iGEM 2019 — Team USTC

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# Team:USTC/Notebook

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* Team

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* Project

  [Description](https://2019.igem.org/Team%3AUSTC/Description)
  [Design](https://2019.igem.org/Team%3AUSTC/Design)
  [Experiment](https://2019.igem.org/Team%3AUSTC/Experiment)
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* Safety

  [Lab](https://2019.igem.org/Team%3AUSTC/Safety)
  [Kill Switch](https://2019.igem.org/Team%3AUSTC/Kill_Switch)
* [Model](https://2019.igem.org/Team%3AUSTC/Model)
* Human Practices

  [General HP](https://2019.igem.org/Team%3AUSTC/Human_Practices)
  [Integrated HP](https://2019.igem.org/Team%3AUSTC/Integrated_HP)
  [Collaborations](https://2019.igem.org/Team%3AUSTC/Collaborations)
* [Parts](https://2019.igem.org/Team%3AUSTC/Parts)
* [Awards](https://2019.igem.org/Team%3AUSTC/Awards)
* [**Judging Form**](https://igem.org/2019_Judging_Form?id=3099)

![Card image](https://static.igem.org/mediawiki/2019/e/e6/T--USTC--Notebook.jpg)

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#### monthly diary

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August

##### **August 26, 2019**

Experiment: PCR and Gibson Assembly

People: Guangtao Xu, Rundong Ye, Zhenhang Lu

##### **August 27, 2019**

Experiment: Plasmid extraction

People: Guangtao Xu, Rundong Ye, Huayu Pan

##### **August 28, 2019**

Experiment: Plasmid extraction

People: Guangtao Xu, Rundong Ye

##### **August 29, 2019**

Experiment: Preparation of the sensitive state of S. oneidensis MR-1

People: Huayu Pan

##### **August 30, 2019**

Experiment: Selection of a single colony of S. oneidensis MR-1 and sequencing

People: Guangtao Xu

A single colony of S. oneidensis MR-1 was selected on solid medium for colony PCR to verify whether the
transformation was successful, and the verified single colony was kept for further culture and DNA sequencing.

##### **August 31, 2019 – September 7, 2019**

Experiment: verification of electronic production ability by MFC (microbial fuel cell)

People: Guangtao Xu, Rundong Ye

September

##### **September 8, 2019 – September 16, 2019**

Experiment: Reduction of methyl orange (MO); Using GFP to test the QS system

People: Guangtao Xu, Rundong Ye

Result:

![Card image](https://static.igem.org/mediawiki/2019/0/0e/T--USTC--Notebook-1.jpg)

Figure 1: Reduction result (Sep 8, 2019)

##### **September 2, 2019 – September 6, 2019**

Experiment: Construction of aNAT expression vector

People: Sihao Yuan, Wujianan Sun

Result:

![Card image](https://static.igem.org/mediawiki/2019/8/86/T--USTC--Notebook-2.jpg)

Figure 2: Gel result (Sep 4, 2019)

##### **September 7, 2019 – September 16, 2019**

Experiment: Characterization of aromatic amine resistance

People: Sihao Yuan

![](https://static.igem.org/mediawiki/2019/4/4b/T--USTC--footer.png)


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# Team:USTC/Experiments

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AWARDS

[Entrepreneurship](https://2019.igem.org/Team%3AUSTC/Entrepreneurship)
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[JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=USTC)

# 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)

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# Team:USTC/Results

[![](https://static.igem.org/mediawiki/2019/b/b5/T--USTC--logo1.png)](https://2019.igem.org/Team%3AUSTC)

* Team

  [Team Members](https://2019.igem.org/Team%3AUSTC/Team)
  [Attributions](https://2019.igem.org/Team%3AUSTC/Attributions)
* Project

  [Description](https://2019.igem.org/Team%3AUSTC/Description)
  [Design](https://2019.igem.org/Team%3AUSTC/Design)
  [Experiment](https://2019.igem.org/Team%3AUSTC/Experiment)
  [Results](https://2019.igem.org/Team%3AUSTC/Results)
  [Notebook](https://2019.igem.org/Team%3AUSTC/Notebook)
  [Protocols](https://2019.igem.org/Team%3AUSTC/Protocals)
* Safety

  [Lab](https://2019.igem.org/Team%3AUSTC/Safety)
  [Kill
  Switch](https://2019.igem.org/Team%3AUSTC/Kill_Switch)
* [Model](https://2019.igem.org/Team%3AUSTC/Model)
* Human Practices

  [General HP](https://2019.igem.org/Team%3AUSTC/Human_Practices)
  [Integrated HP](https://2019.igem.org/Team%3AUSTC/Integrated_HP)
  [Collaborations](https://2019.igem.org/Team%3AUSTC/Collaborations)
* [Parts](https://2019.igem.org/Team%3AUSTC/Parts)
* [Awards](https://2019.igem.org/Team%3AUSTC/Awards)
* [**Judging Form**](https://igem.org/2019_Judging_Form?id=3099)

![Card image](https://static.igem.org/mediawiki/2019/0/05/T--USTC--Results.jpg)

[Electron
Transfer System](#ETS)
[aNAT](#aNAT)

# **Electron Transfer System**

### 1. DNA sequencing result and colony PCR

In order to verify the plasmid that we constructed, we designed 8 sequence-primers AF, AR,
BF,
BR, CF, CR, VF, VR and sequenced our plasmid, each one for 1000bp. The results are laid in
appendix/DNA\_sequencing/AF~VR.

Here is the result of plasmid extraction and colony PCR. Both are positive.

![Card image](https://static.igem.org/mediawiki/2019/c/c9/T--USTC--ETS.jpg)

Figure 1. The result of plasmid
extraction and colony PCR

### 2. Microbial Fuel Cell(MFC)

MFC device consists of cathode cell and anode cell separated by cation exchange membrane and
carbon felt soaked in cathode solution and anode solution respectively. The voltage between
the two carbon felt can be read by the detection device connected to the carbon felt.

When the bacteria that can express Mtr protein attached to the carbon felt at the cathode,
Mtr transmit electrons to the anode through the carbon felt. The protons in the solution
also pass through the cation exchange membrane to form a circuit and generate voltage
between the cathode and anode of the battery. So the voltage is positively related to the
ability of bacteria to release electrons.

We added K3[Fe(CN)6] cathode solution to the anode of MFC, added
aerobic mineral salt culture medium to the cathode, aerated the cathode with nitrogen and
connect the monitoring device. Then, we recorded the voltage between the positive and
negative electrodes every ten minutes after the bacterial solution was injected. The raw
data are laid in appendix/MFC. After ploted with the voltage as the ordinate and the time as
the abscissa, we got this graph.

![Card image](https://static.igem.org/mediawiki/2019/a/a1/T--USTC--MFC.jpg)

Figure 2. MFC

The diagram of the experimental results shows that the voltage produced by MTR
overexpression strain is stronger than that produced by empty plasmid strain, which shows
that MTR overexpression brings stronger electron releasing ability to *S. oneidensis*
MR-1.

### 3. Reduction of MO

The raw data are laid in appendix/reduction\_of\_MO/… . After necessary integration, we got an
excel as follows.

![Card image](https://static.igem.org/mediawiki/2019/8/83/T--USTC--xgt2.png)

Figure 3. Reduction of MO

Well H1~H8 were injected for MO concentration standard curve. After linear fitting, we got
the relation between OD465 and the concentration of MO.

![Card image](https://static.igem.org/mediawiki/2019/7/77/T--USTC--xgt1.png)

Figure 4.

According to this equation, we got the change of MO concentration of each group as time went
by. Then, we calculated the average concentration as final data and standard deviation as
error bar. After exponential fitting, we got this graph.

![Card image](https://static.igem.org/mediawiki/2019/d/d6/T--USTC--res_5.png)

Figure 5.

The exact reduction rate makes little sense because the incubation condition and original
concentration of azo dye are far from identical between lab experiment and factory practice.
The relative improvement, however, can prove that our engineered strain is a successful
system to reduce azo dye. From the fitting equations, we can get that the ratio of slopes at
the same MO concentration is
k=bExp/bWT=0.01362/0.0014=9.73. Thus, considering wildtype *S.
oneidensis* MR-1 itself is a wonderful “azo-dye-discolor strain”, we did construct a very
powerful azo dye reduction system with its amazing reduction efficiency as about 10 times
higher as the original strain.

### 4. Verification of QS system

In order to verify our QS system, we have done the QS verification experiment
(you can click [here](https://2019.igem.org/Team%3AUSTC/Experiment) to see how we
did the experiment).

We measured bacterial concentration (OD600 of 200μL medium in GREINER 96
F-BOTTOM) and the GFP intensity (excitation wavelength=485nm, emission wavelength=525nm,
gain=800) of 2X YT bacteria medium (with 50ng/ml Kan), engineered *S. oneidensis*
MR-1（with PYYDT-GFP plasmid）and wild type *S. oneidensis* MR-1(with empty PYYDT
plasmid) with time. The raw data are laid in appendix/QS\_verification/raw\_data/… .

On the data sheet, we can see an interesting thing that the GFP intensity of WT is reducing
with bacterial concentration growing. But it doesn’t matter, we use relative GFP intensity,
whose value equal to the value of the GFP intensity of engineered
*S. oneidensis* MR-1(with PYYDT-GFP plasmid)minus the value of the GFP intensity of
wild type *S. oneidensis* MR-1(with empty PYYDT plasmid). We also use relative
bacterial concentration, whose value is equal to the value of the OD600 of
engineered
*S. oneidensis* MR-1(with PYYDT-GFP plasmid)minus the value of the OD600 of
YT bacteria medium (with 50ng/ml Kan).

We use MatLab software to manage the above process and deal with the data and make fitted
curves. Firstly, we make fitted curves of relative concentration change with time(picture a)
and relative GFP intensity change with time(picture b). After that, we calculated the slope
of the curve of picture b as the relative value of expression and made it divided by the
value of relative concentration at each time, to get the value of relative expression
efficiency of GFP. Then, we make fitted curves of relative expression efficiency change of
GFP with relative concentration (picture c). Here are the picture a, b, c and the codes we
have programmed are laid in appendix/QS\_verification/matlab\_code/ PYYDT\_GFP\_code.

![Card image](https://static.igem.org/mediawiki/2019/5/51/T--USTC--QS.jpg)

Figure 6. Verification of QS system

On the picture(c), we can see that when the concentration is low (OD600 lower
than about 0.2), the relative expression efficiency of GFP is 0. And when the concentration
is higher than a certain value (OD600>0.2), the relative expression efficiency of
GFP starts to rise until the concentration reach another certain value (OD600=0.5).
Therefore, we can draw a conclusion that our QS system works well.

---

# **aNAT**

### Construction of aNAT expression vector

We clone the gene and vector with primers aNAT-F-SWJN, aNAT-R-SWJN, aNAT-backbone-F-SWJN and
aNAT-backbone-R-SWJN.

![Card image](https://static.igem.org/mediawiki/2019/d/d7/T--USTC--ysh_3.png)

Figure 7. PCR result of vectors

![Card image](https://static.igem.org/mediawiki/2019/9/93/T--USTC--ysh_4.png)

Figure 8. PCR result of aNAT

The bacteria PCR result is as follows

![Card image](https://static.igem.org/mediawiki/2019/9/94/T--USTC--ysh_5.png)

Figure 9. The bacteria PCR result

### Characterization of aromatic amine resistance

![Card image](https://static.igem.org/mediawiki/2019/d/da/T--USTC--Result-table-n.png)

Table 1. The average OD600 of *S.oneidensis*

![Card image](https://static.igem.org/mediawiki/2019/a/a0/T--USTC--Result-2.jpg)

Figure 10. The growing curve of different types of *S.oneidensis*

The result significantly demonstrates that *S.oneidensis* with aNAT grows better in
40ug/mL sulfanilamide than wild type and empty pYYDT controlling groups. The shaking of aNAT
and empty pYYDT growing curves is possibly because of the lack of nutrition in aerobic
mineral salt medium, and the pressure of kanamycin makes the growth of bacteria with pYYDT
unstable.

---

##### Experiment data

[Experiment data](https://static.igem.org/mediawiki/2019/3/36/T--USTC--data.zip)

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