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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                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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                <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-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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            <h2 class="pageContent-main__title pageContent-main__subtitle">
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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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Revision as of 16:45, 6 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℃.