iGEM 2018 — Team NKU_CHINA

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# Team:NKU CHINA/Notebook

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  + [Lab Notes](https://2018.igem.org/Team%3ANKU_CHINA/Notebook)
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# Lab Notes

## Dry Lab

## Wet Lab

![](https://static.igem.org/mediawiki/2018/8/84/T--NKU_CHINA--lab1.png)

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![](https://static.igem.org/mediawiki/2018/7/7e/T--NKU_CHINA--lab2.png)

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![](https://static.igem.org/mediawiki/2018/4/45/T--NKU_CHINA--lab3.png)

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![](https://static.igem.org/mediawiki/2018/6/68/T--NKU_CHINA--lab4.png)

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![](https://static.igem.org/mediawiki/2018/5/5e/T--NKU_CHINA--lab5.png)

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![](https://static.igem.org/mediawiki/2018/c/cc/T--NKU_CHINA--lab6.png)

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![](https://static.igem.org/mediawiki/2018/f/f3/T--NKU_CHINA--lab7.png)

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![](https://static.igem.org/mediawiki/2018/d/dc/T--NKU_CHINA--lab8.png)

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## 2018 Mar. 18th-June. 30th

Wiki: Learning the HyperText Markup Language (HTML) and Cascading Style Sheets (CSS). Learning how to use Javascript and jQuery to add animations to the webpage and the usage of Bootstrap.

HP: Participating in Lotus Festival in Nankai University, propagating biosynthesis and iGEM

## 2018 Mar. 18th-Apr. 7th

Construction of vector P*gltAB*(LL3)-GFP；construction of pHT01 without P*lacI*

Preparation of competent cells of LL3/168/JM110.

Construction of vector LL3-H.

[More details](#first)

## 2018 July. 1st-July. 28th

Model: Modeling the process of partI using Logistic formula and Luedeking-Pieret formula.

Wiki: Designing the page of InterLab and the Home page.

HP: Holding iGEM Summit in China, communicating with another nine teams

## 2018 Mar. 25th-May. 5th

Construction of *lacI* with different promoters including P*gltAB*-*lacI*, P*gltX*-*lacI*, P*accd*-*lacI*, P*ldh*-*lacI*, P*rps*-*lacI*, P*suc*-*lacI*.

Transformation of LL3 genome (Silent the P*gltAB* funtion-LL3 HPT)

[More details](#second)

## 2018 July. 29th-Aug. 18th

Wiki: Building the introduction page of our team and all of the Human\_Practices pages.

Wiki: Building the introduction page of our team and all of the Human\_Practices pages.

Art: Designing team logo, characters, story. Drawing up the experiment flow chart. Making a folding of our project for Human practice group to conduct propaganda. Designing and ordering our team T-shirt. Making poster and PPT for CCiC.

HP: Social practice in Yinchuan, Ningxia to investigate biology education in western China. Conducting questionnaire survey to investigate the public awareness and attitude towards our project.

## 2018 Apr. 22th-July. 14th

Genome editing--adding P*ahp* ahead of *gltAB* and adding a terminater ahead of P*ahp* to eliminate the effect of intrinsic P*gltAB* to *gltAB*.

Construction of pHT01: P*gltAB*-LacI-P*lacO*-TetA by inserting fused P*lacO*-*tetA*, then tried the rest cnstruction with different promoters.

Preparation of interlab protocol and did experiment.

Transformation of LL3 genome (Silent the P*gltAB* funtion); Construction of vector 168H; Manufacture of LL3(HPT) competent cells.

[More details](#third)

## 2018 Aug. 12th-Sept. 1st

Model: Modeling part3 using partition function.

Wiki: Finishing the design of the Attribution and Safety page.

Art: Collecting ideas and pictures from experimental group and prepare for CCiC.Complete poster and PPT for CCiC.

HP: Conducting questionnaire survey to investigate the public awareness and attitude towards our project. Consulting in Tianjin Institute of Industrial Biotechnology and Tianjin International Joint Acadmy Biomedicine,and discuss with another iGEM team NPU-China. Attending the CCiC conference.

## 2018 July. 8th-July. 28th

Submission of interlab data and writing wiki page for interlab.

Construction of vector 168HPT.

Double changed of *icd* in LL3.

[More details](#fourth)

## 2018 Sept. 2nd-Sept. 15th

Model: Using flow cytometry to test GFP distribution type and analyse the result.

Wiki: Collecting the content and pictures of the Project pages including the Description, Experiments, Model, Proof of Concept and Demonstrate pages.

Art: Helping wiki group to establish website. Designing our team costume ——Jacket, refrigerator magnet of our characters and logo.

HP: Holding a science popularization activity for senior high school students with another two teams in Tianjin No. 13 Middle School.

## 2018 July. 15th-Aug. 18th

Competence cell of LL3 and *icd* double-changed LL3.

Construction of pHT01: P*lacO*-TetA-LacI.

Construction of pHT01: P*lacO*-TetA-promoter-LacI, the promoter includes *ugt, ldh, gltAB, gltX, accd, arg.*

Transformation of 168 genome (Silent the P*gltAB* funtion).

[More details](#fifth)

## 2018 Sept. 16th-Sept. 29th

Wiki: Finishing designing the Collaboration page and Parts page.

Art: Preparing for team video, collect pictures and record voice-over. Designing and upload team banner.

HP: Participating in the iGEM league conference in Beijing-Tianjin region. Visiting a famous biotech company named Novozymes and communicating with their engineers.

## 2018 Aug. 19th-Sept. 8th

Construction of pHT01: P*gltAB*-LacI-P*grac*-TetA, pHT01: P*gltAB*-LacI-P*grac*-TetA-GFP.

Construction of vector P*gltAB* (168).

[More details](#sixth)

## 2018 Sept. 23th-Oct. 6th

Model: Predict the total γ-PGA content by conbining part1 and part3 and write down the whole model part.

Wiki: Finishing designing the Collaboration page and Parts page. Finding bugs of our wiki and solving them. Designing the final two pages— Lab Notes and Protocols.

Art: Completing the motion graph team video

HP: Visiting a famous biotech company named Novozymes and communicating with their engineers.

## 2018 Sept. 9th-Sept. 29th

Transformation of pHT01: P*gltAB*-LacI-P*grac*-TetA into LL3,measurement of its γ-PGA yield.

Construction of standard parts vectors: P*gltAB*-LacI-P*grac*-TetA; *lacI*; P*gltAB*; P*grac*; *tetA*; GFP; P*gltAB*-GFP; P*liaG*&43; P*liaG*&43-1; P*liaG*&43-2; P*liaG*&43-3(Unsuccess); *tetA* Optimized.

[More details](#seventh)

## 2018 Oct. 7th-Oct. 17th

Wiki: Modulating and modifying the wiki to make it better and run smoothly.

Art: Modifying and beautifying our image, typography and Experimental designs. Helping our wiki group complete the website.Make our team poster and PPT for presentation.

## 2018 Sept. 30th-Oct. 17th

Fermentation of P43、P*glt* for measuring their OD600 and the fluorescence of GFP.

Measurement of the OD600 and FI of mCherry and GFP and qPCR in P*gltAB*-LacI-mCherry-GFP LL3 per 8 hours of 48 hours.

Construction of pHT01-P43-TetA (condon optimized) and pHT01-P43-TetA (kit) and then testing the fuction of the codon optimized one.

[More details](#eighth)

![](https://static.igem.org/mediawiki/2018/0/0b/T--NKU_CHINA--duihui_final.png)

### Contacts

### Address

Nankai University
No.94 Weijin Road, Nankai District
Tianjin, P.R.China 300071

Copyright © Team NKU\_CHINA 2018

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# Team:NKU CHINA/Experiments

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* [HP](https://2018.igem.org/Team%3ANKU_CHINA/Human_Practices "Human Practices")
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* Parts
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* [InterLab](https://2018.igem.org/Team%3ANKU_CHINA/InterLab)
* Notebook
  + [Lab Notes](https://2018.igem.org/Team%3ANKU_CHINA/Notebook)
  + [Protocols](https://2018.igem.org/Team%3ANKU_CHINA/Protocols)

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# Experiments

## Part 0 Construction of High-glutamate-yield *B. amyloliquefaciens* LL3

In order to get a *Bacillus amyloliquefaciens* LL3 strain for a higher glutamate yield, we edited the genome by constructing promoters in tandem to overexpress glutamate related genes, *icd* and *gltAB*, which are responsible for 2-ketoglutarate and glutamate, separately. Due to the difficulties of genome manipulation, we only succeeded in inserting P*c2up* ahead of *icd* and get a high yield strain.

## Part 1 Verification of P*gltAB* Responding with Glutamate and GltC

In *Bacillus amyloliquefaciens* LL3, there is a *glt* operon which is responsible for the intracellular glutamate synthesis. Since we need to use the regulatory role of glutamate and GltC to P*gltAB*, we need to verify the effect of promoter P*gltAB* under different glutamate concentrations. So we constructed pHT01-P*gltAB*-GFP and pHT01-P43-GFP 2 plasmids and transformed them into *Bacillus amyloliquefaciens* LL3 *icd* strain. We fermented under different glutamate concentrations and tested the fluorescence intensity(FI) of GFP every 6 hours in M9 medium. For the strain transformed with pHT01-P*gltAB*-GFP, as the glutamate concentration goes up, the FI of GFP first went up and came down. On the other hand, for the strain transformed with pHT01-P43-GFP, FI is uncorrelated to the glutamate concentration.

## Part 2 Verification of the Repression Effect of LacI to P*grac*

To make our circuit work, we need to test the inhibition effect to P*grac* which can respond to LacI protein at different *lacI* expression levels. So we expected to construct vectors pHT01-LacI-Pdifferent-P*grac*-TetA.

## Part 3 Constrction of the Whole PopQC System and Functional Test

Our project is aiming to construct a PopQC system in a single plasmid. So we construct the vector pHT01-LacI-P*gltAB*-P*grac*-TetA and ferment to test the function of our system. To furthermore simplify the procedures of functional test, we constructed another vector pHT01-mCherry-LacI-P*gltAB*-P*grac*-TetA-GFP(int) to test the fluorescence intensity instead of mRNA. The results are roughly in accordance with our expectation.

## Parts Creation

We constructed 12 standard parts, 1. P*gltAB*-LacI-P*grac*-TetA (BBa\_K2705006); 2. LacI(BBa\_K2705001); 3. P*gltAB*(BBa\_K2705000); 4. P*grac*(BBa\_K2705002); 5. TetA(BBa\_K2705003); 6. GFP(BBa\_K2705004) 7. P*gltAB*-GFP(BBa\_K2705005); 8. PliaG&43(BBa\_K2705008); 9. PliaG&43-1(BBa\_K2705009); 10. PliaG&43-2(BBa\_K2705010); 11. PliaG&43-3(BBa\_K2705011); 12. TetA(optimized)(BBa\_K2705003). The promoter P*gltAB*, P*grac*, *lacI*, *tetA*, and *gfp* genes are cloned by PCR. Then both these segments and pSB1C3 vector were treated with restriction enzymes. After that, these parts were separately ligated to linear pSB1C3 by ligase or recombinase, and the products were separately transformed into *E.coli* JM110. Verification PCR was performed to select the positive clones. The positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced.

## Parts Improvements

We improved 2 parts, BBa\_K823000 and BBa\_J31006. For BBa\_K823000 the weak, constitutive promoter from *Bacillus subtilis*, we chose to mimic the structure of P*43*, the relatively strong constitutive promoter which can be recognized by both sigma factor 55 (the major sigma factor A) and sigma factor 37 (the lag phase sigma factor B). After designing the new sequences of promoters, we send them to the company GenScript Biotech Corp. to have them synthesized. The P*liaG&43*-0, P*liaG&43*-1, P*liaG&43*-2, P*liaG&43*-3 are cloned by PCR. Then both these segments and pHT01-GFP vector were treated with restriction enzymes. After that, these parts were separately ligated to linear pHT01-GFP by recombinase, and the products were separately transformed into *E.coli* JM110. Verification PCR was performed to select the positive clones. The positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced. Object plasmids are transformed into *Bacillus subtilis* 168 by electroporation transformation and verification PCR was performed. After that, we use the Microplate Reader to analyze the FI of the cultures after 2 hours. For BBa\_J31006, we send the sequence to the company GenScript Biotech Corp. and get a codon optimized one for *Bacillus subtilis*. And then we constructed pHT01-P43-TetA plasmid and transformed into *Bacillus subtilis* LL3 to test its function by measuring the MIC.

![](https://static.igem.org/mediawiki/2018/0/0b/T--NKU_CHINA--duihui_final.png)

### Contacts

### Address

Nankai University
No.94 Weijin Road, Nankai District
Tianjin, P.R.China 300071

Copyright © Team NKU\_CHINA 2018

[Top](#0)

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# Team:NKU CHINA/Results

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TEAM

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

PROJECT

[Description](https://2018.igem.org/Team%3ANKU_CHINA/Description)
[Design](https://2018.igem.org/Team%3ANKU_CHINA/Design)
[Experiments](https://2018.igem.org/Team%3ANKU_CHINA/Experiments)
[Notebook](https://2018.igem.org/Team%3ANKU_CHINA/Notebook)
[InterLab](https://2018.igem.org/Team%3ANKU_CHINA/InterLab)
[Model](https://2018.igem.org/Team%3ANKU_CHINA/Model)
[Results](https://2018.igem.org/Team%3ANKU_CHINA/Results)
[Demonstrate](https://2018.igem.org/Team%3ANKU_CHINA/Demonstrate)
[Improve](https://2018.igem.org/Team%3ANKU_CHINA/Improve)
[Attributions](https://2018.igem.org/Team%3ANKU_CHINA/Attributions)

PARTS

[Parts Overview](https://2018.igem.org/Team%3ANKU_CHINA/Parts)
[Basic Parts](https://2018.igem.org/Team%3ANKU_CHINA/Basic_Part)
[Composite Parts](https://2018.igem.org/Team%3ANKU_CHINA/Composite_Part)
[Part Collection](https://2018.igem.org/Team%3ANKU_CHINA/Part_Collection)

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

HUMAN PRACTICES

[Human Practices](https://2018.igem.org/Team%3ANKU_CHINA/Human_Practices)
[Education & Engagement](https://2018.igem.org/Team%3ANKU_CHINA/Public_Engagement)

AWARDS

[Applied Design](https://2018.igem.org/Team%3ANKU_CHINA/Applied_Design)
[Entrepreneurship](https://2018.igem.org/Team%3ANKU_CHINA/Entrepreneurship)
[Hardware](https://2018.igem.org/Team%3ANKU_CHINA/Hardware)
[Measurement](https://2018.igem.org/Team%3ANKU_CHINA/Measurement)
[Model](https://2018.igem.org/Team%3ANKU_CHINA/Model)
[Plant](https://2018.igem.org/Team%3ANKU_CHINA/Plant)
[Software](https://2018.igem.org/Team%3ANKU_CHINA/Software)

[JUDGING FORM ⇗](https://igem.org/2018_Judging_Form?team=NKU_CHINA)

# 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.

### 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)