iGEM 2016 — Team JSNU-China

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# Team:JSNU-China/Notebook

notebook

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[·Home](https://2016.igem.org/Team%3AJSNU-China)

[·Project **(Background | Experiment | Future work | Protocal)**](https://2016.igem.org/Team%3AJSNU-China/Description)

[· Parts](https://2016.igem.org/Team%3AJSNU-China/Parts)

[·Team **(Members | Attributions | PI&Advisor | Acknowledgements)**](https://2016.igem.org/Team%3AJSNU-China/Team)

[·Human Partice **(Sliver | Quick-flashing | Collaboration)**](https://2016.igem.org/Team%3AJSNU-China/Human_Practices)

[·Notebook](https://2016.igem.org/Team%3AJSNU-China/Notebook)

[·Safety](https://2016.igem.org/Team%3AJSNU-China/Safety)

[![](https://static.igem.org/mediawiki/2016/b/b3/Jsnuchina_safety.png)](https://2016.igem.org/Team%3AJSNU-China/Safety)

[Safety](https://2016.igem.org/Team%3AJSNU-China/Safety#safety)

[![](https://static.igem.org/mediawiki/2016/8/82/Jsnuchina_notebookchange.png)](https://2016.igem.org/Team%3AJSNU-China/Notebook)

[Notebook](https://2016.igem.org/Team%3AJSNU-China/Notebook#notebook)

[![](https://static.igem.org/mediawiki/2016/d/df/Jsnuchina_human.png)](https://2016.igem.org/Team%3AJSNU-China/Human_Practices)

[Silver](https://2016.igem.org/Team%3AJSNU-China/HP/Silver)
[Quick-flashing](https://2016.igem.org/Team%3AJSNU-China/HP/Gold)
[Collaboration](https://2016.igem.org/Team%3AJSNU-China/Collaborations)

[![](https://static.igem.org/mediawiki/2016/2/2c/Jsnuchina_team.png)](https://2016.igem.org/Team%3AJSNU-China/Team)

[Members](https://2016.igem.org/Team%3AJSNU-China/Team#member)
[Attributions](https://2016.igem.org/Team%3AJSNU-China/Attributions)
[PI&Advisor](https://2016.igem.org/Team%3AJSNU-China/Team#advisors)
[Acknowledgements](https://2016.igem.org/Team%3AJSNU-China/Team#acknow)

[![](https://static.igem.org/mediawiki/2016/9/98/Jsnuchina_parts.png)](https://2016.igem.org/Team%3AJSNU-China/Parts)

[Parts](https://2016.igem.org/Team%3AJSNU-China/Parts#parts)

[![](https://static.igem.org/mediawiki/2016/b/b2/Jsnuchina_project.png)](https://2016.igem.org/Team%3AJSNU-China/Description)

[Background](https://2016.igem.org/Team%3AJSNU-China/Description#background)
[Experiment](https://2016.igem.org/Team%3AJSNU-China/Description#experience)
[Future work](https://2016.igem.org/Team%3AJSNU-China/Description#future)
[Protocal](https://2016.igem.org/Team%3AJSNU-China/Description#protocal)

[![](https://static.igem.org/mediawiki/2016/8/8f/Jsnuchina_home.png)](https://2016.igem.org/Team%3AJSNU-China)

[Home](https://2016.igem.org/Team%3AJSNU-China)

[![](https://static.igem.org/mediawiki/2016/4/45/Jsnuchina_jiaojuan.png)](https://www.youtube.com/watch?v=bmXjtAJiko4)

## Notebook

July 10th~12th

![](https://static.igem.org/mediawiki/2016/e/e5/Jsnuchina_r1.png)

1)  Be familiar with experimental steps(including the configuration of soft AGAR,western blot)and listed experiment required list.

2)  Revived cells,inoculation and expansion.

3)  Select an appropriate auxin.

July 14th

![](https://static.igem.org/mediawiki/2016/d/da/Jsnuchina_r2.png)

1)  Configurate the lower gum of SDS-PAGE.

2)  Observing cell metamorphosis.

July 16th

![](https://static.igem.org/mediawiki/2016/e/e5/Jsnuchina_r1.png)

1)  Reextracting protein and do western blot required list .

2)  Observing cell metamorphosis.

3)  Finish the configuration of soft AGAR.

4)  Preparation of solid medium, in preparation for the transfection.

July 18th

![](https://static.igem.org/mediawiki/2016/d/da/Jsnuchina_r2.png)

1)  Tried to make Tprotein band by western blot, but it failed.

July 20th

![](https://static.igem.org/mediawiki/2016/e/e5/Jsnuchina_r1.png)

1)  Did two western blot experiments, which were 193, 231,hela and hela, 231, to the end of primary antibody incubation.

2)Transformed competent cells into two boards, PX300F and PX300.

July 22th

![](https://static.igem.org/mediawiki/2016/d/da/Jsnuchina_r2.png)

Did two western blot. The first experiment, did two pieces of gel to test T protein and KLF4 protein in gastric cancer cell lines. The other one, did two pieces of gel to test T protein and actin in hybrid system cancer cells.

Because concentration was too low, the extraction of PX300 plasmid failed. Cultivated cells by shaking cells, prepared to extract plasmids. Coated PX300F plates succeeded, selected cloning cells to cultivate by shaking cells.

July 24th

![](https://static.igem.org/mediawiki/2016/e/e5/Jsnuchina_r1.png)

1)  Finished the rest of yesterday’s western blot, analyzed the result of the experiment.

July 26th

![](https://static.igem.org/mediawiki/2016/d/da/Jsnuchina_r2.png)

1)  Finished the rest of yesterday’s work, observed the result of the experiment.

July 28th

![](https://static.igem.org/mediawiki/2016/e/e5/Jsnuchina_r1.png)

1)  Tested the content of actin and KLF4 protein in gastric cancer cell lines.

August 4th

![](https://static.igem.org/mediawiki/2016/d/da/Jsnuchina_r2.png)

1)  Tested the content of KLF4 protein in gastric cancer cell lines.

August 9th

![](https://static.igem.org/mediawiki/2016/e/e5/Jsnuchina_r1.png)

1)  PCR amplification.

August 11th~12th

![](https://static.igem.org/mediawiki/2016/d/da/Jsnuchina_r2.png)

1)  We obtained target fragments of KLF4 mutant by PCR amplification which called Ⅳunder an appropriate Temperature.

August 15th

![](https://static.igem.org/mediawiki/2016/e/e5/Jsnuchina_r1.png)

1)  We did the purification of PCR products.Second,we tested our results by electrophoresis and took photos,then did a diagraph analysis.

Result : There was some impurity in the first gel ,so it was not a good result. We made errors when taking photos,so it was not a good result as well.

August 17th

![](https://static.igem.org/mediawiki/2016/d/da/Jsnuchina_r2.png)

1)  We use the agarose gel select rophorosis to amplify 5 specific gene fragment in KLF4 and do the gel purification.

2)  Construct the accessible system.

August 19th

![](https://static.igem.org/mediawiki/2016/e/e5/Jsnuchina_r1.png)

1)  Find whether it has the clonal phenomene or not ,it failed.

2)  Do the PCR, digestion and purification.

August 21th

![](https://static.igem.org/mediawiki/2016/d/da/Jsnuchina_r2.png)

1)  Extracting the plasmid, finally no clonal phenomenon ,It failed.

2)  Dectet the 293,293-klf4 52 cell under different connection survival rate.

August 24th

![](https://static.igem.org/mediawiki/2016/e/e5/Jsnuchina_r1.png)

1)  Gel purification and transfer.

2)  Coating the plate.

July 13th

![](https://static.igem.org/mediawiki/2016/9/99/Jsnuchina_l1.png)

1)  Configuration reagents used in this experiment.

2)  Autoclaving reagent and experiment apparatus.

3)  Observing cell metamorphosis.

July 15th

![](https://static.igem.org/mediawiki/2016/a/a2/Jsnuchina_l2.png)

1)  Configurate the supernatant gum of SDS-PAGE ,extract protein, electrophoresis, trarsmembrane, primary antibody, secondary antibody, visualization and observe.

2)  the configuration of soft AGAR.

3)  Observing cell metamorphosis.

July 17th

![](https://static.igem.org/mediawiki/2016/9/99/Jsnuchina_l1.png)

1)  Finished the rest of western blot, got the result of the experience.

2)  Collected T293 cells and saving them by freezing.

July 19th

![](https://static.igem.org/mediawiki/2016/a/a2/Jsnuchina_l2.png)

1)  Collected protein extraction cancer cells we need urgently.

2)  Did western blot twice to test the actin of gastric cancer cell and Hybrid system cancer cells separately.

3)  Collected the information of Competent cells.

July 21th

![](https://static.igem.org/mediawiki/2016/9/99/Jsnuchina_l1.png)

1)  The transformation of PX300F failed, transformed it again.

2)  The transformation of PX300F succeeded, shaked bacteria for the night, prepared extract plasmid.

3)  Finished yesterday’s rest of western blot, second incubation resistance enhancement, analyzed the result of the experiment.

4)  Observed the cells, collected T293 cells and saved them by freezing.

July 23th

![](https://static.igem.org/mediawiki/2016/a/a2/Jsnuchina_l2.png)

1)  The result of yesterday’s western blot was unsatisfactory, tested partly T protein and actin in hybrid system cancer cells again.

2)  Extracted PX300 and PX300F plasmids.

July 25th

![](https://static.igem.org/mediawiki/2016/9/99/Jsnuchina_l1.png)

1)  Retested actin in hybrid system cancer cells

2)  Tested the content of KLF4 protein in gastric cancer cell lines.

3)  Tested the content of T protein in gastric cancer cell lines.

4)  Tested the content of T protein of 231 under 3 treatments.

July 27th

![](https://static.igem.org/mediawiki/2016/a/a2/Jsnuchina_l2.png)

1)  Tested the content of KLF4 protein in gastric cancer cell lines.

August 3th

![](https://static.igem.org/mediawiki/2016/9/99/Jsnuchina_l1.png)

1)  Tested the content of actin and KLF4 protein in gastric cancer cell lines.

August 7th

![](https://static.igem.org/mediawiki/2016/a/a2/Jsnuchina_l2.png)

1)  Tested the content of actin and KLF4 protein in cell 293 and cell 293KLF4.

August 10th

![](https://static.igem.org/mediawiki/2016/9/99/Jsnuchina_l1.png)

1)  Obtain target fragments of KLF4 mutant by PCR amplification which called ⅠⅡⅢ Ⅳ and Ⅴ.

RESULT : ⅠⅡⅢ and Ⅴ succeeded. However, Ⅳ failed because of the inappropriate temperature.

August 13th~14th

![](https://static.igem.org/mediawiki/2016/a/a2/Jsnuchina_l2.png)

1)  We obtained target fragments of KLF4 mutant by PCR amplification which called ⅠⅡⅢ Ⅳ and Ⅴ for 6 tubes,and each tube contained 120 ml.

August 16th

![](https://static.igem.org/mediawiki/2016/9/99/Jsnuchina_l1.png)

1)  We did electrophoresis and took photos successfully.

2)  We did double digestion and stayed overnight.

August 18th

![](https://static.igem.org/mediawiki/2016/a/a2/Jsnuchina_l2.png)

1)  Transform the palsmid into cell.

2)  Coating to the plate.

August 20th

![](https://static.igem.org/mediawiki/2016/9/99/Jsnuchina_l1.png)

1)  Transfer the plasmid,coating to the plate.

August 23th

![](https://static.igem.org/mediawiki/2016/a/a2/Jsnuchina_l2.png)

1)  Do the PCR, F158-R385, digestion and purification.

August 25th

![](https://static.igem.org/mediawiki/2016/9/99/Jsnuchina_l1.png)

1)  No clonal phenomenon, it failed.

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Address:Jiangsu Normal University, 101 Shanghai Rd, Tongshan District, Xuzhou, China

Mail:jsnuchina@163.com

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