iGEM 2017 — Team Lanzhou

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# Team:Lanzhou/Notebook

Lanzhou

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Lanzhou 2017

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Project
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[Background](https://2017.igem.org/Team%3ALanzhou/Background)
[Tandem RNAi](https://2017.igem.org/Team%3ALanzhou/Tandem_RNAi)
[Design](https://2017.igem.org/Team%3ALanzhou/Design)
[Results](https://2017.igem.org/Team%3ALanzhou/Demonstrate)
[Device](https://2017.igem.org/Team%3ALanzhou/Applied_Design)
[Safety](https://2017.igem.org/Team%3ALanzhou/Safety)
Wet Lab
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[Notebook](https://2017.igem.org/Team%3ALanzhou/Notebook)
[Protocols](https://2017.igem.org/Team%3ALanzhou/Protocols)
[Model](https://2017.igem.org/Team%3ALanzhou/Model)
*face*
Human Pratice
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[HP Silver](https://2017.igem.org/Team%3ALanzhou/HP/Silver)
[Gold Integrated](https://2017.igem.org/Team%3ALanzhou/HP/Gold_Integrated)
[Engagement](https://2017.igem.org/Team%3ALanzhou/Engagement)
*group*
Team
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[About us](https://2017.igem.org/Team%3ALanzhou/Team)
[Attributions](https://2017.igem.org/Team%3ALanzhou/Attributions)
[Collaborations](https://2017.igem.org/Team%3ALanzhou/Collaborations)
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Achievements
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[Parts](https://2017.igem.org/Team%3ALanzhou/Parts)
[Interlab](https://2017.igem.org/Team%3ALanzhou/InterLab)
[Medal requirements](https://2017.igem.org/Team%3ALanzhou/Medal)

#

### April

* Week 1
  Apr 1
  *keyboard\_arrow\_down*

• Establish research project in group meeting.

* Week 2
  Apr 2 ~ Apr 8
  *keyboard\_arrow\_down*

• Prepare for experiment and confirm members of the division of labor.* Antibiotic(Amp,Kana,Chl,Tet).
* prepare 5M IPTG.
* prepare double distilled water, centrifuge tube, PCR tube sterilization, of alcohol cotton.
* Preparation of competent state.

* Week 3
  Apr 9 ~ Apr 15
  *keyboard\_arrow\_down*

• New members acquaint the laboratory,and do some basic experimental to prepare for the IGEM.

* Week 4
  Apr 16 ~ Apr 22
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• Verify the plasmids and primer in group meeting.

* Week 5
  Apr 23 ~ Apr 29
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• Gene synthesis

---

### May

* Week 1
  Apr 30 ~ May 6
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• Direct mutagenesis* Site-directed mutagenesis Lon1, Lon2.and run the gel to proving the verification of the mutation.
• Extract plasmids
• Digestion* Digested pUC19 (EcoRI, PstI), LacI and gel extraction.

* Week 2
  May 7 ~ May 13
  *keyboard\_arrow\_down*

• Site-directed mutagenesis Lon1, Lon2.* make bacterium Lon1, Lon2, pET28a (+), pUc19, pETDuet amplified.
• Transformation* Transform the plasmids into E.Coli DH5a.
• Culture* pick a single clone on the transformed plate and culture in fluid culture medium.

* Week 3
  May 14 ~ May 20
  *keyboard\_arrow\_down*

• Ligation* Use protocol of the Quick Connect Enzyme Kit to connect pUC19+GFP，pUC19+LacI.
• Transformation* Transform the mutation,connect product and L4440 plasmids.

* Week 4
  May 21 ~ May 27
  *keyboard\_arrow\_down*

• Plasmids extraction* Extract the plasmids of Gmix and P with plasmids Mini Kit,then run the gel electrophoresis (Figure 1).
Gel electrophoresis* run the gel electrophoresis of Ga(1~5) (Figure2).
* run the gel electrophoresis of Gb (1~5) (Figure3).
• Gel purification of Ga（Figure4）and Gb (Figure4).

![](https://static.igem.org/mediawiki/2017/1/1c/Lanzhou_wiki_Notebook_figure1.png)

Figure 1.

![](https://static.igem.org/mediawiki/2017/3/35/Lanzhou_wiki_Notebook_figure2.png)

Figure 2.

![](https://static.igem.org/mediawiki/2017/0/09/Lanzhou_wiki_Notebook_figure3.png)

Figure 3.

![](https://static.igem.org/mediawiki/2017/4/4a/Lanzhou_wiki_Notebook_figure4.png)

Figure 4.

* Week 5
  May 28 ~ Jun 3
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• Digestion* digest Gain E/S,Gbin E/S.
• Ligation* Ligate Ga with pUC19,Gb with pUC19,Ga+Gb with pUC19.
• Transformation* Transform Ga+pUC19 into DH5a,Gb+pUC19 into DH5a,Ga+Gb+pUC19 into DH5a.
• Run gel* Extract pUC19 into 6 tubes.
* Gel purification of puc19 (Figure 6&7), analyse the failure of digestion.
`

![](https://static.igem.org/mediawiki/2017/5/5f/Lanzhou_wiki_Notebook_figure6.png)

Figure 6.

![](https://static.igem.org/mediawiki/2017/3/3c/Lanzhou_wiki_Notebook_figure7.png)

Figure 7.

• Ligation* Ligated GaE/P with Puc19 E/P, GbE/P with pUC19 E/P, GaE/S+GbX/P with pUC19 E/P.then Transfer ligated product into the plate,and Cultivate them overnight.

---

### June

* Week 1
  Jun 4 ~ Jun 10
  *keyboard\_arrow\_down*

• PCR* PCR Ga, Gb, P, then gel purification (Figure 8&9).
* PCR protocol
![](https://static.igem.org/mediawiki/2017/a/a7/Lanzhou_wiki_Notebook_table1.png)

![](https://static.igem.org/mediawiki/2017/0/09/Lanzhou_wiki_Notebook_figure8.png)

Figure 8.

![](https://static.igem.org/mediawiki/2017/e/e5/Lanzhou_wiki_Notebook_figure9.png)

Figure 9.

• Plasmids extraction* Plasmids extraction of pUC19+Ga，pUC19+Gb.

* Week 2
  Jun 11 ~ Jun 17
  *keyboard\_arrow\_down*

• Repeat-ligation* Ligate GaE/P with Puc19 E/P,GbE/P with pUC19 E/P,GaE/S+GbX/P with pUC19 E/P.
  • Gel purification
  • Repeat digestion* Repeat pUC19+Ga.E/S,pUC19+Gb.X/P. (Figure10)
  • Digestion* Restriction digest P+EP6,ECR+EP10,ECR+EP6,SHT+EP6,SHT+EP8 (Figure11&12&13).

  ![](https://static.igem.org/mediawiki/2017/4/40/Lanzhou_wiki_Notebook_figure10.png)

  Figure 10.

  ![](https://static.igem.org/mediawiki/2017/2/2a/Lanzhou_wiki_Notebook_figure11.png)

  Figure 11.

  ![](https://static.igem.org/mediawiki/2017/c/c5/Lanzhou_wiki_Notebook_figure12.png)

  Figure 12.

  ![](https://static.igem.org/mediawiki/2017/f/f6/Lanzhou_wiki_Notebook_figure13.png)

  Figure 13.

  • Repeat PCR* Repeat Gb PCR then gel purification

* Week 3
  Jun 18 ~ Jun 24
  *keyboard\_arrow\_down*

• Transformation* Transform ECR+L4440 and SHT+L4440into DH5a.
• Repeat PCR* repeat Gb PCR and P digest (Figure14&15).

![](https://static.igem.org/mediawiki/2017/3/37/Lanzhou_wiki_Notebook_figure14.png)

Figure 14.

![](https://static.igem.org/mediawiki/2017/4/4b/Lanzhou_wiki_Notebook_figure15.png)

Figure 15.

• Ligation* ligate ECR+pUC19 (Figure16)

![](https://static.igem.org/mediawiki/2017/a/aa/Lanzhou_wiki_Notebook_figure16.png)

Figure 16.

* Week 4
  Jun 25 ~ Jul 1
  *keyboard\_arrow\_down*

• Culture* Culture bacterium in glycerol:MIX,GMIX, B1.
• plasmids extraction* MIX,GMIX, B1.
• Digestion* Digest MIX in P/E product and gel purification.
• Transformation* Transform the produce into puC19,then pick the positive.

---

### July

* Week 1
  Jul 2 ~ Jul 8
  *keyboard\_arrow\_down*

• PCR* Do GMIX PCR
• Transformation* Transform PCR GMIX and into puC19.
* Tansform RcsA，RBS，pUC19 into BL21.
• Digestion* L4440，GbEXSP，GaEXSP and B1EXSP pick the positive and digest.
* GbEXSP，GaEXSP and B1EXSP.
• Plasmids extraction* GbEXSP，GaEXSP and B1EXSP extract plasmids.
* Kil，GFP，RBS，OmpT.

* Week 2
  Jul 9 ~ Jul 15
  *keyboard\_arrow\_down*

• Transformation:OmpT，pUC19.* Culture 17P: 06 (0.47), Ga, Ga2, Gb, Gb2, B1, B2, SHT, Ecr, Terminator, pUC19, RcsA, pSB3K3, pSB4K5.
* PCR loop120
• plasmids extraction* 17P: 06 (0.47), Ga, Ga2, Gb, Gb2, B1, B2, SHT, Ecr, Terminator, pUC19, RcsA, pSB3K3, pSB4K5.
• Digestion* Ga, Ga2, Gb, Gb2, B1, B2, SHT, SHT2, Ecr, Ecr2, RcsA, pUC19，pSB3K3, pSB4K5, Loop120, Lysis1, lysis2, Terminator.

* Week 3
  Jul 16 ~ Jul 22
  *keyboard\_arrow\_down*

• Ligation* Ga2+T——Ga2T
* Gb2+T——Gb2T
* B2+T——B2T
* SHT2+T——S2T
* Ecr2+T——E2T
* Loop120+pUC19——Loop120
* BBa\_B0030+17P: 06 (0.47)——0.47x0.6
* BBa\_B0064+17P: 06 (0.47)——0.47x0.35
* RcsA+T——RcsAT
* Lysis1+pUC19——lysis1
* Lysis2+pUC19——lysis2
• Plasmids extraction* Extract plasmids of pSB1T3，pSB1K3，pSB1C3，pSB1A3，SHT.

* Week 4
  Jul 23 ~ Jul 29
  *keyboard\_arrow\_down*

• PCR* Ga2T,Gb2T,B2T,S2T,E2T,Loop120,RcsAT,lysis1,lysis2.
* Prepare the transform cell of M bacterium,and all the plasmids need to transform would be gone and well.

---

### August

* Week 1
  Jul 30 ~ Aug 5
  *keyboard\_arrow\_down*

• Human practice* Confirm the form for human practice in group meeting.

* Week 2
  Aug 6 ~ Aug 12
  *keyboard\_arrow\_down*

• Human practice* Design a questionnaire of transgenosis in public.

* Week 3
  Aug 13 ~ Aug 19
  *keyboard\_arrow\_down*

• Human practice* Go to some schools advertise our work.

* Week 4
  Aug 20 ~ Aug 26
  *keyboard\_arrow\_down*

• We went to FAFU for CCIC.

* Week 5
  Aug 27 ~ Sep 2
  *keyboard\_arrow\_down*

• Gene synthesis* Synthesize the gene sequence act on arabidopsis thaliana.
• Vector construction* Construct 3hree vectors producing dsRNA (Ecr-L4440, ECRL, ECRN)
• Experiment on aphids.

---

### September

* Week 1
  Sep 3 ~ Sep 9
  *keyboard\_arrow\_down*

• Transformation* Transform carrier(Ecr-L4440, ECRL, ECRN) into E.coli M-JLAC19 (Dicer deficit type).
• Induce expression
• Try to express dsRNA form trxzDP.

* Week 2
  Sep 10 ~ Sep 16
  *keyboard\_arrow\_down*

• Induce expression* trxzDP:product dsRNA by T7 bi-directional promoter.
* trxzL: product hpRNA by normal loop construction.
* trxzN: product hpRNA by intron loop construction.

* Week 3
  Sep 17 ~ Sep 23
  *keyboard\_arrow\_down*

• Induce expression* Ecr-L4440:product dsRNA by T7 bi-directional promoter.
* ECRL:product hpRNA by normal loop construction.

* Week 4
  Sep 24 ~ Sep 30
  *keyboard\_arrow\_down*

• Express of dsRNA and hpRNA.

---

### October

* Week 1
  Oct 1 ~ Oct 7
  *keyboard\_arrow\_down*

• Culture arabidopsis thaliana (Col 0)
• Vector construction* Construct vectors of dsRNA which act on arabidopsis thaliana.
• Transformation* Transform carrier(trxzDP, trxzL, trxzN) into E.coli M-JLAC19 (Dicer deficit type).
• Induce expression* induce the M-JLAC19 express dsRNA.

* Week 2
  Oct 8 ~ Oct 14
  *keyboard\_arrow\_down*

• Express dsRNA and hpRNA.
• Prepare for the macro experiments validation.

* Week 3
  Oct 15 ~ Oct 21
  *keyboard\_arrow\_down*

• Improve parts
• Modelling work

* Week 4
  Oct 22 ~ Oct 28
  *keyboard\_arrow\_down*

• Modelling work
• Presentation work

* Week 5
  Oct 29 ~ Oct 31
  *keyboard\_arrow\_down*

• Modelling work
• Wiki & design work
• Presentation work

Lanzhou 2017

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* About Lanzhou 2017
* copyright
* [Made by L. with Material Components for the web
  Made by L.](http://lawliet.pw)

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# Team:Lanzhou/Experiments

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MENU

[HOME](https://2017.igem.org/Team%3ALanzhou)

TEAM

[Team](https://2017.igem.org/Team%3ALanzhou/Team)
[Collaborations](https://2017.igem.org/Team%3ALanzhou/Collaborations)

PROJECT

[Description](https://2017.igem.org/Team%3ALanzhou/Description)
[Design](https://2017.igem.org/Team%3ALanzhou/Design)
[Experiments](https://2017.igem.org/Team%3ALanzhou/Experiments)
[Notebook](https://2017.igem.org/Team%3ALanzhou/Notebook)
[InterLab](https://2017.igem.org/Team%3ALanzhou/InterLab)
[Contribution](https://2017.igem.org/Team%3ALanzhou/Contribution)
[Model](https://2017.igem.org/Team%3ALanzhou/Model)
[Results](https://2017.igem.org/Team%3ALanzhou/Results)
[Demonstrate](https://2017.igem.org/Team%3ALanzhou/Demonstrate)
[Improve](https://2017.igem.org/Team%3ALanzhou/Improve)
[Attributions](https://2017.igem.org/Team%3ALanzhou/Attributions)

PARTS

[Parts](https://2017.igem.org/Team%3ALanzhou/Parts)
[Basic Parts](https://2017.igem.org/Team%3ALanzhou/Basic_Part)
[Composite Parts](https://2017.igem.org/Team%3ALanzhou/Composite_Part)
[Part Collection](https://2017.igem.org/Team%3ALanzhou/Part_Collection)

[SAFETY](https://2017.igem.org/Team%3ALanzhou/Safety)

HUMAN PRACTICES

[Silver HP](https://2017.igem.org/Team%3ALanzhou/HP/Silver)
[Integrated and Gold](https://2017.igem.org/Team%3ALanzhou/HP/Gold_Integrated)
[Public Engagement](https://2017.igem.org/Team%3ALanzhou/Engagement)

AWARDS

[Applied Design](https://2017.igem.org/Team%3ALanzhou/Applied_Design)
[Entrepreneurship](https://2017.igem.org/Team%3ALanzhou/Entrepreneurship)
[Hardware](https://2017.igem.org/Team%3ALanzhou/Hardware)
[Measurement](https://2017.igem.org/Team%3ALanzhou/Measurement)
[Model](https://2017.igem.org/Team%3ALanzhou/Model)
[Plant](https://2017.igem.org/Team%3ALanzhou/Plant)
[Software](https://2017.igem.org/Team%3ALanzhou/Software)

[JUDGING FORM](https://igem.org/2017_Judging_Form?team=Lanzhou)

# Lanzhou

# 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:Lanzhou/Results

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MENU

[HOME](https://2017.igem.org/Team%3ALanzhou)

TEAM

[Team](https://2017.igem.org/Team%3ALanzhou/Team)
[Collaborations](https://2017.igem.org/Team%3ALanzhou/Collaborations)

PROJECT

[Description](https://2017.igem.org/Team%3ALanzhou/Description)
[Design](https://2017.igem.org/Team%3ALanzhou/Design)
[Experiments](https://2017.igem.org/Team%3ALanzhou/Experiments)
[Notebook](https://2017.igem.org/Team%3ALanzhou/Notebook)
[InterLab](https://2017.igem.org/Team%3ALanzhou/InterLab)
[Contribution](https://2017.igem.org/Team%3ALanzhou/Contribution)
[Model](https://2017.igem.org/Team%3ALanzhou/Model)
[Results](https://2017.igem.org/Team%3ALanzhou/Results)
[Demonstrate](https://2017.igem.org/Team%3ALanzhou/Demonstrate)
[Improve](https://2017.igem.org/Team%3ALanzhou/Improve)
[Attributions](https://2017.igem.org/Team%3ALanzhou/Attributions)

PARTS

[Parts](https://2017.igem.org/Team%3ALanzhou/Parts)
[Basic Parts](https://2017.igem.org/Team%3ALanzhou/Basic_Part)
[Composite Parts](https://2017.igem.org/Team%3ALanzhou/Composite_Part)
[Part Collection](https://2017.igem.org/Team%3ALanzhou/Part_Collection)

[SAFETY](https://2017.igem.org/Team%3ALanzhou/Safety)

HUMAN PRACTICES

[Silver HP](https://2017.igem.org/Team%3ALanzhou/HP/Silver)
[Integrated and Gold](https://2017.igem.org/Team%3ALanzhou/HP/Gold_Integrated)
[Public Engagement](https://2017.igem.org/Team%3ALanzhou/Engagement)

AWARDS

[Applied Design](https://2017.igem.org/Team%3ALanzhou/Applied_Design)
[Entrepreneurship](https://2017.igem.org/Team%3ALanzhou/Entrepreneurship)
[Hardware](https://2017.igem.org/Team%3ALanzhou/Hardware)
[Measurement](https://2017.igem.org/Team%3ALanzhou/Measurement)
[Model](https://2017.igem.org/Team%3ALanzhou/Model)
[Plant](https://2017.igem.org/Team%3ALanzhou/Plant)
[Software](https://2017.igem.org/Team%3ALanzhou/Software)

[JUDGING FORM](https://igem.org/2017_Judging_Form?team=Lanzhou)

# Lanzhou

# 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.

##### You should also 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)

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