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

2.1 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℃.

Usage and Biology

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 41
  Illegal NgoMIV site found at 176
  
• 1000
  COMPATIBLE WITH RFC[1000]