iGEM 2018 — Team Jilin_China

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Jilin China/Notebook

This Page Will Be Evaluated!
What should this page have?
Chronological notes of what your team is doing.
Brief descriptions of daily important events.
Pictures of your progress.
Mention who participated in what task.

[ ]

* Background
  [ ]
  + [Thermoregulation](/Team%3AJilin_China/Background)
  + [Thermosensor](/Team%3AJilin_China/Background2)
* Project
  [ ]
  + [Overview](/Team%3AJilin_China/Project/Overview)
  + [Design](/Team%3AJilin_China/Design)
  + [Measurement](/Team%3AJilin_China/Measurement)
  + [Construction](/Team%3AJilin_China/Construction)
  + [Improve](/Team%3AJilin_China/Improve)
* Achievement
  [ ]
  + [Results](/Team%3AJilin_China/Result/Version_1)
    - [Version 1.0](/Team%3AJilin_China/Result/Version_1)
    - [Version 2.0](/Team%3AJilin_China/Result/Version_2)
    - [Version 3.0](/Team%3AJilin_China/Result/Version_3)
  + [Toolkit Explorer](/Team%3AJilin_China/Part/Search_Engine)
  + [Demonstrate](/Team%3AJilin_China/Demonstrate)
  + [For Judgement](/Team%3AJilin_China/For_Judgement)
* Model
  [ ]
  + [Overview](/Team%3AJilin_China/Model)
  + [Curve Fitting](/Team%3AJilin_China/Model/Curve_Fitting)
  + [Screening System](/Team%3AJilin_China/Model/Screening_System)
  + [Thermol Loss](/Team%3AJilin_China/Model/For_Practices)
* Lab
  [ ]
  + [Notebook](/Team%3AJilin_China/Notebook)
  + [Protocol](/Team%3AJilin_China/Experiments)
  + [Interlab](/Team%3AJilin_China/InterLab)
* Parts
  [ ]
  + [Overview](/Team%3AJilin_China/Parts)
  + [Basic Part](/Team%3AJilin_China/Basic_Part)
  + [Composite Part](/Team%3AJilin_China/Composite_Part)
  + [Part Collection](/Team%3AJilin_China/Part_Collection)
* Practices
  [ ]
  + [Integrated HP](/Team%3AJilin_China/Human_Practices)
  + [Engagement](/Team%3AJilin_China/Public_Engagement)
  + [Safety](/Team%3AJilin_China/Safety)
  + [Meetup](https://2018.igem.org/Team%3AJilin_China/Meetup)
  + [Collaborations](/Team%3AJilin_China/Collaborations)
* Team
  [ ]
  + [Team](/Team%3AJilin_China/Team)
  + [Attributions](/Team%3AJilin_China/Attributions)

LAB JOURNAL
NOTEBOOK

## Notebook

# CHOOSE A MONTH

* July
* Aug
* Sep

* Click a month above then details will be shown here. :)

* ![]()

  ![](https://static.igem.org/mediawiki/2018/a/ad/T--Jilin_China--Notebook--July.png)

  ![](#)

* ![](https://static.igem.org/mediawiki/2018/c/c4/T--Jilin_China--Notebook--August01.png)

  ![](https://static.igem.org/mediawiki/2018/3/34/T--Jilin_China--Notebook--August02.png)

  ![](#)

* ![](https://static.igem.org/mediawiki/2018/2/25/T--Jilin_China--Notebook--September01.png)

  ![](#)

[Jilin University](http://www.jlu.edu.cn/)

[Office of Undergraduate Education](http://jwc.jlu.edu.cn/)

[Office of Global Engagement](http://http://oic.jlu.edu.cn/)

[School of Life Science](http://life.jlu.edu.cn/)

[National Engineering Laboratory for AIDS Vaccine](http://nlv.jlu.edu.cn/)

[Key Laboratory for
Molecular Enzymology and Engineering](http://www.mee.jlu.edu.cn/)

CONTACT US

[![Weixin](https://static.igem.org/mediawiki/2018/d/d1/T--Jilin_China--Footer--Contacts--Weixin.png)](http://www.aladdin-e.com/)
[![Twitter](https://static.igem.org/mediawiki/2018/3/37/T--Jilin_China--Footer--Contacts--Twitter.png)](https://twitter.com/iGEM_JLU_China?s=17)

Jilin\_China@Outlook.com

ACKNOWLEDGEMENTS

[![Taimei](https://static.igem.org/mediawiki/2018/b/bd/T--Jilin_China--Footer--Companies--Taimei.png)](https://www.mobilemd.cn/)
[![Comate](https://static.igem.org/mediawiki/2018/d/dc/T--Jilin_China--Footer--Companies--Comate.png)](http://www.comatebio.com/)
[![MathWorks](https://static.igem.org/mediawiki/2018/c/c5/T--Jilin_China--Footer--Companies--MathWorks.png)](http://www.mathworks.com)
[![GeneScript](https://static.igem.org/mediawiki/2018/7/7a/T--Jilin_China--Footer--Companies--GeneScript.png)](https://www.genscript.com/)
[![GitHub](https://static.igem.org/mediawiki/2018/2/25/T--Jilin_China--Footer--Companies--GitHub.png)](https://github.com/)
[![Transgen](https://static.igem.org/mediawiki/2018/d/d8/T--Jilin_China--Footer--Companies--Transgen.png)](http://www.transgen.com.cn/)


[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Jilin China/Experiments

[ ]

* Background
  [ ]
  + [Thermoregulation](/Team%3AJilin_China/Background)
  + [Thermosensor](/Team%3AJilin_China/Background2)
* Project
  [ ]
  + [Overview](/Team%3AJilin_China/Project/Overview)
  + [Design](/Team%3AJilin_China/Design)
  + [Measurement](/Team%3AJilin_China/Measurement)
  + [Construction](/Team%3AJilin_China/Construction)
  + [Improve](/Team%3AJilin_China/Improve)
* Achievement
  [ ]
  + [Results](/Team%3AJilin_China/Result/Version_1)
    - [Version 1.0](/Team%3AJilin_China/Result/Version_1)
    - [Version 2.0](/Team%3AJilin_China/Result/Version_2)
    - [Version 3.0](/Team%3AJilin_China/Result/Version_3)
  + [Toolkit Explorer](/Team%3AJilin_China/Part/Search_Engine)
  + [Demonstrate](/Team%3AJilin_China/Demonstrate)
  + [For Judgement](/Team%3AJilin_China/For_Judgement)
* Model
  [ ]
  + [Overview](/Team%3AJilin_China/Model)
  + [Curve Fitting](/Team%3AJilin_China/Model/Curve_Fitting)
  + [Screening System](/Team%3AJilin_China/Model/Screening_System)
  + [Thermol Loss](/Team%3AJilin_China/Model/For_Practices)
* Lab
  [ ]
  + [Notebook](/Team%3AJilin_China/Notebook)
  + [Protocol](/Team%3AJilin_China/Experiments)
  + [Interlab](/Team%3AJilin_China/InterLab)
* Parts
  [ ]
  + [Overview](/Team%3AJilin_China/Parts)
  + [Basic Part](/Team%3AJilin_China/Basic_Part)
  + [Composite Part](/Team%3AJilin_China/Composite_Part)
  + [Part Collection](/Team%3AJilin_China/Part_Collection)
* Practices
  [ ]
  + [Integrated HP](/Team%3AJilin_China/Human_Practices)
  + [Engagement](/Team%3AJilin_China/Public_Engagement)
  + [Safety](/Team%3AJilin_China/Safety)
  + [Meetup](https://2018.igem.org/Team%3AJilin_China/Meetup)
  + [Collaborations](/Team%3AJilin_China/Collaborations)
* Team
  [ ]
  + [Team](/Team%3AJilin_China/Team)
  + [Attributions](/Team%3AJilin_China/Attributions)

PROTOCOLS

## Protocols

* ## Protocols

  ### 1. Annealing Oligos

  **Experimental procedure：**

  |  |  |
  | --- | --- |
  | Reagent | volume |
  | Oligo1 (50μM) | 9μl |
  | Oligo2 (50μM) | 9μl |
  | T4 DNA Ligase Buffer | 2μl |
  | Total | 20μl |

  1. Label the 0.2 ml eppendorf tubes.

  2. Add all the reagents to the labeled eppendorf tube in ice bath.

  3. Blend the reagents in the 0.2 ml eppendorf tube by short centrifugation.

  4. Place the 0.2 ml eppendorf tubes into PCR instrument and run the annealing program.

  **Instrument flow：**

  step1：95℃——3min

  step2：94℃——1min

  step3：93℃——1min-1.0℃/cycle

  step4：56 times to step3

  step5：37℃——1min

  step6：4℃——∞

  step7：END

  ### 2. Phosphorylation

  **Experimental procedure：**

  |  |  |
  | --- | --- |
  | Reagent | volume |
  | Oligo Annealing Products | 4µl |
  | PNK | 0.4µl |
  | T4 DNA Ligase Buffer | 1.0µl |
  | ddH2O | 4.6µl |
  | Total | 10μl |

  MIX=PNK+T4 DNA Ligase Buffer+ddH2O

  1. Label the 1.5 ml eppendorf tubes and the 0.2ml eppendorf tubes.

  2. First configure enough MIX according to the amount of experiment requirement.

  3. Add all the reagents to a 0.2 ml eppendorf tube in ice bath.

  4. Blend the reagents in the 0.2 ml eppendorf tube by short centrifugation.

  5. Place the 0.2 ml eppendorf tubes into PCR instrument and run the phosphorylation program.

  **Instrument Flow:**

  Step1: 37℃——60min

  Step2: 4℃——∞

  Step3: END

  ### 3. Golden Gate Assembly

  **Experimental Procedure:**

  |  |  |
  | --- | --- |
  | Reagent | volume |
  | Phosphorylation Products | 1µl |
  | T4 DNA Ligase | 0.2µl |
  | T4 DNA Ligase Buffer | 0.5µl |
  | Type IIS Restriction Endonuclease | 0.2µl |
  | Backbone Plasmid | 0.2µl (~200ng) |
  | ddH2O | 2.9µl |
  | Total | 5μl |

  MIX=T4 DNA Ligase+T4 DNA Ligase Buffer+Type IIS Restriction Endonuclease+Backbone Plasmid+ddH2O

  1. Label the 1.5 ml eppendorf tubes and the 0.2 ml eppendorf tubes.

  2. First configure enough MIX according to the amount of experiment requirement.

  3. Add all the reagents to a 0.2 ml eppendorf tube in ice bath.

  4. Blend the reagents in the 0.2 ml eppendorf tube by short centrifugation.

  5. Place the 0.2 ml eppendorf tubes in PCR instrument and run the Golden Gte program.

  **Instrument Flow**

  Step1: 37℃——5min

  Step2: 16℃——5min

  Step3: 20 times to step1

  Step4: 50℃——5min

  Step5: 80℃——5min

  Step6: 4℃——∞

  Step7: END

  ### 4. Preparetion of Competent Cell

  1. Prepare the sterile Transformation & Storage Solution(TSS)

  Filter the 2\*TSS with a 0.22μm filter

  2. Making Competent cell

  Apparatus: test tubes, 50ml eppendorf tubes, 0.5ml eppendorf tubes, 150ml conical flasks, ice box.

  Day 1: Take a DH5α streak on antibiotic-free plates and overnight culture (12-18h).

  Day 2: Pick a colony and inoculate in 5ml LB. Grow the cells overnight (12-16h) at 37℃ and 220 rpm.

  Day 3: Make a 1:100 dilution of overnight culture in LB. Grow the cells at 37 ° C and 225 rpm until the OD600 is about 0.3~0.4.

  Transfer the culture to a clean 50 ml eppendorf tube. Put the tube in ice bath for 30 minutes to cool the culture to 0℃. Centrifugation in 4℃, 1000g for 10 minutes, then discard the supernatant and gather the bacterium.

  Add 2.5 ml pre-cooled LB. Then add 2.5 ml pre-cooled 2\*TSS and blend them gently. Put the tube in ice bath and get competent cells 10 minutes later.

  ### 5. Transformation

  1. Label the 1.5 ml eppendorf tubes.

  2. Let competent cells melt naturally in ice bath, and pre-cool the eppendorf tubes.

  3. Blend 50μL competent cells into an eppendorf tube with 5μL Golden Gate products gentlely.

  4. Place the mixed plasmid and the competent cells in ice bath for 30 minutes.

  5. Heat shock 90s in a 42℃ water bath

  5. Remove the water bath, adjust the temperature to 42℃, heat shock 90s, pay attention to timing.

  6. Transfer the tubes quickly back to ice for 10 minutes.

  7. Incubate the cells in 450μL LB medium + Chloramphenical.

  8. Grow the cells at 37 °C and 220rpm for 1 hour

  9. Aseptically spread plate on LB(with Chloramphenical) + Cam plates.

  10. Incubate at 37°C overnight.

  ### 6. Pick a Monoclonal Colony:

  Pick a colony from each of the transformation plates and inoculate in 5mL LB medium + Chloramphenicol. Let the cells grow overnight at 37°C and 220 rpm.

  ### 7. Culture Preservation

  1. Make a 1:1 dilution of each overnight culture in glycerinum (500μL of culture into 500μL of glycerinum).

  2. Store the bacterium in -20℃.

  ### 8. Kit extraction plasmid

  Strictly refer to the instruction manual of the kit and follow the procedure.

  1. Take 1-4 ml of the overnight culture solution, add to the 1.5ml eppendorf tube, and centrifuge for 1 minute at 12,000 rpm (~13,400×g) using a conventional benchtop centrifuge. Try to remove the supernatant as much as possible. (Centrifuge the bacterial pellet into a centrifuge tube and collect the bacterial pellet into a centrifuge tube).

  2. Add 150μL of buffer P1 tao the 1.5ml eppendorf tube containing the bacterial pellet, and completely suspend the bacterial pellet using a pipette or vortex shaker.

  3. Add 150μL of buffer P2 to the 1.5ml eppendorf tube and gently invert it 6-8 times to fully lyse the cells.

  4. Add 350μL of buffer P5 to the 1.5ml eppendorf tube and quickly flip it up and down 12–20 times, mix thoroughly, and a white flocculent precipitate will appear. Centrifuge at 12,000 rpm (~13,400 xg) for 2 minutes at which time a precipitate formed at the bottom of the 1.5ml eppendorf tube.

  5. Transfer the supernatant collected in the previous step to the adsorption column CP3 with a pipette (the adsorption column is placed in the collection tube), taking care not to aspirate the precipitate as much as possible. Centrifuge at 12,000 rpm (~13,400×g) for 30 seconds, drain the liquid from the collection tube, and place the adsorption column CP3 into the collection tube.

  6. Add 300 μL of buffer PWT to the adsorption column CP3 (please check if anhydrous ethanol has been added), centrifuge at 12,000 rpm (~13,400×g) for 30 sec, pour off the waste liquid in the collection tube, and place the adsorption column CP3. Into the collection tube.

  7. Place the adsorption column CP3 in a collection tube and centrifuge at 12,000 rpm (~13,400×g) for 1 minute to remove the residual buffer from the adsorption column.

  8. Place the adsorption column CP3 in a clean centrifuge tube, add 50μL ddH2O to the middle of the adsorption membrane, leave it at room temperature for 3 minutes, centrifuge at 12,000 rpm (~13,400×g) for 30 sec, and collect the plasmid solution into the centrifuge tube.

  ### 9. DNA concentration measurement

  Use NanoDrop2000 measure the plasmid concentration.

  ### 10. Digestion

  Single digestion: 6μL plasmid+0μL EcoRI+0.2μL PstI+1μL Cutsmart+2.8μLddH2O

  Double digestion: 6μL plasmid+0.2μL EcoRI+0.2μL PstI+1μL Cutsmart+2.6μLddH2O

  Digest the plasmid at 37℃ for an hour.

  ### 11. Agarose gel electrophoresis

  1. Add 0.7% agarose to 35ml TAE and dissolve them by microwave.

  2. After the solution cooled, add 7μLEB to 35mlTAE. Blend and pour it into a board. Then insert a sample comb and wait until it form a gel.

  3. Add sample: 3μL marker, 6μL sample. Run at 120V for about 25 minutes.

  ### 12. Fluorescence measurement

  1. Take 100μL glycerol bacteria and inoculate in 5ml LB medium + chloramphenicol in test tube.

  2. Grow the bacteria at 37 °C and 220 rpm until the Abs600 is about 0.1.

  3. Dilute the cultures further to a target Abs600 of 0.02 in a final volume of 8 ml LB medium + Chloramphenicol in test tube.

  4. Add 100μL of each sample to a black 96-well plate, prior to incubation.

  5. Incubate the remainder of the cultures at the target temperature and 220 rpm.

  6. Measure the fluorescence and Abs600. The fluorescence is measured in sensitivity 75. The measurement is protected from light.

  7. At each time point 0 hours, 4 hours, 6 hours, 8 hours and 10 hours, repeat Step 4-6.

[Jilin University](http://www.jlu.edu.cn/)

[Office of Undergraduate Education](http://jwc.jlu.edu.cn/)

[Office of Global Engagement](http://http://oic.jlu.edu.cn/)

[School of Life Science](http://life.jlu.edu.cn/)

[National Engineering Laboratory for AIDS Vaccine](http://nlv.jlu.edu.cn/)

[Key Laboratory for
Molecular Enzymology and Engineering](http://www.mee.jlu.edu.cn/)

CONTACT US

[![Weixin](https://static.igem.org/mediawiki/2018/d/d1/T--Jilin_China--Footer--Contacts--Weixin.png)](http://www.aladdin-e.com/)
[![Twitter](https://static.igem.org/mediawiki/2018/3/37/T--Jilin_China--Footer--Contacts--Twitter.png)](https://twitter.com/iGEM_JLU_China?s=17)

Jilin\_China@Outlook.com

ACKNOWLEDGEMENTS

[![Taimei](https://static.igem.org/mediawiki/2018/b/bd/T--Jilin_China--Footer--Companies--Taimei.png)](https://www.mobilemd.cn/)
[![Comate](https://static.igem.org/mediawiki/2018/d/dc/T--Jilin_China--Footer--Companies--Comate.png)](http://www.comatebio.com/)
[![MathWorks](https://static.igem.org/mediawiki/2018/c/c5/T--Jilin_China--Footer--Companies--MathWorks.png)](http://www.mathworks.com)
[![GeneScript](https://static.igem.org/mediawiki/2018/7/7a/T--Jilin_China--Footer--Companies--GeneScript.png)](https://www.genscript.com/)
[![GitHub](https://static.igem.org/mediawiki/2018/2/25/T--Jilin_China--Footer--Companies--GitHub.png)](https://github.com/)
[![Transgen](https://static.igem.org/mediawiki/2018/d/d8/T--Jilin_China--Footer--Companies--Transgen.png)](http://www.transgen.com.cn/)

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Jilin China/Results

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[![](http://placehold.it/350x150/e4dede/c4baba)](https://2018.igem.org/Team%3AJilin_China)
[HOME](https://2018.igem.org/Team%3AJilin_China)

TEAM

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

PROJECT

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

PARTS

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

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

HUMAN PRACTICES

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

AWARDS

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

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