Difference between revisions of "Part:BBa K4585013"
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+ | __NOTOC__ | ||
+ | <partinfo>BBa_K4585013 short</partinfo> | ||
+ | The pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS plasmid, which could express GAL4-KRAB, was used for Luciferase detection experiment. GAL4 is a protein that can find and bind UAS (upstream activation sequence). KRAB is a transcription factor that represses downstream gene expression when combined with GAL4. | ||
+ | <html> | ||
+ | |||
+ | <head> | ||
+ | |||
+ | </head> | ||
+ | |||
+ | <body> | ||
+ | <h2 class="pageContent-main__title"> | ||
+ | <!--put tile here, <h2>title</h2>, class="pageContent-main__title" means it is the main title--> | ||
+ | pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS | ||
+ | </h2> | ||
+ | <div class="pageContent-main__textBox"> | ||
+ | <!--all the content must included in this div--> | ||
+ | <p>The pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS plasmid was obtained through homologous recombination of the KRAB homologous recombination insert (BBa_K4585003) with pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS linearized vector (BBa_K4585008). The homologous recombination plasmid product was identified as the target product by sequencing and enzyme cutting and agarose gel electrophoresis. | ||
+ | </p> | ||
+ | <!--put text here, <p>content</p>--> | ||
+ | </div> | ||
+ | <h2 class="pageContent-main__title pageContent-main__subtitle"> | ||
+ | <!--class="pageContent-main__title" means it is the sub title--> | ||
+ | 1 Pattern Diagram | ||
+ | </h2> | ||
+ | <div class="pageContent-main__textBox"> | ||
+ | <!--all the content must included in this div--> | ||
+ | <p style="text-align: center;"> | ||
+ | <img width="400px" src="https://static.igem.wiki/teams/4585/wiki/the-model-diagram-of-pgl4-35-3-ha-9-gal4uas-krab-nls.png"></p> | ||
+ | <br /> | ||
+ | <!--put image's url here--> | ||
+ | <p style="width: 80%;text-align:center;font-size: .9rem; margin: -1rem auto 1rem auto; color: #888;">Fig.1 The model diagram of pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS</p> | ||
+ | </div> | ||
+ | <h2 class="pageContent-main__title pageContent-main__subtitle"> | ||
+ | <!--class="pageContent-main__title" means it is the sub title--> | ||
+ | 2 Experiment | ||
+ | </h2> | ||
+ | <h2 class="pageContent-main__title pageContent-main__subtitle"> | ||
+ | <!--class="pageContent-main__title" means it is the sub title--> | ||
+ | 2.1 Method | ||
+ | </h2> | ||
+ | <div class="pageContent-main__textBox"> | ||
+ | <!--all the content must included in this div--> | ||
+ | <p>The pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS plasmid could express GAL4-KRAB, and GAL4-KRAB could bind 9×UAS and inhibit downstream gene expression, therefore GAL4-KRAB could inhibit GnRH expression. Considering that the plasmid itself contains a 9×UAS sequence, GAL4-KRAB can also suppress its own expression. | ||
+ | </p> | ||
+ | <!--put text here, <p>content</p>--> | ||
+ | </div> | ||
+ | <h2 class="pageContent-main__title pageContent-main__subtitle"> | ||
+ | <!--class="pageContent-main__title" means it is the sub title--> | ||
+ | 2.2 Results | ||
+ | </h2> | ||
+ | <div class="pageContent-main__textBox"> | ||
+ | <!--all the content must included in this div--> | ||
+ | <p>HEK 293T cells were transiently transfected with GAL-VP64 and GAL-KRAB plasmids, and an appropriate amount of Luciferase plasmids were transfected to simulate GnRH. The experiment showed that the GAL-VP64 plasmid could initiate the expression of GAL4-KRAB and Luciferase. | ||
+ | </p> | ||
+ | <p style="text-align: center;"> | ||
+ | <img width="400px" src="https://static.igem.wiki/teams/4585/wiki/biofluorescence-intensity-when-gal4-vp64-gal4-krab-400-ng.png"></p> | ||
+ | <br /> | ||
+ | <!--put image's url here--> | ||
+ | <p style="width: 80%;text-align:center;font-size: .9rem; margin: -1rem auto 1rem auto; color: #888;">Fig 2. Bioluminescence intensity when GAL4-VP64=GAL4-KRAB=400 ng</p> | ||
+ | </div> | ||
+ | <h2 class="pageContent-main__title pageContent-main__subtitle"> | ||
+ | <!--class="pageContent-main__title" means it is the sub title--> | ||
+ | 3.Caution | ||
+ | </h2> | ||
+ | <div class="pageContent-main__textBox"> | ||
+ | <!--all the content must included in this div--> | ||
+ | <p>After sequencing and ensuring the sequence was correct, we applied it to the experiments. Store at 4℃. | ||
+ | </p> | ||
+ | </div> | ||
+ | </body> | ||
+ | </html> |
Revision as of 16:18, 6 October 2023
pGL4.35-3XHA-9XGAL4UAS-KRAB-NLS
The pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS plasmid, which could express GAL4-KRAB, was used for Luciferase detection experiment. GAL4 is a protein that can find and bind UAS (upstream activation sequence). KRAB is a transcription factor that represses downstream gene expression when combined with GAL4.
pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS
The pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS plasmid was obtained through homologous recombination of the KRAB homologous recombination insert (BBa_K4585003) with pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS linearized vector (BBa_K4585008). The homologous recombination plasmid product was identified as the target product by sequencing and enzyme cutting and agarose gel electrophoresis.
1 Pattern Diagram
Fig.1 The model diagram of pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS
2 Experiment
2.1 Method
The pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS plasmid could express GAL4-KRAB, and GAL4-KRAB could bind 9×UAS and inhibit downstream gene expression, therefore GAL4-KRAB could inhibit GnRH expression. Considering that the plasmid itself contains a 9×UAS sequence, GAL4-KRAB can also suppress its own expression.
2.2 Results
HEK 293T cells were transiently transfected with GAL-VP64 and GAL-KRAB plasmids, and an appropriate amount of Luciferase plasmids were transfected to simulate GnRH. The experiment showed that the GAL-VP64 plasmid could initiate the expression of GAL4-KRAB and Luciferase.
Fig 2. Bioluminescence intensity when GAL4-VP64=GAL4-KRAB=400 ng
3.Caution
After sequencing and ensuring the sequence was correct, we applied it to the experiments. Store at 4℃.