Difference between revisions of "Team:Tongji Shanghai/Parts"

Favorite Parts

Team Fudan need tet-on or tet-of promoter to get tetO element and miniCMV as the promoter to drive their genes of interest. Vice professor Wenwen Jia in our school works on apparent heredity field, happens to have tet-on kit for his work. We made contact with Pro. Wenwen Jia and gained 3μl.

We also asked him for the associating kit that responses to panmycin and send the signal to promoter. Team Fudan used these two kits to PCR and continued their experiments. At the same time, Team Fudan had provided great help either. They built their kit ahead of us, and they generously provided almost all of the materials that we gonna need for kit submitting.(15 ml of chloromycetin powder,8 chloramphenicol resistance plates,80ml chloramphenicol resistance LB,Enzyme of XbaI and SpeI each 3μl)

Basic Parts

BBa_K1922001

Approximately 80% of cancers are immortalized by constitutive activa-tion of telomerase to maintain telomeres throughout rapid cellular prolif-eration. HTert promoter is the promoter we cloned from human cell, which can drive the transcription of telomerase in human. We hoped that it can promote the transcription of the certain gene following it in tumor cells specifically, instead of in normal cells.

BBa_K1922003

The cells of all tissues react to various stresses through the quick synthesization of HSPs.As the most important HSPs family. HSP70 has the highest temperature sensitivity and conservation. Having synthesized human hsp70 promoter, we hope it can drive the transcription of selected gene after heat shock at certain tem-perature. From the literature we finally set the preform condition as 42C for 1h

BBa_K1922004

The cells of all tissues react to various stresses through the quick synthesization of HSPs.As the most important HSPs family. HSP70 has the highest temperature sensitivity and conservation. Having synthesized human hsp70 promoter, we hope it can drive the transcription of selected gene after heat shock at certain tem-perature. From the literature we finally set the preform condition as 42C for 1h

BBa_K1922008

To test if it works, we also constructed a plasmid linking a GFP as a re-porter gene linked with it. So when we transfected it into cancer cells like hcc 1937 or hell, and telomerase negative cells, we could observe GFP fluorescent in cancer cells instead of normal cells.

BBa_K1922010

We hope we can optimize the conventional theotherapy into a responsive system, which means if the laser is working, we want to know it. It will be more convenient and efficient for researchers to set appropriate condi-tion of the equipment, such as the power of laser. So a gene of luciferase was linked after p53 as a reporter.

Composite Parts

BBa_K1922006

With the combination of hTert promoter and p53, if we deliver the plasmid into mouse by virus packaging, p53 will be overexpressed in tumor cells specifically, so the thermosensitivity of tumor cells would be increased rather than of normal cells. When tumor is treated by laser with certain-length wave, with the heat induced by our golden nano rods(GNRs), the apoptosis of tumor cells whose thermosensitivity has been increased would start more efficiently. Thus, because of the tumor-targeted hTert promoter and the tumor suppressor following it, we improved the targeting of conventional thermotherapy.

BBa_K1922007

As we mentioned above, heat shock protein 70(hsp 70) will be synthesized rapidly after heat shock. While convention-al thermotherapy has already got great efficiency in clinical appli-cation, we wondered if we can make the whole system responsive, which means there can be a reporter system telling people that the heat treatment of laser is working. Hsp70 promoter can drive the transcription of p53 gene after heat shock which is done by laser and GNRs in thermotherapy.

BBa_K1922009

GFP gene was linked after hTert promoter,after transfecting it into different cell lines, we can examine if the promoter can drive the transcription of the gene after it in hTert positive cells but not nega-tive ones.

BBa_K1922012

After inserting the hsp70-p53 fraction into pGL3 vector which con-tains a luciferase gene, we could observe if the tumor cells had been heat treated in in vivo bioluminescence imaging machine with the injection of luciferase substance. If the result can be re-flected from mouse direcly, it will be easier to set experimental condition and save the use of model animals. At the same time, p53 can work to increase the thermosesitivity of tumor cells. We hope hsp70-p53-luciferase can be a novel reporter system to make conventional thermotherapy a responsive method for us to treat cancer in a more efficient way.