

pNH604-PDH3/TEF1/Ura3/Tet07-phyB-yEGFP-HOTag3
pNH605-PDH3/TEF1/Ura3/Tet07-PIF-yEGFP-HOTag6
pNH604-PDH3/TEF1/Ura3/Tet07-VVDn-yEGFP-HOTag3
pNH605-PDH3/TEF1/Ura3/Tet07-VVDp-yEGFP-HOTag6

14

### 2018.8.13-2018.8.19

Carotene production and extraction test.

Construction yeast transformation plasmids of carotene production with SPOT:
pNH603--PDH3/TEF1/Ura3/Tet07-Frb-crtI-HOTag6
pNH605--PDH3/TEF1/Ura3/Tet07-Frb-crtE-HOTag6
pNH606--PDH3/TEF1/Ura3/Tet07-Frb-crtYB-HOTag6
pNH603--PDH3/TEF1/Ura3/Tet07-SIM-crtI-HOTag6
pNH605--PDH3/TEF1/Ura3/Tet07-SIM-crtE-HOTag6
pNH606--PDH3/TEF1/Ura3/Tet07-SIM-crtYB-HOTag6

15

### 2018.8.20-2018.8.26

Carotene production and extraction test.

Construction yeast transformation plasmids of carotene production with SPOT:
pNH603--PDH3/TEF1/Ura3/Tet07-Frb-crtI-HOTag6
pNH605--PDH3/TEF1/Ura3/Tet07-Frb-crtE-HOTag6
pNH606--PDH3/TEF1/Ura3/Tet07-Frb-crtYB-HOTag6
pNH603--PDH3/TEF1/Ura3/Tet07-SIM-crtI-HOTag6
pNH605--PDH3/TEF1/Ura3/Tet07-SIM-crtE-HOTag6
pNH606--PDH3/TEF1/Ura3/Tet07-SIM-crtYB-HOTag6

16

### 2018.8.27-2018.9.2

Carotene production and extraction test.

Construction the control group plasmids of SPOT:

pNH604-PDH3/TEF1/Ura3/Tet07-FKBP-yEGFP
pNH605-PDH3/TEF1/Ura3/Tet07-Frb-yEGFP
pNH604-PDH3/TEF1/Ura3/Tet07-SUMO-yEGFP
pNH605-PDH3/TEF1/Ura3/Tet07-SIM-yEGFP
pNH604-PDH3/TEF1/Ura3/Tet07-FKBP-mCherry
pNH605-PDH3/TEF1/Ura3/Tet07-Frb-mCherry
pNH604-PDH3/TEF1/Ura3/Tet07-SUMO-mCherry
pNH605-PDH3/TEF1/Ura3/Tet07-SIM-mCherry
pNH603-H2A-CFP

Joined the 5th CCiC at ShanghaiTech.

17

### 2018.9.3-2018.9.9

Growth curve measurement of the SPOT system yeast.
Characterization of the carotene production by SPOT system.

18

### 2018.9.10-2018.9.16

The ability of phase separation induced by rapamycin in FKBP-Frb SPOT system test and longer time observation.
Yeast fermentation test.

19

### 2018.9.17-2018.9.23

Completed the two kinds of fluorescence repeat in both FKBP-Frb and SUMO-SIM SPOT system.
Carotene production and quantification test.

Construction yeast transformation plasmids of ABA induced SPOT system:
pNH604-PDH3/TEF1/Ura3/Tet07-ABI-yEGFP-HOTag3
pNH605-PDH3/TEF1/Ura3/Tet07-PYL1-yEGFP-HOTag6

Construction yeast transformation plasmids of GA induced SPOT system:
pNH604-PDH3/TEF1/Ura3/Tet07-GAI-yEGFP-HOTag3
pNH605-PDH3/TEF1/Ura3/Tet07-GID-yEGFP-HOTag6

20

### 2018.9.24-2018.9.30

FRAP test of FKBP-Frb SPOT system.
BioBrick submitted bioparts construction.

21

### 2018.10.1-2018.10.7

Construction yeast transformation plasmids of GFP nanobody SPOT system:
pNH605-PDH3/TEF1/Ura3/Tet07-Frb-GFPnanobody-HOTag3

Construction yeast transformation plasmids of NAD sensor SPOT system:
pNH604-PDH3/TEF1/Ura3/Tet07-NAD1A-yEGFP-HOTag3
pNH605-PDH3/TEF1/Ura3/Tet07-NAD1B-yEGFP-HOTag6

22

### 2018.10.8-2018.10.14

Data collection and modeling processing.
HPLC quantification of carotene.

23

### 2018.10.15-2018.10.17

WIKI and poster making.
Presentation preparation.

* [![](https://static.igem.org/mediawiki/2018/7/7a/T--Peking--images_PKU_Administration_logo.png)](http://dean.pku.edu.cn/pkudean/index.html)
* [![](https://static.igem.org/mediawiki/2018/0/04/T--Peking--images_PKU_SLS_logo.png)](http://www.bio.pku.edu.cn/)
* [![](https://static.igem.org/mediawiki/2018/e/e1/T--Peking--images_PKU_CQB_logo.png)](http://cqb.pku.edu.cn/en/)

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# Team:Peking/Experiments

[**Peking iGEM 2018**](https://2018.igem.org/Team%3APeking)
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[Home](https://2018.igem.org/Team%3APeking)

[Project](https://2018.igem.org/Team%3APeking/Project)

[Description](https://2018.igem.org/Team%3APeking/Description)
[Design](https://2018.igem.org/Team%3APeking/Design)
[Demonstrate](https://2018.igem.org/Team%3APeking/Demonstrate)
[Future Plan](https://2018.igem.org/Team%3APeking/Future_Plan)

[Data Page](https://2018.igem.org/Team%3APeking/Results)

[Phase Separation](https://2018.igem.org/Team%3APeking/Phase_Separation_D)
[Function](https://2018.igem.org/Team%3APeking/Function_D)

[Model](https://2018.igem.org/Team%3APeking/Model)

[Phase Separation](https://2018.igem.org/Team%3APeking/Phase_Separation_M)
[Function](https://2018.igem.org/Team%3APeking/Function_M)

[Software](https://2018.igem.org/Team%3APeking/Software)

[Human Practices](https://2018.igem.org/Team%3APeking/Human_Practices)

[Human Practices](https://2018.igem.org/Team%3APeking/HumanPractices)
[Public Engagement](https://2018.igem.org/Team%3APeking/Public_Engagement)

[**Team**](https://2018.igem.org/Team%3APeking/Team)

[Team Members](https://2018.igem.org/Team%3APeking/Team)
[Attributions](https://2018.igem.org/Team%3APeking/Attributions)
[Safety](https://2018.igem.org/Team%3APeking/Safety)
[Notebook](https://2018.igem.org/Team%3APeking/Notebook)
[Experiments](https://2018.igem.org/Team%3APeking/Experiments)

[Collaborations](https://2018.igem.org/Team%3APeking/Collaborations)

[Acknowledgement](https://2018.igem.org/Team%3APeking/Acknowledgement)
[InterLab](https://2018.igem.org/Team%3APeking/InterLab)

[Parts](https://2018.igem.org/Team%3APeking/Parts)

[Improve](https://2018.igem.org/Team%3APeking/Improve)

* [**Team**](https://2018.igem.org/Team%3APeking/Team)

* [◊   Team Members](https://2018.igem.org/Team%3APeking/Team)* * [◊   Attributions](https://2018.igem.org/Team%3APeking/Attributions)* * [◊   Safety](https://2018.igem.org/Team%3APeking/Safety)* * [◊   Notebook](https://2018.igem.org/Team%3APeking/Notebook)* * [>   Experiments](https://2018.igem.org/Team%3APeking/Experiments)

Welcome to our wiki!

Experiments

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

# Team:Peking/Results

[![](https://static.igem.org/mediawiki/2018/e/e2/T--Peking--_toolbar.jpeg)](https://2018.igem.org/Team%3APeking)

* Project
  + [Project](https://2018.igem.org/Team%3APeking/Project)
  + [Description](https://2018.igem.org/Team%3APeking/Description)
  + [Design](https://2018.igem.org/Team%3APeking/Design)
  + [Demonstration](https://2018.igem.org/Team%3APeking/Demonstrate)
  + [Future Plan](https://2018.igem.org/Team%3APeking/Future_Plan)
* Data Page
  + [Data Page](https://2018.igem.org/Team%3APeking/Results)
  + [Phase Separation](https://2018.igem.org/Team%3APeking/Phase_Separation_D)
  + [Functions](https://2018.igem.org/Team%3APeking/Functions_D)
* Model
  + [Model](https://2018.igem.org/Team%3APeking/Model)
  + [Phase Separation](https://2018.igem.org/Team%3APeking/Phase_Separation_M)
  + [Functions](https://2018.igem.org/Team%3APeking/Functions_M)
* [Software](https://2018.igem.org/Team%3APeking/Software)
* Human Practices
  + [Human Practices](https://2018.igem.org/Team%3APeking/Human_Practices/Gold)
  + [Public Engagement](https://2018.igem.org/Team%3APeking/Public_Engagement)
* Team
  + [Team Members](https://2018.igem.org/Team%3APeking/Team_Members)
  + [Attributions](https://2018.igem.org/Team%3APeking/Attributions)
  + [Safety](https://2018.igem.org/Team%3APeking/Safety)
  + [Notebook](https://2018.igem.org/Team%3APeking/Notebook)
* Collaborations
  + [Collaborations](https://2018.igem.org/Team%3APeking/Collaborations)
  + [Acknowledgement](https://2018.igem.org/Team%3APeking/Acknowledgement)
  + [InterLab](https://2018.igem.org/Team%3APeking/InterLab)
* Parts
  + [Parts](https://2018.igem.org/Team%3APeking/Parts)
  + [Favorite Parts](https://2018.igem.org/Team%3APeking/Favorite_Parts)
  + [Part Table](https://2018.igem.org/Team%3APeking/Part_Table)
  + [Part Collection](https://2018.igem.org/Team%3APeking/Part_Collection)

# Results

In this section, you could find what we achieved during this summer. For detailed lab results, models or human practices, please use the navigation bar for a quick webpage-redirecting.

#### To A

#### To B

#### To C

Project Achievements

1.

Constructed a multifunctional protein of interest via molecular biological methods, and introduced the constructed plasmids into the engineered bacteria for protein expression. (Learn more)

2.

Searched for methods and the best conditions for the extraction of each protein. (Learn more)

3.

Demonstrated a quick and stable crosslinking process of Triple SpyTag-SUP and Triple SpyTag-mSA with Triple SpyCatcher via covalent bonds. We also optimized this reaction concerning the relevant parameters such as temperature, pH, etc.. (Learn more)

4.

Demonstrated effective adsorption of uranyl ions by monomeric Triple SpyTag-SUP or polymer network containing the SUP module under a number of conditions. The adsorption was highly efficient and fast, not only under experimental conditions but also in simulated seawater or lake water containing uranium pollution. (Learn more)

5.

Attached biotin to amino-coated magnetic beads and achieved clearance of the polymer network formed via the crosslinking of Triple SpyTag-SUP and Triple SpyTag-mSA with Triple SpyCatcher with a magnet. (Learn more)

6.

Set up a signal peptide library and screened for optimally suited signal peptides in order to efficiently secrete the proteins of interest. We found two signal peptides of high efficiency - those derived from OmpA and LtIIb. (Learn more)

7.

Used all the above-mentioned experiments together to demonstrate that the complete Uranium Reaper system, consisting of Triple SpyTag-SUP, Triple SpyTag-mSA, Triple SpyCatcher and biotin-coated magnetic beads, could effectively handle uranium pollution under simulated real-life conditions in about 2 hours. We aimed to optimize this strategy and hoped it could be implemented as a uranyl removal kit. (Learn more)

8.

We exchanged the Triple SpyTag-SUP monomer for Triple SpyTag-LBP or Triple SpyTag-CBP, and tried using the same strategy to adsorb lead and cadmium. The results were remarkable, clearly demonstrating that the Uranium Reaper strategy has much potential to be expanded to other heavy metals. (Learn more)

![](https://static.igem.org/mediawiki/2016/a/af/T--Peking--image_demotable1.png)

Even though the efficiency of the Uranium Reaper system may be somewhat lower than current methods, it could certainly be optimized through further development work. Importantly, Uranium Reaper is much better in other aspects. In the future, we plan to optimize the entire Uranium Reaper strategy in order to enhance the adsorption efficiency.

For an overall view of our project, please redirect to Overview Page or Design Page. Links to detailed lab results could also be found on these pages.

Beyond Experiment

1.

We submitted 53 high-quality and well-characterized Standard BioBricks, including a set of derivatives of Triple SpyTag and Triple SpyCatcher, such as the Triple SpyTag-SUP and Triple SpyTag-mSA. (Learn more)

2.

We developed a special software which could be used to calculate the molecular weight distribution of protein polymers using Flory’s theory. The results of testing have demonstrated that the software is accurate and useful. (Learn more)

3.

We visited experts from the College of Chemistry and Molecular Engineering and School of Physics of Peking University, respectively, to learn about the current situation surrounding uranium pollution in the real world and how people could control the situation. After finishing the main work, we presented them with the achievements of the project and got their feedback. (Learn more)

4.

We did an interview with the Hunan Nuclear Geology 311 Brigade and gained thorough insights into the treatment of uranyl pollution used by the people on the firing line. This way we could compare the methods they were using with the Uranium Reaper strategy. (Learn more)

5.

We helped and collaborated with other iGEM teams by guiding a new team (BHU\_China), as well as discussing about project design and technical skills and sharing DNA materials (OUC-China, BIT-China, Tianjin, UCAS, Jinlin\_China and BNU-China). (Learn more)

6.

We attended the CCiC (Central China iGEM Consortium), which is a large-scale competition-free jamboree of about 50 teams, providing participants with an opportunity for meaningful exchanges of ideas and problem solving. (Learn more)

Our future plan

1.

We should reproduce all of the experiments that we have done this summer to make sure the results are credible.

2.

We will optimize the whole strategy to enhance the adsorption efficiency by changing pH, temperature, reaction time of crosslinking and clearance. (The efficiency is only about 60% without further optimization)

3.

According to the results for the adsorption of 13nM uranyl, the polymer network exhibited a good ability in a simulated seawater environment. We could thus also look into other usage scenarios of Uranium Reaper, such as bio-mining and uranium enrichment.

4.

Exchange of the SUP module for other functional proteins. For example, we could integrate proteins which could bind other heavy metals such as mercury so that the polymer network could be used to treat other kinds of pollution as well.

5.

We could assemble enzyme systems behind the SpyTag backbone to create a production plant in vitro. In the protein polymeric network, the concentration of enzymes could be increased and the efficiency of biocatalysis may consequently also be enhanced.

6.

If we optimize the number of SpyTag or SpyCatcher modules per protein monomer, as well as the working concentrations of proteins, we may make protein-3D printing using the Spy Crosslinking Network come true.

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