Plasmid

Part:BBa_K4585014

Designed by: Zijun Zhang   Group: iGEM23_CSU-CHINA   (2023-10-04)

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 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℃.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1772
    Illegal XbaI site found at 1591
    Illegal XbaI site found at 1811
    Illegal SpeI site found at 249
    Illegal PstI site found at 1734
    Illegal PstI site found at 1777
    Illegal PstI site found at 3206
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1772
    Illegal SpeI site found at 249
    Illegal PstI site found at 1734
    Illegal PstI site found at 1777
    Illegal PstI site found at 3206
    Illegal NotI site found at 1798
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1772
    Illegal BglII site found at 12
    Illegal BamHI site found at 1448
    Illegal BamHI site found at 1637
    Illegal XhoI site found at 1218
    Illegal XhoI site found at 1805
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1772
    Illegal XbaI site found at 1591
    Illegal XbaI site found at 1811
    Illegal SpeI site found at 249
    Illegal PstI site found at 1734
    Illegal PstI site found at 1777
    Illegal PstI site found at 3206
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1772
    Illegal XbaI site found at 1591
    Illegal XbaI site found at 1811
    Illegal SpeI site found at 249
    Illegal PstI site found at 1734
    Illegal PstI site found at 1777
    Illegal PstI site found at 3206
    Illegal NgoMIV site found at 2316
    Illegal NgoMIV site found at 3657
    Illegal NgoMIV site found at 3942
    Illegal AgeI site found at 1869
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1137
    Illegal BsaI.rc site found at 882
    Illegal BsaI.rc site found at 5470
    Illegal SapI site found at 4387
    Illegal SapI.rc site found at 3506
    Illegal SapI.rc site found at 3716


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