iGEM 2018 — Team NEU_China_A

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# Team:NEU China A/Notebook

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TEAM

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

PROJECT

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

PARTS

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

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

HUMAN PRACTICES

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

AWARDS

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

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

[Home](https://2018.igem.org/Team%3ANEU_China_A)
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+ *folder\_open*Project

  [Introduction](https://2018.igem.org/Team%3ANEU_China_A/Introduction)
  [Design](https://2018.igem.org/Team%3ANEU_China_A/Design)
  [Model](https://2018.igem.org/Team%3ANEU_China_A/Model)
  [Results & Demonstrate](https://2018.igem.org/Team%3ANEU_China_A/Demonstrate)
  [InterLab](https://2018.igem.org/Team%3ANEU_China_A/InterLab)
+ *place*Lab

  [Notebook](https://2018.igem.org/Team%3ANEU_China_A/Notebook)
  [Protocols](https://2018.igem.org/Team%3ANEU_China_A/Protocols)
  [Safety](https://2018.igem.org/Team%3ANEU_China_A/Safety)
+ *details*Parts

  [Basic
  And Composite Parts](https://2018.igem.org/Team%3ANEU_China_A/BasicAndCompositeParts)
  [Improved
  Biobricks](https://2018.igem.org/Team%3ANEU_China_A/Improve)
+ *lightbulb\_outline*HP

  [Human
  Practices](https://2018.igem.org/Team%3ANEU_China_A/Human_Practices)
  [Collaborations](https://2018.igem.org/Team%3ANEU_China_A/Collaborations)
  [Engagement](https://2018.igem.org/Team%3ANEU_China_A/Public_Engagement)
+ *people*Team

  [Team Member](https://2018.igem.org/Team%3ANEU_China_A/TeamMember)
  [Attributions](https://2018.igem.org/Team%3ANEU_China_A/Attributions)
  [Gallery](https://2018.igem.org/Team%3ANEU_China_A/Gallery)

* [Introduction](https://2018.igem.org/Team%3ANEU_China_A/Introduction)
* [Design](https://2018.igem.org/Team%3ANEU_China_A/Design)
* [Model](https://2018.igem.org/Team%3ANEU_China_A/Model)
* [Results & Demonstrate](https://2018.igem.org/Team%3ANEU_China_A/Demonstrate)
* [Interlab](https://2018.igem.org/Team%3ANEU_China_A/InterLab)

* [Notebook](https://2018.igem.org/Team%3ANEU_China_A/Notebook)
* [Protocols](https://2018.igem.org/Team%3ANEU_China_A/Protocols)
* [Safety](https://2018.igem.org/Team%3ANEU_China_A/Safety)

* [Basic And Composite Parts](https://2018.igem.org/Team%3ANEU_China_A/BasicAndCompositeParts)
* [Improved Biobricks](https://2018.igem.org/Team%3ANEU_China_A/Improve)

* [Human Practices](https://2018.igem.org/Team%3ANEU_China_A/Human_Practices)
* [Collaborations](https://2018.igem.org/Team%3ANEU_China_A/Collaborations)
* [Engagement](https://2018.igem.org/Team%3ANEU_China_A/Public_Engagement)

* [Team member](https://2018.igem.org/Team%3ANEU_China_A/TeamMember)
* [Attributions](https://2018.igem.org/Team%3ANEU_China_A/Attributions)
* [Gallery](https://2018.igem.org/Team%3ANEU_China_A/Gallery)

## Notebook

![](https://static.igem.org/mediawiki/2018/d/d3/T--NEU_China_A--notebook.jpg)

### Notebook

You can download the notebook [here](https://static.igem.org/mediawiki/2018/9/9e/T--NEU_China_A--notebook.pdf)

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Contact us

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# Team:NEU China A/Experiments

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[HOME](https://2018.igem.org/Team%3ANEU_China_A)

TEAM

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

PROJECT

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

PARTS

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

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

HUMAN PRACTICES

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

AWARDS

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

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

# 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/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:NEU China A/Results

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[HOME](https://2018.igem.org/Team%3ANEU_China_A)

TEAM

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

PROJECT

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

PARTS

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

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

HUMAN PRACTICES

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

AWARDS

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

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

[Home](https://2018.igem.org/Team%3ANEU_China_A)
*menu*

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* [Lab*arrow\_drop\_down*](#!)
* [Parts*arrow\_drop\_down*](#!)
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+ *folder\_open*Project

  [Introduction](https://2018.igem.org/Team%3ANEU_China_A/Introduction)
  [Design](https://2018.igem.org/Team%3ANEU_China_A/Design)
  [Model](https://2018.igem.org/Team%3ANEU_China_A/Model)
  [Results & Demonstrate](https://2018.igem.org/Team%3ANEU_China_A/Demonstrate)
  [InterLab](https://2018.igem.org/Team%3ANEU_China_A/InterLab)
+ *place*Lab

  [Notebook](https://2018.igem.org/Team%3ANEU_China_A/Notebook)
  [Protocols](https://2018.igem.org/Team%3ANEU_China_A/Protocols)
  [Safety](https://2018.igem.org/Team%3ANEU_China_A/Safety)
+ *details*Parts

  [Basic
  And Composite Parts](https://2018.igem.org/Team%3ANEU_China_A/BasicAndCompositeParts)
  [Improved
  Biobricks](https://2018.igem.org/Team%3ANEU_China_A/Improve)
+ *lightbulb\_outline*HP

  [Human
  Practices](https://2018.igem.org/Team%3ANEU_China_A/Human_Practices)
  [Collaborations](https://2018.igem.org/Team%3ANEU_China_A/Collaborations)
  [Engagement](https://2018.igem.org/Team%3ANEU_China_A/Public_Engagement)
+ *people*Team

  [Team Member](https://2018.igem.org/Team%3ANEU_China_A/TeamMember)
  [Attributions](https://2018.igem.org/Team%3ANEU_China_A/Attributions)
  [Gallery](https://2018.igem.org/Team%3ANEU_China_A/Gallery)

* [Introduction](https://2018.igem.org/Team%3ANEU_China_A/Introduction)
* [Design](https://2018.igem.org/Team%3ANEU_China_A/Design)
* [Model](https://2018.igem.org/Team%3ANEU_China_A/Model)
* [Results & Demonstrate](https://2018.igem.org/Team%3ANEU_China_A/Demonstrate)
* [Interlab](https://2018.igem.org/Team%3ANEU_China_A/InterLab)

* [Notebook](https://2018.igem.org/Team%3ANEU_China_A/Notebook)
* [Protocols](https://2018.igem.org/Team%3ANEU_China_A/Protocols)
* [Safety](https://2018.igem.org/Team%3ANEU_China_A/Safety)

* [Basic And Composite Parts](https://2018.igem.org/Team%3ANEU_China_A/BasicAndCompositeParts)
* [Improved Biobricks](https://2018.igem.org/Team%3ANEU_China_A/Improve)

* [Human Practices](https://2018.igem.org/Team%3ANEU_China_A/Human_Practices)
* [Collaborations](https://2018.igem.org/Team%3ANEU_China_A/Collaborations)
* [Engagement](https://2018.igem.org/Team%3ANEU_China_A/Public_Engagement)

* [Team member](https://2018.igem.org/Team%3ANEU_China_A/TeamMember)
* [Attributions](https://2018.igem.org/Team%3ANEU_China_A/Attributions)
* [Gallery](https://2018.igem.org/Team%3ANEU_China_A/Gallery)

### Results

1.Nitric oxide sensor

We constructed our NO sensor in the pCDFDuet-1 plasmid (Figure 1)

![](https://static.igem.org/mediawiki/2018/1/14/T--NEU_China_A--results-1.png "Figure 1. The construction of our NO sensor.")

According to the results of the ShanghaiTechChina\_B 2016 team, 100μM SNP aqueous solution can
continuously release NO, and the NO concentration is stable at about 5.5μM. This concentration is the
concentration of NO in patients with IBD. Therefore, in the subsequent test of the NO sensor, the final
concentration of the SNP aqueous solution we added was 100 μM.

We transformed the constructed NO sensor plasmid into DH5α, and cultured at 37 ℃ overnight to dilute to
OD = 0.2. After 1.5 h of growth at 37 ℃, the inducer IPTG and SNP aqueous solution were added. After 6
h at 37 ℃, 1 mL of the bacterial solution was centrifuged at 8000 rpm for 1 min (Figure 2).

![](https://static.igem.org/mediawiki/2018/0/07/T--NEU_China_A--results-2.png "Figure 2. Pellets of bacteria transformed with constructed NO sensor plasmid after induction of 6h.")

From left to right: control, 0.5mM IPTG without SNP, 1mM IPTG without SNP, 0.5mM IPTG with 100μM SNP,
1mM IPTG with 100μM SNP.

We also tested the response of BBa\_K381001 to NO in the Distribution Kit (Figure 3).

![](https://static.igem.org/mediawiki/2018/8/84/T--NEU_China_A--results-3.png "Figure 3. The construction of BBa_K381001.")

We transformed the plasmid containing BBa\_K381001 into DH5α, and cultured at 37 ℃ overnight to dilute
to OD = 0.4. Then we took half as control and the other half added SNP aqueous solution and induced at
37 ℃ for 6 h. Then we detected the fluorescence using a microplate reader and a fluorescence microscope
(Figure 4).

![](https://static.igem.org/mediawiki/2018/b/b3/T--NEU_China_A--results-4.png "Figure 4. The response to NO of BBa_K381001. ")

A, Histogram of GFP fluorescence: LB control, without SNP, with 100μM SNP. B, GFP fluorescence image
from top to bottom: without SNP, with 100μM SNP.

2.Production

At the beginning of the experiment, we directly constructed the plasmids of YebF-IL10 and
YebF-myrosinase, but the effect of extracellular detection was not satisfactory. During the
participation in CCiC, iGEMer suggested that we first start from testing the performance of the
secretory tag, so we constructed the plasmid YebF-GFP shown in Figure 5A. To express IL10 and
myrosinase, we constructed plasmids IL10-flag and myrosinase-his as shown in Figure 5B and Figure 5C.

![](https://static.igem.org/mediawiki/2018/2/2a/T--NEU_China_A--results-5.png "Figure 5. Production module construction.")

**A**, the construction of our YebF-GFP using strong promoter.
**B**, the construction of IL10 production module.
**C**, the construction of myrosinase production module.

When testing the validity of the secretory tag YebF, we transformed the constructed YebF-GFP plasmid
into DH5α and cultured overnight at 37 ℃. The supernatant was centrifuged at 3000 rpm for 5 min, and
the supernatant was taken for fluorescence detection. The results are shown in the Figure 6. But we
forgot to set up a positive control group. Due to time constraints, we have no time to repeat this
experiment.

![](https://static.igem.org/mediawiki/2018/9/92/T--NEU_China_A--results-6.png "Figure 6. The fluorescence of overnight bacterial suspension.")

We transformed the IL10-flag plasmid into BL21, and incubated at 37 ℃ overnight to dilute to OD = 0.2.
After 2 h of growth at 37 ℃, IPTG was added and induced at 30 ℃ for 16 h. Then, the bacterial solution
was lysed and the expression of IL10 was detected by western blot (Figure 7).

![](https://static.igem.org/mediawiki/2018/a/ac/T--NEU_China_A--results-7.png "Figure 7. Western blot analyses using a flag antibody on bacterial lysate to detect IL10.")

Lane 1: Negative control (cell lysate without IPTG induction); Lane 2: 0.5mM IPTG, Lane3: 1mM IPTG
induction for 16h at 30℃.

We transformed the plasmid of myrosinase-his into BL21, and cultured at 37 ℃ overnight and diluted to
OD = 0.2. After growth for 2 h at 37 ℃, different concentrations of IPTG were added and induced at 16 ℃
for 16 h. The bacterial cell lysis was then performed to detect the expression of myrosinase by
SDS-PAGE (Figure 8).

![](https://static.igem.org/mediawiki/2018/a/ae/T--NEU_China_A--results-8.png "Figure 8. SDS-PAGE analyses on bacterial lysate to detect myrosinase.")

Lane 1: Negative control (cell lysate without IPTG induction); Lane 2: 0.25mM IPTG; Lane3: 0.5mM IPTG,
Lane4: 0.75mM IPTG induction for 16h at 16℃.

3.Kill Switch

We inserted the reporter gene amilCP into the pColdI plasmid to characterize the performance of the
cold shock promoter PcspA (Figure 9A). We inserted maz-F into the pColdI plasmid to build our kill
switch (Figure 9B).

![](https://static.igem.org/mediawiki/2018/d/de/T--NEU_China_A--results-9.png "Figure 9. Kill switch module construction. ")

**A**, the construction of PcspA-amilCP plasmid.
**B**, the construction of PcspA-mazF plasmid.

We transformed the PcspA-amilCP plasmid into DH5α and cultured overnight at 37℃. The overnight culture
was diluted to OD = 0.2 and allowed to grow for 2 h at 37℃. It was then divided into different
concentrations of IPTG at 16℃ and 37℃ for 6 h (Figure 10).

![](https://static.igem.org/mediawiki/2018/5/5a/T--NEU_China_A--results-10.png "Figure 10. Pellets of bacteria transformed with constructed PcspA-amilCP plasmid after induction of 6h.")

From left to right: 37℃ without IPTG, 37℃ with 0.5mM IPTG, 37℃ with 1mM IPTG, 16℃ without IPTG, 16℃
with 0.5mM IPTG, 16℃ with 1mM IPTG.

We transformed the constructed PcspA-mazF plasmid into BL21, added 1 mM IPTG to the plate, and cultured at 16℃ for 16 h (Figure 11A). We then cultured BL21 transformed with the PcspA-mazF plasmid overnight at 16℃. After diluting to OD=0.2 on the next day, the cells were cultured at 16℃, and the OD value was measured every hour for 9 hours (Figure 11B).

![](https://static.igem.org/mediawiki/2018/0/05/T--NEU_China_A--results-11.png "Figure 11. The effect of our killer gene under 16℃.")

**A**, the plate of BL21 with and without killer gene under induction. B, the growth curve of BL21 with and without killer gene under induction.