Difference between revisions of "Part:BBa K4016040"
 
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F-Prime：5’CTAGCGTTTAAACTTAAGCTTggtaccATTTAAATGCCA 3’
 
F-Prime：5’CTAGCGTTTAAACTTAAGCTTggtaccATTTAAATGCCA 3’
  
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R-Prime：5’TGCTGGATATCTGCAGAATTCttaGATGTAGTCGGTCTT 3’
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R-Prime：5’TGCTGGATATCTGCAGAATTCttaGGGAGCGGCGCCGATCAT 3’
  
 
The PCR protocol is selected based on the Users Manuel. The Electrophoresis was performed on a 1% Agarose gel.
 
The PCR protocol is selected based on the Users Manuel. The Electrophoresis was performed on a 1% Agarose gel.
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===Enzyme Digestion===
 
===Enzyme Digestion===
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==Functional validation==
 
==Functional validation==
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To test whether our design of changing the GS linker into longer one work, pcDNA3.1 with EGFP, pcDNA3.1 with Trim21-3x GSlinker-CRY2 and pcDNA3.1 with GFPnano-3x GSlinker-CIB1 plasmids were co-transfected into HEK-293T cells, with pcDNA3.1 of the same dose as control group.The transfected cells were cultured with 5mA current, blue 2/28S frequency illumination. Fluorescent images were taken 48 hours post transfection.
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To test whether adjusting the GS linker length can help proteins fold correctly and therefore lead to a better degradation efficiency. To begin with, truncated Trim21 protein was linked with CRY2 with a 3x GGGGS protein linker (Trim21-3x GSlinker-CRY2, BBa_K4016040), GFPnanobody was liked to CIB1 in a similar manner (GFPnano-3x GSlinker-CIB1, BBa_K4016041).  HEK293T cells were co-transfected with pcDNA3.1-eGFP, BBa_K4016040, and BBa_K4016041(LiPrePro 3XGS linker group). Fluorescent images were captured 48 hours post transfection for the quantification of GFP protein level.
  
 
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<figure class="figure">
 
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<img src="https://static.igem.org/mediawiki/parts/3/3b/T--NUDT_CHINA--Part_Validation_Flourescent_48h.png
 
<img src="https://static.igem.org/mediawiki/parts/3/3b/T--NUDT_CHINA--Part_Validation_Flourescent_48h.png
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Figure 1. Experimental validation approach.
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Figure 1. Schematic representation of the experimental process of BBa_K4016040 part validation.
  
 
===Result===
 
===Result===
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Since relative fluorescence intensity showed slight decrease (~10%) of GFP fluorescence in RiPrePro1.0 expressing group comparing to the control group under blue light induction. (With the implications obtained from the model), we changed the 1xGS linker unit into 3xGS linker unit. From the Fluorescence images, we can see a significant GFP degradation. Also, the Fluorescent imaging showed significant decrease (~55%) of GFP fluorescence in LiPrePro(3xGS linker) group compared with the LiPrePro(1xGS linker) group.
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Results showed a significant reduction of GFP levels in cells transfected with LiPrePro (3x GS Linker, using BBa_K4016040 and BBa_K4016041) compared to the eGFP and LiPrePro (1x GS linker) transfected control cells under 24, 48, and 72 h of blue light illumination, indicating an improved protein degradation ability of these designs.
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Figure 2.Fluorescence images (left) and quantified fluorescent intensity(right) of HEK-293T cells co-transfection and blue light stimulation with pcDNA3.1(control group), LiPrePro(1xGS linker) and LiPrePro(3xGS linker)(experiment group).
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Figure 2. Fluorescence images (A) and quantified fluorescent intensity (B) of HEK-293T cells co-transfection with pcDNA3.1 (Control group), LiPrePro (1xGS linker, BBa_K4016035 and BBa_K4016036) and LiPrePro (3xGS linker, BBa_K4016040 and BBa_K4016041) (experimental group), 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 indicated hours. data represent mean ± s.e.m. (n=3 biological replicates); ***P < 0.001; two-tailed unpaired Student’s t-test.
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===Reference===
 
===Reference===

Latest revision as of 01:23, 22 October 2021


Trim21-3xGS linker-CRY2

This composite part consists of truncated Trim21 (Part:BBa_K3396007) fused in the N-terminal, CRY2 fused in the C-terminal and 3 x GS linker in the middle. It is designed to generate protein degradation with other parts contain CIB1 through blue light induced Cry2-CIB1 interaction.


Usage and Biology

Researchers have found that optical dimerizers are a powerful new class of optogenetic tools that allow light-inducible control of protein-protein interactions and such tools allow exquisite spatial, temporal, and dose-dependent control of biological events. The basis of these tools is an interaction between two proteins or domains where one of the interacting partners is a photosensory protein or domain that exists in a ‘ground’ or unexcited state, but undergoes a conformational change with light excitation. The second protein or domain selectively binds either the ground orphotoexcited state of the photosensory protein.[1]


Therefore, as our 2020 igem proved that the [antibody Fc domain – Trim21 PRYSPRY domain] interface can be replaced with other protein dimerization pairs, optical dimerizers was used in our program to achieve blue-light induced protein degradation.


One of the most widely used optical dimerizers is the CRY2/CIB system, based on a light-dependent interaction between Arabidopsis cryptochrome 2 (CRY2) and an interacting partner, CIB1. CRY2 is one of the Cryptochromes(CRYs) that photolyase-related blue light receptors which related to vital movement of cells. CRY2-CIB1 system has been used in a variety of cell lines and model systems to optically regulate transcription, recombinase activity, phosphoinositide levels, signaling, cytoskeletal dynamics, and other cellular functions. [2]


To sum up, we changed the [antibody Fc domain – Trim21 PRYSPRY domain] interface to CIB1-CRY2 interaction, to regulate protein degradation precisely by blue light.


This part is an targeting module, consists of truncated Trim21 (Part:BBa_K3396007) fused in the N-terminal, CRY2 fused in the C-terminal and 3 x GS linker in the middle. The 3 x GS Linker is added to stabilize the connection between Trim21 and CRY2. In addition, we also have a version of 5 x GS Linker(see Part:BBa_K4016042).


Characterization

This part was measured through three ways: PCR, enzyme digestion and sequencing.

PCR

The PCR is performed with Green Taq Mix by Vazyme.

F-Prime：5’CTAGCGTTTAAACTTAAGCTTggtaccATTTAAATGCCA 3’

R-Prime：5’TGCTGGATATCTGCAGAATTCttaGGGAGCGGCGCCGATCAT 3’

The PCR protocol is selected based on the Users Manuel. The Electrophoresis was performed on a 1% Agarose gel.

Enzyme Digestion

After the assembly the plasmid was transferred into the Competent E. coli DH5α). After culturing overnight in LB,we minipreped the plasmid for cutting. The cutting procedure was performed with Hind III EcoR I restriction endonuclease bought. The plasmid was cutted in a 20μL system at 37 ℃ for 2 hours. The Electrophoresis was performed on a 1％ Agarose glu.

Sequecing

The plasmid was sequenced correct.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 204
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1240
    Illegal BglII site found at 1699
    Illegal BamHI site found at 2178
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 161
    Illegal AgeI site found at 1124
    Illegal AgeI site found at 1853
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1476
    Illegal BsaI.rc site found at 885
    Illegal SapI.rc site found at 993



Functional validation

To test whether adjusting the GS linker length can help proteins fold correctly and therefore lead to a better degradation efficiency. To begin with, truncated Trim21 protein was linked with CRY2 with a 3x GGGGS protein linker (Trim21-3x GSlinker-CRY2, BBa_K4016040), GFPnanobody was liked to CIB1 in a similar manner (GFPnano-3x GSlinker-CIB1, BBa_K4016041). HEK293T cells were co-transfected with pcDNA3.1-eGFP, BBa_K4016040, and BBa_K4016041(LiPrePro 3XGS linker group). Fluorescent images were captured 48 hours post transfection for the quantification of GFP protein level.

Figure 1. Schematic representation of the experimental process of BBa_K4016040 part validation.

Result

Results showed a significant reduction of GFP levels in cells transfected with LiPrePro (3x GS Linker, using BBa_K4016040 and BBa_K4016041) compared to the eGFP and LiPrePro (1x GS linker) transfected control cells under 24, 48, and 72 h of blue light illumination, indicating an improved protein degradation ability of these designs.

Figure 2. Fluorescence images (A) and quantified fluorescent intensity (B) of HEK-293T cells co-transfection with pcDNA3.1 (Control group), LiPrePro (1xGS linker, BBa_K4016035 and BBa_K4016036) and LiPrePro (3xGS linker, BBa_K4016040 and BBa_K4016041) (experimental group), 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 indicated hours. data represent mean ± s.e.m. (n=3 biological replicates); ***P < 0.001; two-tailed unpaired Student’s t-test.

Reference

[1] Taslimi A , Zoltowski B , Miranda J G , et al. Optimized second-generation CRY2-CIB dimerizers and photoactivatable Cre recombinase[J]. Nature Chemical Biology, 2016.

[2] Liu Y , Li X , Ma D , et al. CIB1 and CO interact to mediate CRY2‐dependent regulation of flowering[J]. EMBO reports, 2018, 19(10):e45762.