Difference between revisions of "Part:BBa K2980002"
 
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<partinfo>BBa_K2980002 parameters</partinfo>
 
<partinfo>BBa_K2980002 parameters</partinfo>
 
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==Contribution: NUDT_CHINA 2021==
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In our project for iGEM2021, we use this basic part with CIB1([[Part:BBa_K2980002]]) to fuse with other proteins to introduce a blue light-induced switch to our protein degradation system. Herein, we constructed plasmids based on the mechanism of tet-on system, co-transfected the plasmids into HEK293T cells, applied the experiment group with blue light stimulus (480nm, stimulate 2 seconds with a 58 second-interval) for 24/48h and then conducted SEAP assay to validate the interaction between this part and CIB1.
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===Method===
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We separately expressed tetR fused with CIB1 and CRY2 fused with VP64 so that only by applying blue light stimulus would the two proteins bind to each other through CRY2-CIB1 interaction.Meanwhile, we utilized a translational control element (TCE) which consists of seven tetO to enable the transcription of reporter gene (SEAP) downstream once bound with tetR and VP64. Under this assumption, the expression level of SEAP can illustrate whether CRY2 interacts with CIB1 under blue light and we can further demonstrate the interaction level through SEAP assay.
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<figure class="figure">
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<img src="https://static.igem.org/mediawiki/parts/8/8e/T--NUDT_CHINA--Part_Validation_SEAP_CRY2-CIB1.png" class="figure-img img-fluid rounded"  height="350px">
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Figure1. Experimental validation approach of CRY2 and CIB1
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===Result===
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<figure class="figure">
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<img src="https://2021.igem.org/wiki/images/8/89/T--NUDT_CHINA--Contribution_CRY2-CIB1_.png
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" class="figure-img img-fluid rounded"  height="350px">
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Figure2. SEAP activity in the culture medium collect from cells transfected with tetR-Cry2, CIB1-VP64 and tetO7-SEAP expressing plasmids under either blue light illumination or dark condition. Cells were illuminated with 3 mW/cm2 of 405nm blue light, the illumination was programed as the repeat of [2 s ON /58 s OFF] cycle for 48 hours. BDL represents below detection limits, Data represent mean ± s.e.m. (n=3 biological replicates)
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As expected, HEK-293 cells co-transfected with tetR-Cry2, CIB1-VP64 and tetO7-SEAP expressing plasmids showed significantly increased SEAP production upon 24h or 48 h of blue light illumination comparing to control cells transfected with the same plasmids placed in a dark incubator.

Latest revision as of 03:35, 22 October 2021


CIB1

CIB1 will interact with CRY2 (Part:BBa K2980000) under blue light stimulation. It is the main light control element in our system.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 112
  • 1000
    COMPATIBLE WITH RFC[1000]



Contribution: NUDT_CHINA 2021

In our project for iGEM2021, we use this basic part with CIB1(Part:BBa_K2980002) to fuse with other proteins to introduce a blue light-induced switch to our protein degradation system. Herein, we constructed plasmids based on the mechanism of tet-on system, co-transfected the plasmids into HEK293T cells, applied the experiment group with blue light stimulus (480nm, stimulate 2 seconds with a 58 second-interval) for 24/48h and then conducted SEAP assay to validate the interaction between this part and CIB1.

Method

We separately expressed tetR fused with CIB1 and CRY2 fused with VP64 so that only by applying blue light stimulus would the two proteins bind to each other through CRY2-CIB1 interaction.Meanwhile, we utilized a translational control element (TCE) which consists of seven tetO to enable the transcription of reporter gene (SEAP) downstream once bound with tetR and VP64. Under this assumption, the expression level of SEAP can illustrate whether CRY2 interacts with CIB1 under blue light and we can further demonstrate the interaction level through SEAP assay.

Figure1. Experimental validation approach of CRY2 and CIB1


Result

Figure2. SEAP activity in the culture medium collect from cells transfected with tetR-Cry2, CIB1-VP64 and tetO7-SEAP expressing plasmids under either blue light illumination or dark condition. Cells were illuminated with 3 mW/cm2 of 405nm blue light, the illumination was programed as the repeat of [2 s ON /58 s OFF] cycle for 48 hours. BDL represents below detection limits, Data represent mean ± s.e.m. (n=3 biological replicates)

As expected, HEK-293 cells co-transfected with tetR-Cry2, CIB1-VP64 and tetO7-SEAP expressing plasmids showed significantly increased SEAP production upon 24h or 48 h of blue light illumination comparing to control cells transfected with the same plasmids placed in a dark incubator.