Difference between revisions of "Part:BBa K4585015"

 
 
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<partinfo>BBa_K4585015 short</partinfo>
  
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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.
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                pGL4.35-3×HA-9×GAL4UAS-KRAB-Degron-NLS
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                <p>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.
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            <h2 class="pageContent-main__title pageContent-main__subtitle">
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                1 Pattern Diagram
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                    <img width="400px" src="https://static.igem.wiki/teams/4585/wiki/the-model-diagram-of-pgl4-35-3-ha-9-gal4uas-krab-degron-nls111.png"></p>
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                <br />
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                <p style="width: 80%;text-align:center;font-size: .9rem; margin: -1rem auto 1rem auto; color: #888;">Fig.1 The model diagram of pGL4.35-3×HA-9×GAL4UAS-KRAB-Degron-NLS</p>
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            <h2 class="pageContent-main__title pageContent-main__subtitle">
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                2 Experiment
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                2.1 Method
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                <p>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.
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                2.2 Results
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                <p>The bioluminescence period was successfully shortened from T=30.24 hours to T=12.72 hours.
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                <p style="text-align: center;">
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                    <img width="400px" src="https://static.igem.wiki/teams/4585/wiki/biofluorescence-intensity-when-gal4-vp64-to-gal4-krab-1-to-1.png"></p>
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                <p style="width: 80%;text-align:center;font-size: .9rem; margin: -1rem auto 1rem auto; color: #888;">Fig 2. Bioluminescence intensity when GAL4-VP64:GAL4-KRAB=1:1</p>
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                    <img width="400px" src="https://static.igem.wiki/teams/4585/wiki/biofluorescence-intensity-when-gal4-vp64-degron-to-gal4-krab-degron-1-to-1.png"></p>
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                <p style="width: 80%;text-align:center;font-size: .9rem; margin: -1rem auto 1rem auto; color: #888;">Fig 2. Bioluminescence intensity when GAL4-VP64-Degron:GAL4-KRAB-Degron=1:1</p>
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                3.Caution
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                <p>After sequencing and ensuring the sequence was correct, we applied it to the experiments. Store at 4℃.
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4585015 SequenceAndFeatures</partinfo>
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<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameters===
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<partinfo>BBa_K4585015 parameters</partinfo>
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Latest revision as of 03:42, 9 October 2023

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℃.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 188
    Illegal XbaI site found at 25
    Illegal XbaI site found at 260
    Illegal XbaI site found at 1350
    Illegal XbaI site found at 3132
    Illegal SpeI site found at 5218
    Illegal PstI site found at 1177
    Illegal PstI site found at 4307
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 188
    Illegal NheI site found at 216
    Illegal SpeI site found at 5218
    Illegal PstI site found at 1177
    Illegal PstI site found at 4307
    Illegal NotI site found at 4286
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 188
    Illegal BglII site found at 263
    Illegal BamHI site found at 855
    Illegal BamHI site found at 1080
    Illegal BamHI site found at 1612
    Illegal XhoI site found at 68
    Illegal XhoI site found at 625
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 188
    Illegal XbaI site found at 25
    Illegal XbaI site found at 260
    Illegal XbaI site found at 1350
    Illegal XbaI site found at 3132
    Illegal SpeI site found at 5218
    Illegal PstI site found at 1177
    Illegal PstI site found at 4307
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 188
    Illegal XbaI site found at 25
    Illegal XbaI site found at 260
    Illegal XbaI site found at 1350
    Illegal XbaI site found at 3132
    Illegal SpeI site found at 5218
    Illegal PstI site found at 1177
    Illegal PstI site found at 4307
    Illegal NgoMIV site found at 1254
    Illegal NgoMIV site found at 1365
    Illegal NgoMIV site found at 2843
    Illegal NgoMIV site found at 2860
    Illegal NgoMIV site found at 2992
    Illegal NgoMIV site found at 3083
    Illegal NgoMIV site found at 3335
    Illegal AgeI site found at 3138
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 544
    Illegal SapI site found at 1633
    Illegal SapI site found at 3344
    Illegal SapI.rc site found at 1038