Part:BBa_K4585015
pGL4.35-3XHA-9XGAL4UAS-KRAB-Degron-NLS
pGL4.35-3×HA-9×GAL4UAS-KRAB-Degron-NLS, which could express GAL4-KRAB-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-KRAB was shortened when it carries the Degron fragment.
pGL4.35-3×HA-9×GAL4UAS-KRAB-Degron-NLS
The pGL4.35-3×HA-9×GAL4UAS-KRAB-Degron-NLS plasmid was obtained through homologous recombination of the Degron insert (BBa_K4585005) with pGL4.35-3×HA-9×GAL4UAS-KRAB-Degron-NLS linearized vector (BBa_K4585010). 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-Degron-NLS
2 Experiment
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℃.
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