Part:BBa_K4585014

pcDNA3.1(+)-3XHA-GAL4-VP64-Degron-NLS

pcDNA3.1(+)-3×HA-GAL4-VP64-Degron-NLS, which could express GAL4-VP64-Degron, was used for Luciferase detection experiment. Degron is a short peptide of 41 amino acids in length. Protein substrates containing the Degron sequence can be specifically bound by the E3 ubiquitin ligase in the ubiquitin-proteasome system, resulting in the ubiquitinated modification of the substrate for degradation. The half-life of GAL4-VP64 was shortened when it carries the Degron fragment.

pcDNA3.1(+)-3×HA-GAL4-VP64-Degron-NLS

The pcDNA3.1(+)-3×HA-GAL4-VP64-Degron-NLS plasmid was obtained through homologous recombination of the Degron insert (BBa_K4585005) with pcDNA3.1(+)-3×HA-GAL4-VP64-Degron-NLS linearized vector (BBa_K4585009). 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-Degron-NLS

2.1 Method

Degron could increase the rate of protein degradation, that is, shorten the half-life of protein. When the degradation rate of GAL4-VP64 was accelerated, its amount was reduced and the time for it to exert its activation effect on 9×UAS was shortened. Similarly, when the degradation rate of GAL4-KRAB was increased, its amount was decreased, and the time during which it inhibited 9×UAS was shortened. Thus both the activation and inhibition time of Luciferase were shortened, and eventually the period was shortened.

2.2 Results

The bioluminescence period was successfully shortened from T=30.24 hours to T=12.72 hours.

Fig 2. Bioluminescence intensity when GAL4-VP64:GAL4-KRAB=1:1

Fig 2. Bioluminescence intensity when GAL4-VP64-Degron:GAL4-KRAB-Degron=1:1

3.Caution

After sequencing and ensuring the sequence was correct, we applied it to the experiments. Store at 4℃.