iGEM 2020 — Team Xiamen_city

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# Team:Xiamen city/Notebook

非模块化方式使用layui

Entprenuership

![](https://static.igem.org/mediawiki/2020/0/03/T--Xiamen_city--logo.jpg)

**Nap Tuner**

---

* [Home](https://2020.igem.org/Team%3AXiamen_city)
* Project

  :   [Description](https://2020.igem.org/Team%3AXiamen_city/Description)
  :   [Design](https://2020.igem.org/Team%3AXiamen_city/Design)
  :   [Experiments](https://2020.igem.org/Team%3AXiamen_city/Experiment)
  :   [Notebook](https://2020.igem.org/Team%3AXiamen_city/Notebook)
  :   [Result](https://2020.igem.org/Team%3AXiamen_city/Result)
  :   [Safety](https://2020.igem.org/Team%3AXiamen_city/Safety)
* [Contribution](https://2020.igem.org/Team%3AXiamen_city/Contribution)
* [Engineering](https://2020.igem.org/Team%3AXiamen_city/Engineering)
* [Implementation](https://2020.igem.org/Team%3AXiamen_city/Implementation)
* Parts

  :   [Basic Parts](https://2020.igem.org/Team%3AXiamen_city/Basic%20Parts)
  :   [Composite Parts](https://2020.igem.org/Team%3AXiamen_city/Composite%20Parts)
  :   [Parts Collection](https://2020.igem.org/Team%3AXiamen_city/Parts%20Collection)
* [Model](https://2020.igem.org/Team%3AXiamen_city/Model)
* Human Practice

  :   [Integrated Human Practice](https://2020.igem.org/Team%3AXiamen_city/Human_Practices)
  :   [Fundraising](https://2020.igem.org/Team%3AXiamen_city/Fundraising)
  :   [Entrepreneurship](https://2020.igem.org/Team%3AXiamen_city/Entrepreneurship)
* Team

  :   [Team Member](https://2020.igem.org/Team%3AXiamen_city/Team)
  :   [Collaborations](https://2020.igem.org/Team%3AXiamen_city/Collaborations)
  :   [Attributions](https://2020.igem.org/Team%3AXiamen_city/Attributions)

Notebook

●7/18 research topic discussion (led by dr. wang from Chinese Academy of Science)

●7/25 first offline bonding

○established our team name, team logo, and team slogan

○Captain election

○Made several icebreakers to help us get to know each other and build friendship and team soul

●7/26 wet team day 1

○Learn about the use of laboratory apparatus, the principle of reagent dilution, and PCR principal

○Configure PCR reaction system and gain the DNA fragments from the PCR reaction

○Configure agarose electrophoresis and test the DNA fragments from PCR reaction

●7/27 wet team day 2

○PCR products of sod and cat genes were separated and purified from the gel extraction.

○Test the DNA concentration

○Double digestion and connection of 2 genes and plasmid

○purified DNA fragment was digested with restriction enzymes and the digested products was ligated to the linear plasmid.

●7/28 wet team day 3

○Verify the right clones by using PCR and send the verified samples for sequencing

○preparation and transformation of competent cells from Escherichia coli.

○Make solid LB plates

●7/29 wet team & dry team exchange

○Compose WeChat posts

○Advertise our market demand survey of stay-up-late alleviation products

●7/30 wet team day 4

○Verify the sequencing of sod and cat gene

○Extract plasmid

○cultivate the protein expression of bacterial strain

○IPTG overnight induction

●7/31 wet team, day 5

○Centrifugate the inducted bacterial cells

○The target proteins were released from the cells by sonication.

○Purify SOD and CAT protein by nickel chromatography

○Detection of SOD and CAT proteins by using SDS page

●8/1

○Take official portrait for wiki

●8/3 wet team, day 6

○Concentrate and desalinate the SOD and CAT protein

○The enzyme activity of SOD was examined.

●8/4 wet team, day 7

○The enzyme activity of CAT was examined.

○Review the entire experimental ideas

●8/5

○Team report speech

![](https://static.igem.org/mediawiki/2020/1/19/T--Xiamen_city--notebook01.JPG)

Contact Email: ni3222601872@gmail.com

Wechat: Search "NapTuner" in WeChat

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# Team:Xiamen city/Engineering

非模块化方式使用layui

Entprenuership

![](https://static.igem.org/mediawiki/2020/0/03/T--Xiamen_city--logo.jpg)

**Nap Tuner**

---

* [Home](https://2020.igem.org/Team%3AXiamen_city)
* Project

  :   [Description](https://2020.igem.org/Team%3AXiamen_city/Description)
  :   [Design](https://2020.igem.org/Team%3AXiamen_city/Design)
  :   [Experiments](https://2020.igem.org/Team%3AXiamen_city/Experiment)
  :   [Notebook](https://2020.igem.org/Team%3AXiamen_city/Notebook)
  :   [Result](https://2020.igem.org/Team%3AXiamen_city/Result)
  :   [Safety](https://2020.igem.org/Team%3AXiamen_city/Safety)
* [Contribution](https://2020.igem.org/Team%3AXiamen_city/Contribution)
* [Engineering](https://2020.igem.org/Team%3AXiamen_city/Engineering)
* [Implementation](https://2020.igem.org/Team%3AXiamen_city/Implementation)
* Parts

  :   [Basic Parts](https://2020.igem.org/Team%3AXiamen_city/Basic%20Parts)
  :   [Composite Parts](https://2020.igem.org/Team%3AXiamen_city/Composite%20Parts)
  :   [Parts Collection](https://2020.igem.org/Team%3AXiamen_city/Parts%20Collection)
* [Model](https://2020.igem.org/Team%3AXiamen_city/Model)
* Human Practice

  :   [Integrated Human Practice](https://2020.igem.org/Team%3AXiamen_city/Human_Practices)
  :   [Fundraising](https://2020.igem.org/Team%3AXiamen_city/Fundraising)
  :   [Entrepreneurship](https://2020.igem.org/Team%3AXiamen_city/Entrepreneurship)
* Team

  :   [Team Member](https://2020.igem.org/Team%3AXiamen_city/Team)
  :   [Collaborations](https://2020.igem.org/Team%3AXiamen_city/Collaborations)
  :   [Attributions](https://2020.igem.org/Team%3AXiamen_city/Attributions)

![](https://static.igem.org/mediawiki/2020/2/21/T--Xiamen_city--engineering01.jpg)
![](https://static.igem.org/mediawiki/2020/8/83/T--Xiamen_city--engineering02.jpg)
![](https://static.igem.org/mediawiki/2020/b/bf/T--Xiamen_city--engineering03.jpg)
![](https://static.igem.org/mediawiki/2020/0/07/T--Xiamen_city--engineering04.jpg)
![](https://static.igem.org/mediawiki/2020/4/41/T--Xiamen_city--engineering05.jpg)

Contact Email: ni3222601872@gmail.com

Wechat: Search "NapTuner" in WeChat

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Xiamen city/Experiments

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* [Home](https://2020.igem.org/Team%3AXiamen_city)
* [Inspiration](https://2020.igem.org/Team%3AXiamen_city/Inspiration)
* Project
  + [Description](https://2020.igem.org/Team%3AXiamen_city/Description)
  + [Design](https://2020.igem.org/Team%3AXiamen_city/Design)
  + [Experiments](https://2020.igem.org/Team%3AXiamen_city/Experiments)
  + [Notebook](https://2020.igem.org/Team%3AXiamen_city/Notebook)
  + [Result](https://2020.igem.org/Team%3AXiamen_city/Result)
  + [Safety](https://2020.igem.org/Team%3AXiamen_city/Safety)
* [Engineering](https://2020.igem.org/Team%3AXiamen_city/Engineering)
* [Contribution](https://2020.igem.org/Team%3AXiamen_city/Contribution)
* [Model](https://2020.igem.org/Team%3AXiamen_city/Model)
* [Implementation](https://2020.igem.org/Team%3AXiamen_city/Implementation)
* Human Practice
  + [Fundrasing](https://2020.igem.org/Team%3AXiamen_city/Fundrasing)
  + [Integrated Human Practice](https://2020.igem.org/Team%3AXiamen_city/Integrated%20Human%20Practice)
* [Entprenuership](https://2020.igem.org/Team%3AXiamen_city/Entprenuership)
* Team
  + [Members](https://2020.igem.org/Team%3AXiamen_city/Members)
  + [Attributions](https://2020.igem.org/Team%3AXiamen_city/Attributions)
  + [Collaboration](https://2020.igem.org/Team%3AXiamen_city/Collaboration)

## [LB Kanamycin Culture](#collapseOne)

### LB Kanamycin Culture(liquid)

#### 200ml double distilled H2O (ddH2O)

#### 2g Tryptone

#### 2g NaCl

#### 1g Yeast Extract

#### 0.2ml Kanamycin 50mg/ml

### LB Kanamycin Culture (solid)

#### 200ml ddH2O

#### 2g Tryptone

#### 2g NaCl

#### 1g Yeast Extract

#### 0.2ml Kanamycin 50mg/ml

#### 3g Agar

## [PCR Extraction for the gene fragments of SOD and CAT](#collapseTwo)

### SOD/CAT Extraction

#### Material 25ᶙL ddH2O

#### 3ᶙL 25MM MgSO4

#### 5ᶙL 2MM dNTPs

#### 5ᶙL pmol/ᶙL Primer Front

#### 5ᶙL pmol/ᶙL Primer Rear

#### 1ᶙL Template

#### 5ᶙL 10\*PCR Buffer

#### 1ᶙL KOD-Plus-Neo (polymerase)

### Procedure

#### For pre-denaturation--95℃ 5min

#### For denaturation--95℃ 30s

#### For annealer--55℃ 30s

#### For extension--68℃ 4min

#### Repeat above step for 35 times

#### For complete extension--68℃ 5min

#### For preservation-- 4℃

## [Gel test](#collapse3)

### Motive: detect whether the genes of SOD and CAD multiply successfully Material:

#### 1\*TAE buffer

#### 6\*TAE buffer

#### Agarose

#### DNAgreen

#### SOD gene fragments

#### CAT gene fragments

#### Loading marker

### Procedure

#### 1. Produce gel.

#### A. pour 1\*TAE buffer in a conical flask. Weight matched mass of agarose.

#### B. Cover the mouth of flask with plastic wrap. Stick some tiny holes on plastic wrap. Heat the conical flask with microwave oven until agarose completely dissolving.

#### C. Cool flask down to 50-60℃, and then add 1/10000 DNAgreen. Blend the gel.

#### D. Pour gel into mould and insert comb. Wait until solidification.

#### 2. Blend SOD fragments with 6\*TAE buffer (1:5). Blend CAT fragments with 6\*TAE buffer (1:5)

## [Gel Extraction](#collapse4)

### Material:<>

#### Buffer DE-A

#### 6\*TAE buffer

#### Buffer DE-B

#### Washing Buffer 1

#### Washing Buffer 2

#### Spin column

#### Target gel

### Procedure

#### 1. Cut gel containing target DNA fragment and transfer target gel into a 1.5ml Eppendorf (EP tube)

#### 2. Inject Buffer DE-A (Volume of Buffer: Volume of gel = 3:1). Heat EP tube until gel completely melting.

#### 3. Inject Buffer DE-B (Volume of Buffer DE-B : Volume of Buffer DE-A = 2:1)

#### 4. A. Transfer mixed liquid into 2ml EP tube equipped with spin column. Centrifuge tube at the centrifugal force of 12000g for 1min. Pour out the filtrate.

#### B. Inject 500ᶙl Buffer W1. Centrifuge tube for 30s. Pour out the filtrate.

#### C. Inject 700ᶙl Buffer W2. Centrifuge tube for 30s. Pour out the filtrate. Repeat the step C again.

#### Centrifuge tube at the speed of 12000rmp for 1 min.

#### 5. Transfer spin column into a new tube. Inject 25-30ᶙl ddH2O. Wait for 1min and then centrifuge tube. Collect the filtrate containing target DNA fragments.

## [Cleavage](#collapse5)

### Cleavage of SOD

#### 1ᶙg DNA

#### 1ᶙl Nhel

#### 5ᶙl 10\*NE Buffer

#### 44ᶙl ddH2O

### Cleavage of CAT

#### 1ᶙg DNA

#### 1ᶙl XhoI

#### 5ᶙl 10\*NE Buffer

#### 44ᶙl ddH2O

### Cleavage of Plasmid

#### 1ᶙg plasmid

#### 1ᶙl Nhel

#### 1ᶙl XhoI

#### 10ᶙl 10\*NE Buffer

#### 38ᶙl ddH2O

## [Connection](#collapse6)

#### After cleavage, blend plasmids with SOD, and plasmids with CAT together (Cb : Cb = 1:1). Then blend blended groups together. All the step needs involvement of T4 DNA ligase.

## [DNA sequencing](#collapse7)

#### Confer simple of CAT and SOD to sequencing center (Use T7 common primers).

## [Transform and multiplication E. coli(DH5α and BL21DH3)](#collapse8)

#### For melting-1. Take out the four Eppendorfs (two of them contain DH5α competent cells and the other contain BL21DH3 competent cells from -80℃ condition)，and insert these four tubes into ice, wait 5 min.

#### For plasmids attaching to E. coli membrane-2. Inject recombinant plasmids (SOD and CAT) into four tubes (DH5α with SOD, DH5α with CAT, BL21DH3 with SOD, BL21DH3 with CAT), and wait 25 min

#### For accepting plasmids-3. Put these tubes in 42℃ condition for 45s, and then bring back to the ice immediately.

#### For growing cell wall-4. Inject 700ᶙl LB liquid culture (free of antibiotic). Shake tubes at the speed of 200rpm in Shaking Incubator for 60 min

#### For concentrating-5. Centrifuge tubes at the speed of 5000rmp for 1 min. Extract the upper transparent liquid until 100ᶙl and use pipette to blend upper transparent liquid and E. coli in the bottom.

#### Apply each tubes of liquid evenly on the different LB cultures (contain Kanamycin). Make signatures to each LB culture.

#### For propagating-6. Inverse petrie dish in 37℃ condition for 12h.

## [Preliminary screening](#collapse9)

#### 1. Select 10 single E. coli colonies of four LB cultures, and sign 1-10 to these 10 colonies in order to select again. Put colonies into 1.5ml EP tubes

#### 2. Blend each E. coli colony with PCR system for extraction.

### PCR system: 9ᶙl mix (including dNTPs, polymerase, and PCR buffer)

#### 9ᶙl ddH2O

#### 1ᶙl T7 primer

#### 1ᶙl template (E. coli colonies)

#### 3. Take the step of gel test and gel extraction to every colony in order to preliminary screening out target colonies.

## [DNA sequencing for secondary screening](#collapse10)

#### 1. Confer simple of target colonies to sequencing center (Use T7 common primers).

#### 2. Propagate target colonies in BL liquid culture.

## [提取蛋白](#collapse11)

#### 1. Pour target E. coli liquid culture in 50 ml EP tubes.

#### 2. Centrifuge EP tubes and collect bottom E. coli.

#### 3. Use ultrasonic wave to disrupt the E. coli,

#### 4. Centrifuge the disruption of E. coli at the centrifugal force of 12000g. Collect the upper transparent liquid. Filter upper transparent liquid through nickel column.

#### 5. Wash through nickel column by different molar concentration of imidazole. (0mM, 50mM, 100mM, 250mM, 500mM), collect and sign each filtrate.

### The imidazole systems:

#### 0mM imidazole: 15ml loading buffer

#### 50mM imidazole: 0.15ml 5M imidazole and 14.85ml loading buffer

#### 100mM imidazole: 0.3ml 5M imidazole and 14.7ml loading buffer

#### 250mM imidazole: 0.75ml 5M imidazole and 14.25ml loading buffer

#### 500mM imidazole: 1.5ml 5M imidazole and 13.5ml loading buffer

#### 6. Blend filtrate with gel buffer system for protein gel test.

#### 7. The result indicates that filtrate with 100mM imidazole contains SOD/CAT.

## [Test SOD enzyme activity](#collapse12)

### Material:

#### SOD testing buffer（buffer）

#### NBT

#### Xanthine Oxidase (XO) solution

#### 40\*Primer fluid

#### SOD solution

#### 1. Preparation

#### A. Make working solution. Blend XO solution with NBT and SOD testing buffer together (1:1:158)

#### B. Blend 40\*primer fluid with SOD testing buffer (1:39)

#### C. Dilute SOD solution to five ranks (20U/ml, 10U/ml, 5U/ml, 2U/ml, 1U/ml, 0.5U/ml)

#### 2. A. Set up 8 groups in 96-hole plate.

#### Samples: 20ᶙl SOD solution (20U/ml, 10U/ml, 5U/ml, 2U/ml, 1U/ml, 0.5U/ml)+160ᶙl working solution+20ᶙl primer fluid

#### Blank1: 20ᶙl buffer+160ᶙl working solution+20ᶙl primer fluid

#### Blank2: 40ᶙl buffer+160ᶙl working solution

#### Blank3: 20ᶙl SOD solution+20ᶙl buffer+160ᶙl working solution

#### B. Incubate the groups for 30min in 37℃.

#### C. Test absorbance in 560nm.

#### 3. Calculate SOD enzyme activity

#### Inhibition percentage=[(Ablank1-Ablank2) - (Asample-Ablank3)]/(Ablank1-Ablank2) \* 100%

#### IP (SOD solution:0.5U/ml) = [(0.236-0.041) - (0.101-0.043)]/(0.236-0.041)\*100% =0.137/0.197\*100% =69.543%(30% < IP < 70%)

#### Enzyme activity percentage=inhibition percentage / (1-inhibition percentage) units =69.543% / (1-69.543%) units =2.283units (SOD solution:0.5U/ml)

## [Test CAT enzyme activity](#collapse13)

### Material:

#### CAT testing buffer

#### H2O2(1M) solution

#### CAT termination solution

#### Chromogenic substrate

#### Peroxidase

#### CAT

#### 1. Preparation

#### A. Dilute H2O2 solution to 5mM and 250mM.

#### B. Make 4ml chromogenic working solution on the ice (peroxidase : chromogenic substrate=1:1000).

#### 2. determination of standard curve

#### A. Inject 0, 12.5, 25, 50, 75ᶙl 5mM H2O2 solution in 1.5ml EP tubes. Inject CAT testing buffer to each tube to 100ᶙl.

#### B. Transfer 4ᶙl liquid in each tube into 96-hole plate. Incubate plate for 20min in 25℃ and test the absorbance in A520.

#### C. Calculate standard curve.

#### 3. determination of sample

#### A. Set up 5 samples and blank in EP tubes, incubate EP tubes for 5min in 25℃.S1: 0.5ᶙl CAT + 39.5ᶙl buffer + 10ᶙl 250mM H2O2S2: 1ᶙl CAT + 39ᶙl buffer + 10ᶙl 250mM H2O2S3: 2ᶙl CAT + 38ᶙl buffer + 10ᶙl 250mM H2O2S4: 4ᶙl CAT + 36ᶙl buffer + 10ᶙl 250mM H2O2S5: 8ᶙl CAT + 32ᶙl buffer + 10ᶙl 250mM H2O2Blank: 40ᶙl buffer + 10ᶙl 250mM H2O2B. Inject 450ᶙl termination solution. Bland the tubes gently.C. Inject 40ᶙl buffer in 6 new EP tubes. Inject 10ᶙl of aforementioned six systems separately in new tubes. Bland the tubes gently.D. Transform 10ᶙl solution from six tubes to 96-hole plat. Inject 200ᶙl chromogenic working solution in 6 holes.E. Incubate plate for 15min in 25℃. Test the absorbance in A520.4. CalculationStandard curve: A520=84.976[mM H202] + 0.0605The rest H2O2 in sample= (Asample-Ablank)/kCAT enzyme activity= (H2O2 Moles consumed × Dilution times) / (Time×Volume×Concentration of protein) units

电话号码：+333-444-5555

地址：湖北省武汉市江夏区流芳大道404号

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# Team:Xiamen city/Results

# Results

You can describe the results of your project and your future plans here.

### 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 also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* 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)