iGEM 2021 — Team NWU-CHINA-A

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:NWU-CHINA-A/Notebook

响应式导航

响应式导航

[![](https://static.igem.org/mediawiki/2021/4/48/T--NWU-CHINA-A--logo.png)](https://2021.igem.org/Team%3ANWU-CHINA-A)
[![](https://static.igem.org/mediawiki/2021/4/48/T--NWU-CHINA-A--logo.png)](https://2021.igem.org/Team%3ANWU-CHINA-A)

* [HOME](https://2021.igem.org/Team%3ANWU-CHINA-A)
* PROJECT

  [DESIGN](https://2021.igem.org/Team%3ANWU-CHINA-A/Design)
  [DESCRIPTION](https://2021.igem.org/Team%3ANWU-CHINA-A/Description)
  [EXPERIMENTS](https://2021.igem.org/Team%3ANWU-CHINA-A/Experiments)
  [IMPLEMENTATION](https://2021.igem.org/Team%3ANWU-CHINA-A/Implementation)
  [PROOF OF CONCEPT](https://2021.igem.org/Team%3ANWU-CHINA-A/Proof_Of_Concept)
  [NOTEBOOK](https://2021.igem.org/Team%3ANWU-CHINA-A/Notebook)
  [SAFETY](https://2021.igem.org/Team%3ANWU-CHINA-A/Safety)
  [RESULTS](https://2021.igem.org/Team%3ANWU-CHINA-A/Results)
* MODEL

  [MODEL](https://2021.igem.org/Team%3ANWU-CHINA-A/Model)
* PARTS

  [PARTS](https://2021.igem.org/Team%3ANWU-CHINA-A/Parts)
  [CONTRIBUTION](https://2021.igem.org/Team%3ANWU-CHINA-A/Contribution)
  [ENGINEERING](https://2021.igem.org/Team%3ANWU-CHINA-A/Engineering)
* HP

  [HUMAN PRACTICES](https://2021.igem.org/Team%3ANWU-CHINA-A/Human_Practices)
  [EDUCATION](https://2021.igem.org/Team%3ANWU-CHINA-A/Education)
  [MEETUPS](https://2021.igem.org/Team%3ANWU-CHINA-A/Meetups)
  [COMMUNICATION](https://2021.igem.org/Team%3ANWU-CHINA-A/Communication)
  [SHADOW PUPPETS](https://2021.igem.org/Team%3ANWU-CHINA-A/Excellence_in_Another_Area)
* TEAM

  [MEMBERS](https://2021.igem.org/Team%3ANWU-CHINA-A/Team)
  [ATTRIBUTIONS](https://2021.igem.org/Team%3ANWU-CHINA-A/Attributions)
  [COLLABORATIONS](https://2021.igem.org/Team%3ANWU-CHINA-A/Collaborations)
  [PARTNERSHIP](https://2021.igem.org/Team%3ANWU-CHINA-A/Partnership)
* [Judging Form](https://igem.org/2021_Judging_Form?id=3722)

![](https://static.igem.org/mediawiki/2021/9/9f/T--NWU-CHINA-A--closed.png)

![](https://static.igem.org/mediawiki/2021/4/48/T--NWU-CHINA-A--logo.png)

* [HOME](https://2021.igem.org/Team%3ANWU-CHINA-A)
* PROJECT

  [DESIGN](https://2021.igem.org/Team%3ANWU-CHINA-A/Design)
  [DESCRIPTION](https://2021.igem.org/Team%3ANWU-CHINA-A/Description)
  [EXPERIMENTS](https://2021.igem.org/Team%3ANWU-CHINA-A/Experiments)
  [IMPLEMENTATION](https://2021.igem.org/Team%3ANWU-CHINA-A/Implementation)
  [PROOF OF CONCEPT](https://2021.igem.org/Team%3ANWU-CHINA-A/Proof_Of_Concept)
  [NOTEBOOK](https://2021.igem.org/Team%3ANWU-CHINA-A/Notebook)
  [SAFETY](https://2021.igem.org/Team%3ANWU-CHINA-A/Safety)
  [RESULTS](https://2021.igem.org/Team%3ANWU-CHINA-A/Results)
* MODEL

  [MODEL](https://2021.igem.org/Team%3ANWU-CHINA-A/Model)
* PARTS

  [PARTS](https://2021.igem.org/Team%3ANWU-CHINA-A/Parts)
  [CONTRIBUTION](https://2021.igem.org/Team%3ANWU-CHINA-A/Contribution)
  [ENGINEERING](https://2021.igem.org/Team%3ANWU-CHINA-A/Engineering)
* HP

  [HUMAN PRACTICES](https://2021.igem.org/Team%3ANWU-CHINA-A/Human_Practices)
  [EDUCATION](https://2021.igem.org/Team%3ANWU-CHINA-A/Education)
  [MEETUPS](https://2021.igem.org/Team%3ANWU-CHINA-A/Meetups)
  [COMMUNICATION](https://2021.igem.org/Team%3ANWU-CHINA-A/Communication)
  [SHADOW PUPPETS](https://2021.igem.org/Team%3ANWU-CHINA-A/Excellence_in_Another_Area)
* TEAM

  [MEMBERS](https://2021.igem.org/Team%3ANWU-CHINA-A/Team)
  [ATTRIBUTIONS](https://2021.igem.org/Team%3ANWU-CHINA-A/Attributions)
  [COLLABORATIONS](https://2021.igem.org/Team%3ANWU-CHINA-A/Collaborations)
  [PARTNERSHIP](https://2021.igem.org/Team%3ANWU-CHINA-A/Partnership)
* [Judging Form](https://igem.org/2021_Judging_Form?id=3722)

N
o
t
e
b
o
o
k

* [🌞PDF](#dev)

###

---

Hello Model,

Best,

[Point Me](https://2021.igem.org/Team%3ANWU-CHINA-A/Model)

Hello HP,

Best,

[Point Me](https://2021.igem.org/Team%3ANWU-CHINA-A/Human_Practices)

Hello Project,

Best,

[Point Me](https://2021.igem.org/Team%3ANWU-CHINA-A/Description)

Hello Members,

Best,

[Point Me](https://2021.igem.org/Team%3ANWU-CHINA-A/Team)

### Follow us

[![](https://static.igem.org/mediawiki/2021/3/3a/T--NWU-CHINA-A--bilibili.png)](https://space.bilibili.com/504559990?spm_id_from=333.788.b_765f7570696e666f.1)
[![](https://static.igem.org/mediawiki/2021/9/9e/T--NWU-CHINA-A--weixin.jpg)](https://static.igem.org/mediawiki/2021/a/a5/T--NWU-CHINA-A--gaoqiweixin.jpg)
[![](https://static.igem.org/mediawiki/2021/4/48/T--NWU-CHINA-A--logo.png)](https://static.igem.org/mediawiki/2021/7/72/T--NWU-CHINA-A--weixingongzhonghao.png)

![](https://static.igem.org/mediawiki/2021/0/03/T--NWU-CHINA-A--zanzhushang.png)

Northwest University 2021 iGEM Team

igem\_nwu\_a@outlook.com

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:NWU-CHINA-A/Engineering

响应式导航

响应式导航

[![](https://static.igem.org/mediawiki/2021/4/48/T--NWU-CHINA-A--logo.png)](https://2021.igem.org/Team%3ANWU-CHINA-A)
[![](https://static.igem.org/mediawiki/2021/4/48/T--NWU-CHINA-A--logo.png)](https://2021.igem.org/Team%3ANWU-CHINA-A)

* [HOME](https://2021.igem.org/Team%3ANWU-CHINA-A)
* PROJECT

  [DESIGN](https://2021.igem.org/Team%3ANWU-CHINA-A/Design)
  [DESCRIPTION](https://2021.igem.org/Team%3ANWU-CHINA-A/Description)
  [EXPERIMENTS](https://2021.igem.org/Team%3ANWU-CHINA-A/Experiments)
  [IMPLEMENTATION](https://2021.igem.org/Team%3ANWU-CHINA-A/Implementation)
  [PROOF OF CONCEPT](https://2021.igem.org/Team%3ANWU-CHINA-A/Proof_Of_Concept)
  [NOTEBOOK](https://2021.igem.org/Team%3ANWU-CHINA-A/Notebook)
  [SAFETY](https://2021.igem.org/Team%3ANWU-CHINA-A/Safety)
  [RESULTS](https://2021.igem.org/Team%3ANWU-CHINA-A/Results)
* MODEL

  [MODEL](https://2021.igem.org/Team%3ANWU-CHINA-A/Model)
* PARTS

  [PARTS](https://2021.igem.org/Team%3ANWU-CHINA-A/Parts)
  [CONTRIBUTION](https://2021.igem.org/Team%3ANWU-CHINA-A/Contribution)
  [ENGINEERING](https://2021.igem.org/Team%3ANWU-CHINA-A/Engineering)
* HP

  [HUMAN PRACTICES](https://2021.igem.org/Team%3ANWU-CHINA-A/Human_Practices)
  [EDUCATION](https://2021.igem.org/Team%3ANWU-CHINA-A/Education)
  [MEETUPS](https://2021.igem.org/Team%3ANWU-CHINA-A/Meetups)
  [COMMUNICATION](https://2021.igem.org/Team%3ANWU-CHINA-A/Communication)
  [SHADOW PUPPETS](https://2021.igem.org/Team%3ANWU-CHINA-A/Excellence_in_Another_Area)
* TEAM

  [MEMBERS](https://2021.igem.org/Team%3ANWU-CHINA-A/Team)
  [ATTRIBUTIONS](https://2021.igem.org/Team%3ANWU-CHINA-A/Attributions)
  [COLLABORATIONS](https://2021.igem.org/Team%3ANWU-CHINA-A/Collaborations)
  [PARTNERSHIP](https://2021.igem.org/Team%3ANWU-CHINA-A/Partnership)
* [Judging Form](https://igem.org/2021_Judging_Form?id=3722)

![](https://static.igem.org/mediawiki/2021/9/9f/T--NWU-CHINA-A--closed.png)

![](https://static.igem.org/mediawiki/2021/4/48/T--NWU-CHINA-A--logo.png)

* [HOME](https://2021.igem.org/Team%3ANWU-CHINA-A)
* PROJECT

  [DESIGN](https://2021.igem.org/Team%3ANWU-CHINA-A/Design)
  [DESCRIPTION](https://2021.igem.org/Team%3ANWU-CHINA-A/Description)
  [EXPERIMENTS](https://2021.igem.org/Team%3ANWU-CHINA-A/Experiments)
  [IMPLEMENTATION](https://2021.igem.org/Team%3ANWU-CHINA-A/Implementation)
  [PROOF OF CONCEPT](https://2021.igem.org/Team%3ANWU-CHINA-A/Proof_Of_Concept)
  [NOTEBOOK](https://2021.igem.org/Team%3ANWU-CHINA-A/Notebook)
  [SAFETY](https://2021.igem.org/Team%3ANWU-CHINA-A/Safety)
  [RESULTS](https://2021.igem.org/Team%3ANWU-CHINA-A/Results)
* MODEL

  [MODEL](https://2021.igem.org/Team%3ANWU-CHINA-A/Model)
* PARTS

  [PARTS](https://2021.igem.org/Team%3ANWU-CHINA-A/Parts)
  [CONTRIBUTION](https://2021.igem.org/Team%3ANWU-CHINA-A/Contribution)
  [ENGINEERING](https://2021.igem.org/Team%3ANWU-CHINA-A/Engineering)
* HP

  [HUMAN PRACTICES](https://2021.igem.org/Team%3ANWU-CHINA-A/Human_Practices)
  [EDUCATION](https://2021.igem.org/Team%3ANWU-CHINA-A/Education)
  [MEETUPS](https://2021.igem.org/Team%3ANWU-CHINA-A/Meetups)
  [COMMUNICATION](https://2021.igem.org/Team%3ANWU-CHINA-A/Communication)
  [SHADOW PUPPETS](https://2021.igem.org/Team%3ANWU-CHINA-A/Excellence_in_Another_Area)
* TEAM

  [MEMBERS](https://2021.igem.org/Team%3ANWU-CHINA-A/Team)
  [ATTRIBUTIONS](https://2021.igem.org/Team%3ANWU-CHINA-A/Attributions)
  [COLLABORATIONS](https://2021.igem.org/Team%3ANWU-CHINA-A/Collaborations)
  [PARTNERSHIP](https://2021.igem.org/Team%3ANWU-CHINA-A/Partnership)
* [Judging Form](https://igem.org/2021_Judging_Form?id=3722)

Engineeri
ng

* [🌞Engineer cycle](#cycle)
* [🌞First iteration](#intro)
  + [🌏Design](#dev)
    - [🌕Research](#Research)
  + [🌏Build](#css)
    - [🌕Plasmid construction](#Plasmid)
    - [🌕Strain establishment](#Strain)
  + [🌏Test](#Test)
    - [🌕Expression verification](#Expression1)
    - [🌕Growth curve measurement](#Growth)
    - [🌕Indigo production in vivo](#Indigo)
  + [🌏Learn](#Learn)
* [🌞Second iteration](#Second)
  + [🌏Design](#Design)
  + [🌏Build](#Build)
  + [🌏Test](#Test1)
    - [🌕Expression verification](#Expression2)
    - [🌕Whole-cell reaction](#Whole-cell)
  + [🌏Learn](#Learn2)
  + [🌏Improvement](#Improvement1)
* [🌞References](#References)

## Engineer cycle

### ![](https://static.igem.org/mediawiki/2021/9/92/T--NWU-CHINA-A--eng1.png)

## First iteration

## Design

### Research

The main chemical constituent of Tyrian Purple: 6, 6'-dibromoindigo(6BrIG), is a halide derivative of indigo. Therefore, we tried finding a way to produce indigo at first. Fortunately, we found that some previous iGEM
teams(
 [2013 iGEM team Berkeley](https://2013.igem.org/Team%3ABerkeley), 2019 iGEM team GreatBay\_SZ, 2020 iGEM team SZU-China et al.) have already built the pathway from tryptophan
to indigo. So we decided to reconstruct the indigo pathway based on their work.

### ![](https://static.igem.org/mediawiki/2021/8/8f/T--NWU-CHINA-A--eng2.png)Figure 1 Scheme of indigo production pathway

## Build

### Plasmid construction

We synthesize the sequence of TnaA and MaFMO with pET28a vectors through Genscript.

### ![](https://static.igem.org/mediawiki/2021/7/71/T--NWU-CHINA-A--eng3.png)Figure 2 Plasmid Maps of pET28a-TnaA and pET28a-MaFMO

### Strain establishment

Once received the DNA dry powder of pET28a-TnaA and pET28a-MaFMO from Genscript, we diluted them with ddH2O and use DH5alpha to clone the plasmid. Then we transformed BL21 strains with the cloned plasmids to establish
expression strains.

### ![](https://static.igem.org/mediawiki/2021/d/d1/T--NWU-CHINA-A--eng4.png)Figure 3 Transformant colony on the LB agar

## Test

### Expression verification

To gain the enzyme we need to produce indigo, the BL21 strain which harbored pET28a-TnaA and pET28a-MaFMO was inoculated to LB medium and incubated. Continue the culture for about 4 hours after induction with IPTG
when the OD reach 0.6-0.8. SDS-PAGE was loaded with the denatured culture sample and results are as follows. We also determine the optimal induction time of these enzymes.

### ![](https://static.igem.org/mediawiki/2021/6/6f/T--NWU-CHINA-A--eng5.11.png)![](https://static.igem.org/mediawiki/2021/2/2d/T--NWU-CHINA-A--eng5.2.png)Figure 4 MaFMO(right) expression results under induction time gradient:1: 0h, 2: 9h, 3: 10.5h, 4: 12h, 5: 13.5h, 6: 15h, 7: 16.5h, 8: 18h, 9: 19.5h; TnaA(left) expression results under induction time gradient: 0: 0h, 1: 4h, 2: 5h, 3: 6h, 4: 7h, 5: 8h, 6: 9h, 7: 10h, 8: 11h

### Growth curve measurement

We measured the growth curve of the two strain to get the optimal growth condition. In this experiment, we took samples every half an hour and tracked the growth of the strain continuously for 36 hours.

### ![](https://static.igem.org/mediawiki/2021/8/8f/T--NWU-CHINA-A--eng6.png)Figure 5 Growth curve of TnaA and MaFMO

### Indigo production in vivo

For MaFMO need reducing power like NADH to exert its catalytic function1, we take a strategy of in vivo reaction. We centrifuged the culture of logarithmic period to cells, and use their resuspension to conduct
the in vivo reaction. You could find more experiment details here.The results is listed below.

### ![](https://static.igem.org/mediawiki/2021/4/41/T--NWU-CHINA-A--eng7.jpg)Figure 6 LB medium with produced indigo in culturetube

## Learn

In our first iteration, we have successfully construct a pathway to produce indigo using **E.coli**as chassis. Although we hadn't measured the actual yield of the strain,
the result of the in vivo experiment show that our strategy is feasible. The second iteration would realize the preliminary production of 6,6'-dibromoindigo.

## Second iteration

## Design

After we produced Tyrian purple, we want to simplify the reaction process and improve the yield of producing. We bought 6-Br-indole and did a series of solubility test, found 6-Br-indole is very difficult to dissolute
even in absolute ethyl alcohol(only dissolved in DMSO). Based on this we suspected that due to insolubility of 6-Br-indole, the transmembrane transport was limited，so we used high-efficiency assembly method——In-Fusion
to connect TnaA and MaFMO in one plasmid.

Here is the brief reaction steps:

### ![](https://static.igem.org/mediawiki/2021/c/c6/T--NWU-CHINA-A--egq1.jpeg)

## Bulid

We connected the sequence of TnaA and MaFMO with pET28a vectors through In-Fusion.

### ![](https://static.igem.org/mediawiki/2021/d/d8/T--NWU-CHINA-A--AAA.png)Figure 7 Plasmid Maps of pET28a-TnaA- MaFMO

## Test

First, we verified the expression of TnaA-MaFMO at 30℃

### ![](https://static.igem.org/mediawiki/2021/2/2b/T--NWU-CHINA-A--egq3.jpeg)Figure 8 SDS-PAGE analysis of TnaA-MaFMO

Second, we added 6-Br-Trp to whole cell reaction and obtained excellent production.

### ![](https://static.igem.org/mediawiki/2021/e/ea/T--NWU-CHINA-A--egq4.jpeg)Figure 9 Some image to verify the infusion reaction

Third ,We compared two reaction systems by Microplate Reader, and found this improvement was more productive.

### ![](https://static.igem.org/mediawiki/2021/d/d1/T--NWU-CHINA-A--egq5.jpeg)Figure 10 Microplate Reader analysised of produced Tyrian purple.

### TnaA knockout strain

Potential side reaction in the first step may reduce the final productivity, which we have explained in the [our description page](https://2021.igem.org/Team%3ANWU-CHINA-A/Description). Hence we knocked out
TnaA gene of the **E.coli** strain we used in the first steps by CRISPR/Cas9. Here are the PCR result to validate the knock-out.

### ![](https://static.igem.org/mediawiki/2021/5/5d/T--NWU-CHINA-A--egq6.png)Figure 11 PCR products using the upstream and downstream sequence contained primers

## Learn

We had achieved our goal to simply the second step of the whole-cell reaction and increase 6BrIG production.

## Safety and risk assessment

On April 15, 2021, a new biosecurity law was passed in China, making biological information security to the national level. We strictly obey the legal approval process, carry out a series of safety monitoring such as
testing chemical drugs in dye process before putting our production into market. We should try our best to ensure that our products don’t do harm to humans and environment.

## References

[1] Fabara, A.N., Fraaije, M.W. An overview of microbial indigo-forming enzymes. Appl Microbiol Biotechnol 104, 925–933 (2020). doi.org/10.1007/s00253-019-10292-5

[2] Lee J, Kim J, Song JE, et al. Production of Tyrian purple indigoid dye from tryptophan in Escherichia coli. Nat Chem Biol. 2021; 17(1):104-112. doi:10.1038/s41589-020-00684-4

---

Hello Model,

Best,

[Point Me](https://2021.igem.org/Team%3ANWU-CHINA-A/Model)

Hello HP,

Best,

[Point Me](https://2021.igem.org/Team%3ANWU-CHINA-A/Human_Practices)

Hello Project,

Best,

[Point Me](https://2021.igem.org/Team%3ANWU-CHINA-A/Description)

Hello Members,

Best,

[Point Me](https://2021.igem.org/Team%3ANWU-CHINA-A/Team)

### Follow us

[![](https://static.igem.org/mediawiki/2021/3/3a/T--NWU-CHINA-A--bilibili.png)](https://space.bilibili.com/504559990?spm_id_from=333.788.b_765f7570696e666f.1)
[![](https://static.igem.org/mediawiki/2021/9/9e/T--NWU-CHINA-A--weixin.jpg)](https://static.igem.org/mediawiki/2021/a/a5/T--NWU-CHINA-A--gaoqiweixin.jpg)
[![](https://static.igem.org/mediawiki/2021/4/48/T--NWU-CHINA-A--logo.png)](https://static.igem.org/mediawiki/2021/7/72/T--NWU-CHINA-A--weixingongzhonghao.png)

![](https://static.igem.org/mediawiki/2021/0/03/T--NWU-CHINA-A--zanzhushang.png)

Northwest University 2021 iGEM Team

igem\_nwu\_a@outlook.com

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:NWU-CHINA-A/Experiments

响应式导航

响应式导航

[![](https://static.igem.org/mediawiki/2021/4/48/T--NWU-CHINA-A--logo.png)](https://2021.igem.org/Team%3ANWU-CHINA-A)
[![](https://static.igem.org/mediawiki/2021/4/48/T--NWU-CHINA-A--logo.png)](https://2021.igem.org/Team%3ANWU-CHINA-A)

* [HOME](https://2021.igem.org/Team%3ANWU-CHINA-A)
* PROJECT

  [DESIGN](https://2021.igem.org/Team%3ANWU-CHINA-A/Design)
  [DESCRIPTION](https://2021.igem.org/Team%3ANWU-CHINA-A/Description)
  [EXPERIMENTS](https://2021.igem.org/Team%3ANWU-CHINA-A/Experiments)
  [IMPLEMENTATION](https://2021.igem.org/Team%3ANWU-CHINA-A/Implementation)
  [PROOF OF CONCEPT](https://2021.igem.org/Team%3ANWU-CHINA-A/Proof_Of_Concept)
  [NOTEBOOK](https://2021.igem.org/Team%3ANWU-CHINA-A/Notebook)
  [SAFETY](https://2021.igem.org/Team%3ANWU-CHINA-A/Safety)
  [RESULTS](https://2021.igem.org/Team%3ANWU-CHINA-A/Results)
* MODEL

  [MODEL](https://2021.igem.org/Team%3ANWU-CHINA-A/Model)
* PARTS

  [PARTS](https://2021.igem.org/Team%3ANWU-CHINA-A/Parts)
  [CONTRIBUTION](https://2021.igem.org/Team%3ANWU-CHINA-A/Contribution)
  [ENGINEERING](https://2021.igem.org/Team%3ANWU-CHINA-A/Engineering)
* HP

  [HUMAN PRACTICES](https://2021.igem.org/Team%3ANWU-CHINA-A/Human_Practices)
  [EDUCATION](https://2021.igem.org/Team%3ANWU-CHINA-A/Education)
  [MEETUPS](https://2021.igem.org/Team%3ANWU-CHINA-A/Meetups)
  [COMMUNICATION](https://2021.igem.org/Team%3ANWU-CHINA-A/Communication)
  [SHADOW PUPPETS](https://2021.igem.org/Team%3ANWU-CHINA-A/Excellence_in_Another_Area)
* TEAM

  [MEMBERS](https://2021.igem.org/Team%3ANWU-CHINA-A/Team)
  [ATTRIBUTIONS](https://2021.igem.org/Team%3ANWU-CHINA-A/Attributions)
  [COLLABORATIONS](https://2021.igem.org/Team%3ANWU-CHINA-A/Collaborations)
  [PARTNERSHIP](https://2021.igem.org/Team%3ANWU-CHINA-A/Partnership)
* [Judging Form](https://igem.org/2021_Judging_Form?id=3722)

![](https://static.igem.org/mediawiki/2021/9/9f/T--NWU-CHINA-A--closed.png)

![](https://static.igem.org/mediawiki/2021/4/48/T--NWU-CHINA-A--logo.png)

* [HOME](https://2021.igem.org/Team%3ANWU-CHINA-A)
* PROJECT

  [DESIGN](https://2021.igem.org/Team%3ANWU-CHINA-A/Design)
  [DESCRIPTION](https://2021.igem.org/Team%3ANWU-CHINA-A/Description)
  [EXPERIMENTS](https://2021.igem.org/Team%3ANWU-CHINA-A/Experiments)
  [IMPLEMENTATION](https://2021.igem.org/Team%3ANWU-CHINA-A/Implementation)
  [PROOF OF CONCEPT](https://2021.igem.org/Team%3ANWU-CHINA-A/Proof_Of_Concept)
  [NOTEBOOK](https://2021.igem.org/Team%3ANWU-CHINA-A/Notebook)
  [SAFETY](https://2021.igem.org/Team%3ANWU-CHINA-A/Safety)
  [RESULTS](https://2021.igem.org/Team%3ANWU-CHINA-A/Results)
* MODEL

  [MODEL](https://2021.igem.org/Team%3ANWU-CHINA-A/Model)
* PARTS

  [PARTS](https://2021.igem.org/Team%3ANWU-CHINA-A/Parts)
  [CONTRIBUTION](https://2021.igem.org/Team%3ANWU-CHINA-A/Contribution)
  [ENGINEERING](https://2021.igem.org/Team%3ANWU-CHINA-A/Engineering)
* HP

  [HUMAN PRACTICES](https://2021.igem.org/Team%3ANWU-CHINA-A/Human_Practices)
  [EDUCATION](https://2021.igem.org/Team%3ANWU-CHINA-A/Education)
  [MEETUPS](https://2021.igem.org/Team%3ANWU-CHINA-A/Meetups)
  [COMMUNICATION](https://2021.igem.org/Team%3ANWU-CHINA-A/Communication)
  [SHADOW PUPPETS](https://2021.igem.org/Team%3ANWU-CHINA-A/Excellence_in_Another_Area)
* TEAM

  [MEMBERS](https://2021.igem.org/Team%3ANWU-CHINA-A/Team)
  [ATTRIBUTIONS](https://2021.igem.org/Team%3ANWU-CHINA-A/Attributions)
  [COLLABORATIONS](https://2021.igem.org/Team%3ANWU-CHINA-A/Collaborations)
  [PARTNERSHIP](https://2021.igem.org/Team%3ANWU-CHINA-A/Partnership)
* [Judging Form](https://igem.org/2021_Judging_Form?id=3722)

E
x
p
e
r
i
m
e
n
t
s

* [🌞PDF](#dev)

###

---

Hello Model,

Best,

[Point Me](https://2021.igem.org/Team%3ANWU-CHINA-A/Model)

Hello HP,

Best,

[Point Me](https://2021.igem.org/Team%3ANWU-CHINA-A/Human_Practices)

Hello Project,

Best,

[Point Me](https://2021.igem.org/Team%3ANWU-CHINA-A/Description)

Hello Members,

Best,

[Point Me](https://2021.igem.org/Team%3ANWU-CHINA-A/Team)

### Follow us

[![](https://static.igem.org/mediawiki/2021/3/3a/T--NWU-CHINA-A--bilibili.png)](https://space.bilibili.com/504559990?spm_id_from=333.788.b_765f7570696e666f.1)
[![](https://static.igem.org/mediawiki/2021/9/9e/T--NWU-CHINA-A--weixin.jpg)](https://static.igem.org/mediawiki/2021/a/a5/T--NWU-CHINA-A--gaoqiweixin.jpg)
[![](https://static.igem.org/mediawiki/2021/4/48/T--NWU-CHINA-A--logo.png)](https://static.igem.org/mediawiki/2021/7/72/T--NWU-CHINA-A--weixingongzhonghao.png)

![](https://static.igem.org/mediawiki/2021/0/03/T--NWU-CHINA-A--zanzhushang.png)

Northwest University 2021 iGEM Team

igem\_nwu\_a@outlook.com

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:NWU-CHINA-A/Results

响应式导航

响应式导航

[![](https://static.igem.org/mediawiki/2021/4/48/T--NWU-CHINA-A--logo.png)](https://2021.igem.org/Team%3ANWU-CHINA-A)
[![](https://static.igem.org/mediawiki/2021/4/48/T--NWU-CHINA-A--logo.png)](https://2021.igem.org/Team%3ANWU-CHINA-A)

* [HOME](https://2021.igem.org/Team%3ANWU-CHINA-A)
* PROJECT

  [DESIGN](https://2021.igem.org/Team%3ANWU-CHINA-A/Design)
  [DESCRIPTION](https://2021.igem.org/Team%3ANWU-CHINA-A/Description)
  [EXPERIMENTS](https://2021.igem.org/Team%3ANWU-CHINA-A/Experiments)
  [IMPLEMENTATION](https://2021.igem.org/Team%3ANWU-CHINA-A/Implementation)
  [PROOF OF CONCEPT](https://2021.igem.org/Team%3ANWU-CHINA-A/Proof_Of_Concept)
  [NOTEBOOK](https://2021.igem.org/Team%3ANWU-CHINA-A/Notebook)
  [SAFETY](https://2021.igem.org/Team%3ANWU-CHINA-A/Safety)
  [RESULTS](https://2021.igem.org/Team%3ANWU-CHINA-A/Results)
* MODEL

  [MODEL](https://2021.igem.org/Team%3ANWU-CHINA-A/Model)
* PARTS

  [PARTS](https://2021.igem.org/Team%3ANWU-CHINA-A/Parts)
  [CONTRIBUTION](https://2021.igem.org/Team%3ANWU-CHINA-A/Contribution)
  [ENGINEERING](https://2021.igem.org/Team%3ANWU-CHINA-A/Engineering)
* HP

  [HUMAN PRACTICES](https://2021.igem.org/Team%3ANWU-CHINA-A/Human_Practices)
  [EDUCATION](https://2021.igem.org/Team%3ANWU-CHINA-A/Education)
  [MEETUPS](https://2021.igem.org/Team%3ANWU-CHINA-A/Meetups)
  [COMMUNICATION](https://2021.igem.org/Team%3ANWU-CHINA-A/Communication)
  [SHADOW PUPPETS](https://2021.igem.org/Team%3ANWU-CHINA-A/Excellence_in_Another_Area)
* TEAM

  [MEMBERS](https://2021.igem.org/Team%3ANWU-CHINA-A/Team)
  [ATTRIBUTIONS](https://2021.igem.org/Team%3ANWU-CHINA-A/Attributions)
  [COLLABORATIONS](https://2021.igem.org/Team%3ANWU-CHINA-A/Collaborations)
  [PARTNERSHIP](https://2021.igem.org/Team%3ANWU-CHINA-A/Partnership)
* [Judging Form](https://igem.org/2021_Judging_Form?id=3722)

![](https://static.igem.org/mediawiki/2021/9/9f/T--NWU-CHINA-A--closed.png)

![](https://static.igem.org/mediawiki/2021/4/48/T--NWU-CHINA-A--logo.png)

* [HOME](https://2021.igem.org/Team%3ANWU-CHINA-A)
* PROJECT

  [DESIGN](https://2021.igem.org/Team%3ANWU-CHINA-A/Design)
  [DESCRIPTION](https://2021.igem.org/Team%3ANWU-CHINA-A/Description)
  [EXPERIMENTS](https://2021.igem.org/Team%3ANWU-CHINA-A/Experiments)
  [IMPLEMENTATION](https://2021.igem.org/Team%3ANWU-CHINA-A/Implementation)
  [PROOF OF CONCEPT](https://2021.igem.org/Team%3ANWU-CHINA-A/Proof_Of_Concept)
  [NOTEBOOK](https://2021.igem.org/Team%3ANWU-CHINA-A/Notebook)
  [SAFETY](https://2021.igem.org/Team%3ANWU-CHINA-A/Safety)
  [RESULTS](https://2021.igem.org/Team%3ANWU-CHINA-A/Results)
* MODEL

  [MODEL](https://2021.igem.org/Team%3ANWU-CHINA-A/Model)
* PARTS

  [PARTS](https://2021.igem.org/Team%3ANWU-CHINA-A/Parts)
  [CONTRIBUTION](https://2021.igem.org/Team%3ANWU-CHINA-A/Contribution)
  [ENGINEERING](https://2021.igem.org/Team%3ANWU-CHINA-A/Engineering)
* HP

  [HUMAN PRACTICES](https://2021.igem.org/Team%3ANWU-CHINA-A/Human_Practices)
  [EDUCATION](https://2021.igem.org/Team%3ANWU-CHINA-A/Education)
  [MEETUPS](https://2021.igem.org/Team%3ANWU-CHINA-A/Meetups)
  [COMMUNICATION](https://2021.igem.org/Team%3ANWU-CHINA-A/Communication)
  [SHADOW PUPPETS](https://2021.igem.org/Team%3ANWU-CHINA-A/Excellence_in_Another_Area)
* TEAM

  [MEMBERS](https://2021.igem.org/Team%3ANWU-CHINA-A/Team)
  [ATTRIBUTIONS](https://2021.igem.org/Team%3ANWU-CHINA-A/Attributions)
  [COLLABORATIONS](https://2021.igem.org/Team%3ANWU-CHINA-A/Collaborations)
  [PARTNERSHIP](https://2021.igem.org/Team%3ANWU-CHINA-A/Partnership)
* [Judging Form](https://igem.org/2021_Judging_Form?id=3722)

R
e
s
u
l
t
s

* [🌏Plasmid Construction And Expression](#dev)
* [🌏Expression of Fre-Linker-SttH, TnaA, MaFMO](#css)
* [🌏Tyrian Purple Production](#United1)
  + [🌕Whole-cell reactions](#Plasmid)
  + [🌕Lyophilized Powder](#Strain)
* [🌏The Improvement Production](#PR)
  + [🌕The effect of Fre](#teof)
  + [🌕Improvement through In-Fusion](#iti)
  + [🌕CRISPR Cas9](#CRI)
* [🌏Dyeing Effect](#DE)

## Plasmid Construction And Expression

We constructed three basic expression plasmids to produce Tyrian Purple.(Click [here](https://2021.igem.org/Team%3ANWU-CHINA-A/Design) to know more). After transferring them into E.coli BL21(DE3), we measured the growth curves of each strain (Fig 1. a,b,c).

### ![](https://static.igem.org/mediawiki/2021/c/c3/T--NWU-CHINA-A--R1.png)Fig.1 a. FLS growth curve. b. TnaA growth curve. c. MaFMO growth curve.

As we can see, all of these bacterial growth curves experienced lag phase, logarithmic phase, stationary phase and decline phase. From this, we obtained the best induction time of these strains.

## Expression of Fre-Linker-SttH, TnaA, MaFMO

For FLS，some references and our pre-experiment show that it will express as inclusion body at high temperature (37℃). So, to obtain the best induction condition, we induced FLS at different temperatures and time (Fig.2).
Then, we induced TnaA and MaFMO at different time and temperatures respectively (Fig.3, Fig.4).

### ![](https://static.igem.org/mediawiki/2021/8/80/T--NWU-CHINA-A--R2.png)Fig.2 SDS-PAGE of FLS expression. Induce condition: LB media, OD600 0.6 ~ 0.8, IPTG 0.1 mM. 1:negitive control; 2: 9h 37℃; 3:9h 30℃; 4:9h 23℃; 5:9h 17℃; 6:15h 37℃; 7:15h 30℃; 8:15h 23℃; 9: 15h 17℃.

### ![](https://static.igem.org/mediawiki/2021/c/c9/T--NWU-CHINA-A--R3.png) ![](https://static.igem.org/mediawiki/2021/2/2b/T--NWU-CHINA-A--part084.png)Fig.3 SDS-PAGE of TnaA expression. Induce condition: LB media, OD600 0.6 ~ 0.8, IPTG 0.1 mM, 37 °C and 200 rpm.1:negitive control; 2: 9h; 3:10.5h; 4:12h; 5:13.5h; 6:15h; 7:16.5h; 8: 18h; 9: 19.5h.Fig.4 SDS-PAGE of TnaA expression. Induce condition: LB media, OD600 0.6 ~ 0.8, IPTG 0.1 mM, 18 °C and 200 rpm. 0:negitive control; 1:4h; 2:5h; 3:6h; 4:7h; 5:8h; 6:9h; 7:10h; 8:11h.

## Tyrian Purple Production

### Whole-cell reactions

Limited by Fre-Linker-SttH expression, we decided to use 6-Br-Trp as the substrate to go on the whole-cell reactions. First of all, we expressed TnaA (30℃,16h) and MaFmo (26℃,4h) with 6-Br-Trp containing LB medium and common LB medium respectively. The cells were resuspended in 50mM NPB solution after centrifugating through high-speed freezing centrifuge. Then the TnaA and MaFMO suspension were mixed and incubated in 30℃, 200rpm for 12h. In this way, we successfully obtained the target product Tyrian Purple (Figure.5 a,b). At the same time, we also obtained the optimal mixing ratio of TnaA to MaFmo by comparative experiments. Additionally, we found that the NPB buffer was more effective than the PBS buffer, because Na+ was able to assist transmembrane transport of 6-Br-Trp and 6-Br-indole (Fig.5 e).

### ![](https://static.igem.org/mediawiki/2021/2/20/T--NWU-CHINA-A--R5.png)Fig.5 a.TnaA:MaFMO (1.2:1), LB medium resuspended. b. TnaA:MaFMO (1.2:1), NPB resuspended. c. TnaA:MaFMO(1:1), NPB resuspended. d.TnaA:MaFMO (1.2:1), NPB resuspended. e. TnaA:MaFMO(1:1), PBS resuspended.)

### Lyophilized Powder

Tyrian Purple contained bacterial fluid was frozen at-80℃for 12h, and then made into lyophilized powder by a freeze dryer.

### ![](https://static.igem.org/mediawiki/2021/d/de/T--NWU-CHINA-A--igq7.jpeg)

## The Improvement Production

### The effect of Fre

SttH was successfully overexpressed in E. coli, but the enzyme mostly existed in an insoluble form (Fig.6). To test Fre can improve the bromination reaction rate, we did a comparative experiment between SttH and Fre-Linker-SttH. First ,we did PCR with the primer of SttH, it showed good results.

### ![](https://static.igem.org/mediawiki/2021/b/ba/T--NWU-CHINA-A--part092.png)Fig.6 Agarose-gel electrophoresis of SttH.

Then ,we connected SttH to pET-28a(+), transfered into BL21(DE3) and added IPTG to induce SttH expression.

### Improvement through In-Fusion

To achieve the highest yield of producing and simply the reaction process, we made TnaA and MaFMO in one plasmid use In-Fusion method. After PCR reaction, DNA electrophoresis bands were shown below.

The pET-28a(+) expression vector was chosen, BamHI and Hind III restriction sites were selected.(click here to learn more [protocol](https://2021.igem.org/Team%3ANWU-CHINA-A/Experiments))

### ![](https://static.igem.org/mediawiki/2021/d/dd/T--NWU-CHINA-A--re9.png)Fig.7 1：PCR results of TnaA; 2: negitive control; 3: PCR resulting MaFMO; 4: linear pET-28a(+) after enzyme digestion.

### ![](https://static.igem.org/mediawiki/2021/d/d3/T--NWU-CHINA-A--part112.png)Fig.8 1:negitive control; 2: 3.5h; 3:9h; 4:10h; 5:11h; 6:12h;

We also did a whole cell reaction, this improvement saved us nearly twice as long(Fig.9,10). We suspected that this is due to the transmenmbrane transport of 6-Br-indole was skipped.

### ![](https://static.igem.org/mediawiki/2021/b/b8/T--NWU-CHINA-A--part114.png) ![](https://static.igem.org/mediawiki/2021/9/9b/T--NWU-CHINA-A--re11.jpg)Fig.10 The absorption curves of Tyrian Purple production.Fig.11 In-Fusion Production.

### CRISPR Cas9

Since TnaA is an endogenous enzyme of E.coli, when the strain with engineered TnaA, there will be a competition between TnaA and SttH, and produce indindex (indole). So, we imported the Cas9 plasmid and the Target plasmid into BL21 (Fig.12), and designed primers which contained the upstream and downstream sequence of the E. coli genome TnaA, then we evaluated PCR products by agarose gel electrophoresis (Fig.13) to ensure that we successfully knocked down the TnaA.

### ![](https://static.igem.org/mediawiki/2021/2/25/T--NWU-CHINA-A--re12.jpg)Fig.12 BL21 with Cas9 and Target.

### ![](https://static.igem.org/mediawiki/2021/1/17/T--NWU-CHINA-A--kkkkk.png)Fig.13. PCR products using the upstream and downstream sequence contained primers.

After knockdown of TnaA, we eliminated CRISPR Cas9 plasmids in E.coli BL21 (DE3) and grown them in Kan and Amp contained LB medium for ensurance (Fig.14). Subsequently, we transformed FLS and TnaA-MaFMO plasmids into E.coli BL21 (DE3) TnaA(-) for protein expression.

### ![](https://static.igem.org/mediawiki/2021/4/4b/T--NWU-CHINA-A--re14.jpg)Fig.14

## Dyeing Effect

After obtaining Tyrian Purple, we immediately performed staining experiments on different materials. According to the results, the wool and silk showed the best effect (Fig.15), and common materials can be well colored.

### ![](https://static.igem.org/mediawiki/2021/e/e4/T--NWU-CHINA-A--re15.jpg)Fig. 15 Results of dying experiments.

---

Hello Model,

Best,

[Point Me](https://2021.igem.org/Team%3ANWU-CHINA-A/Model)

Hello HP,

Best,

[Point Me](https://2021.igem.org/Team%3ANWU-CHINA-A/Human_Practices)

Hello Project,

Best,

[Point Me](https://2021.igem.org/Team%3ANWU-CHINA-A/Description)

Hello Members,

Best,

[Point Me](https://2021.igem.org/Team%3ANWU-CHINA-A/Team)

### Follow us

[![](https://static.igem.org/mediawiki/2021/3/3a/T--NWU-CHINA-A--bilibili.png)](https://space.bilibili.com/504559990?spm_id_from=333.788.b_765f7570696e666f.1)
[![](https://static.igem.org/mediawiki/2021/9/9e/T--NWU-CHINA-A--weixin.jpg)](https://static.igem.org/mediawiki/2021/a/a5/T--NWU-CHINA-A--gaoqiweixin.jpg)
[![](https://static.igem.org/mediawiki/2021/4/48/T--NWU-CHINA-A--logo.png)](https://static.igem.org/mediawiki/2021/7/72/T--NWU-CHINA-A--weixingongzhonghao.png)

![](https://static.igem.org/mediawiki/2021/0/03/T--NWU-CHINA-A--zanzhushang.png)

Northwest University 2021 iGEM Team

igem\_nwu\_a@outlook.com