Difference between revisions of "Part:BBa K4839024"

 
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<partinfo>BBa_K4839024 short</partinfo>
 
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This part is design for the validification of our BioPROTC system. In our design, we fused GFP and IRF4 together and thus we can use both anti-GFP or anti-IRF4 BioPROTAC to detect the degradation efficiency of both protein.
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<p>This part is design for the validification of our BioPROTC system. In our design, we fused GFP and IRF4 together and thus we can use both anti-GFP or anti-IRF4 BioPROTAC to detect the degradation efficiency of both protein.</p>
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<p>we transfected HEK293T cells in a 12-well plate with 1 μg of pLVX-TREG3S-FLAG-PU.1-(SSG)3-SPOP-TetOne-Puro and 1 μg of pcDNA3.1-CMV-FLAG-IRF4/pcDNA3.1-CMV-HA-IRF4, induced expression with 1 μg/mL of doxycycline for 48h, and collected the protein for western blot analysis. The results are shown in Figure 3-2, where we observed significant degradation of both HA-IRF4 and FLAG-IRF4.</p>
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<html><img src="https://static.igem.wiki/teams/4839/wiki/parts-files/10-2.png" width="650"</html>
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<p align="center">Figure1.  Western blot signing degrdation of IRF4. "D" represents doxycycline.</p>
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<p>Due to time constraints, we have not been able to conduct this experiment in the THP-1 cell line. However, in the HEK293T cell experiment, we have observed a certain degree of IRF4 degradation, which is a breakthrough that has not been achieved in previous research. We are very excited about these results. We will then continue to optimize the transfection/infection efficiency for THP-1 cells and have also contacted Professor Chong Wu, a macrophage expert from Sun Yat-sen University School of Life Sciences, to explore ways to optimize related experiments for THP-1 cells. Additionally, we will conduct further experimental validation for the designed anti-IRF5 BioPROTAC and optimize anti-IRF4 BioPROTAC using bioinformatics methods to enhance its degradation efficiency.</p>
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 16:49, 3 October 2023


pGK-GFP-IRF4

This part is design for the validification of our BioPROTC system. In our design, we fused GFP and IRF4 together and thus we can use both anti-GFP or anti-IRF4 BioPROTAC to detect the degradation efficiency of both protein.

we transfected HEK293T cells in a 12-well plate with 1 μg of pLVX-TREG3S-FLAG-PU.1-(SSG)3-SPOP-TetOne-Puro and 1 μg of pcDNA3.1-CMV-FLAG-IRF4/pcDNA3.1-CMV-HA-IRF4, induced expression with 1 μg/mL of doxycycline for 48h, and collected the protein for western blot analysis. The results are shown in Figure 3-2, where we observed significant degradation of both HA-IRF4 and FLAG-IRF4.


Figure1. Western blot signing degrdation of IRF4. "D" represents doxycycline.

Due to time constraints, we have not been able to conduct this experiment in the THP-1 cell line. However, in the HEK293T cell experiment, we have observed a certain degree of IRF4 degradation, which is a breakthrough that has not been achieved in previous research. We are very excited about these results. We will then continue to optimize the transfection/infection efficiency for THP-1 cells and have also contacted Professor Chong Wu, a macrophage expert from Sun Yat-sen University School of Life Sciences, to explore ways to optimize related experiments for THP-1 cells. Additionally, we will conduct further experimental validation for the designed anti-IRF5 BioPROTAC and optimize anti-IRF4 BioPROTAC using bioinformatics methods to enhance its degradation efficiency.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal SpeI site found at 257
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 2527
    Illegal SpeI site found at 257
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2627
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 257
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 257
    Illegal NgoMIV site found at 2052
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 99
    Illegal BsaI.rc site found at 2358
    Illegal SapI site found at 1510
    Illegal SapI.rc site found at 1441