Part:BBa_K4016040
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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 204
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1240
Illegal BglII site found at 1699
Illegal BamHI site found at 2178 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 161
Illegal AgeI site found at 1124
Illegal AgeI site found at 1853 - 1000INCOMPATIBLE 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 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.
Result
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.
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.
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