Part:BBa_K3132000
Gal4-VP64
This part is transformed from the part: Gal4-KRAB(BBa_K2446037) of Igem17_Fudan. To turn this TF into an activating one, we replaced the KRAB domain with a VP64. And this approach successfully reversed its effect as we had expected. Gal4-VP64 containing three core domains from N-terminal to C-terminal: GAL4 DNA binding domain, nuclear location sequence and VP64 transcription regulating domain. And a (G4S) linker was added between DBD and NLS for providing region flexibility. GAL4DBD enable binding to specific DNA sequences, so that we can use Gal4-VP64 as a specific transcription factors to activate the expression of our downstream synthetic promoter elements and minimal CMV.
Contribution
Team: CSU-CHINA 2023
pcDNA3.1(+)-3XHA-GAL4-VP64-NLS
The pcDNA3.1(+)-3×HA-GAL4-VP64-NLS plasmid, which could express GAL4-VP64, was used for Luciferase detection experiment. GAL4 is a protein that can find and bind UAS (upstream activation sequence). VP64 is a transcription factor that, when used in combination with GAL4, can activate UAS and initiate the expression of downstream genes.
pcDNA3.1(+)-3×HA-GAL4-VP64-NLS
The pcDNA3.1(+)-3×HA-GAL4-VP64-NLS plasmid was obtained through homologous recombination of the VP64 homologous recombination insert (BBa_K4585002) with pcDNA3.1(+)-3×HA-GAL4-VP64-NLS linearized vector (BBa_K4585006). 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 pcDNA3.1(+)-3×HA-GAL4-VP64-NLS
2 Experiment
2.1 Method
The pcDNA3.1(+)-3×HA-GAL4-VP64-NLS plasmid could express GAL4-VP64, thereby activating 9×UAS, which could activate the expression of its downstream gene, GAL4-KRAB or Luciferase.
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℃.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 623
Illegal XbaI site found at 584
Illegal XbaI site found at 662
Illegal SpeI site found at 5426
Illegal PstI site found at 628
Illegal PstI site found at 2057 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 623
Illegal SpeI site found at 5426
Illegal PstI site found at 628
Illegal PstI site found at 2057
Illegal NotI site found at 649 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 623
Illegal BglII site found at 5189
Illegal XhoI site found at 215
Illegal XhoI site found at 656 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 623
Illegal XbaI site found at 584
Illegal XbaI site found at 662
Illegal SpeI site found at 5426
Illegal PstI site found at 628
Illegal PstI site found at 2057 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 623
Illegal XbaI site found at 584
Illegal XbaI site found at 662
Illegal SpeI site found at 5426
Illegal PstI site found at 628
Illegal PstI site found at 2057
Illegal NgoMIV site found at 1167
Illegal NgoMIV site found at 2508
Illegal NgoMIV site found at 2793
Illegal AgeI site found at 720 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 134
Illegal BsaI.rc site found at 4321
Illegal BsaI.rc site found at 6059
Illegal SapI site found at 3238
Illegal SapI.rc site found at 2357
Illegal SapI.rc site found at 2567
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 41
Illegal NgoMIV site found at 176 - 1000COMPATIBLE WITH RFC[1000]
| None |