Part:BBa_K4016043

GFPnano-5xGS linker-CIB1

This composite part consists of GFP nano fused in the N-terminal, CIB1 fused in the C-terminal and 5 x GS linker in the middle. It is designed to generate GFP degradation through blue light induced Cry2-CIB1 interaction as a verification of our design.

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]

The antibody GFP-nano(see Part :BBa_K2653001) is to bind with target GFP. It works as a core to degrade the proof of concept target GFP as the tool to bind with it. The 5 x GS Linker is added to stabilize the connection between CIB1 and GFPnano. In addition, we also have a version of 3 x GS Linker(see Part:BBa_K4016042).

• Here is the mechanism of the recombined GFPnano-5 x GS linker-CIB1:

1. The Trim21-5 x GS linker tagged with CRY2 combines with CIB1. 2. TRIM21 -5 x GS linker-CRY2 (see BBa_K4016040) connect GFPnano-5 x GS linker-CIB1 target through the CRY2-CIB1 interaction. 3. The GFP is degraded by ubiquitin-proteasome system recruited by TRIM21.

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’TGCTGGATATCTGCAGAATTCttaGATGTAGTCGGTCTT 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 gel.

Sequecing

The plasmid was sequenced correct.

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 541
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 35
  

Functional validation

To test whether our design of changing the GS linker into longer one work, pcDNA3.1 with EGFP, pcDNA3.1 with Trim21-5x GSlinker-CRY2 and pcDNA3.1 with GFPnano-5x 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.

Figure 1. Experimental validation approach.

Result

Since our part TRIM21-CRY2 (BBa_K4016035) and GFPnano-CIB1 (BBa_K4016036) didn’t show great degradation effect compared to the control group, we interviewed experts and find a solution--expend the linerk between two modules. To enhance the degradation performance of parts, we designed part TRIM21-5*GS linker-CRY2 (BBa_K4016042) and part GFPnano-5*GS linker-CIB1 (BBa_K4016043), which expend the linker for three times. The result showed a significant decrease of fluorenscent intensity compared to the control group, indacating high degradation efficiency of our system. The result successfully proved our system can work as we expected and confirmed our resign of expending the linker between two modules .

Figure 2.Fluorescence images (A) and quantified fluorescent intensity(B) of HEK-293T cells co-transfection and blue light stimulation with pcDNA3.1(control group), LiPrePro(1xGS linker) and LiPrePro(5xGS linker)(experiment 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.