Part:BBa_K4016035

Trim21-CRY2

This composite part is designed to generate GFP degradation with GFPnano-CIB1(Part:BBa_K4016036) through CRY2-CIB1 dimerization.

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]

This part is composed of Cryptochrome 2(CRY2) photoreceptor linked to Trim21[3-4]. When induced by blue light, CRY2 dimerizes with its binding partner CIB1, effectively bringing the target site defined antibody GFP-nano[5-6]. While achieving the purpose of optical control, Trim21 bind with antibody GFP-nano to prove that the PREDATOR PRO really works.

Figure 1. Schematic figure of BBa_K4016035 and BBa_K4016036

• Here is the mechanism of the recombined Trim21-CRY2:

1.Trim21-CRY2 connect with GFPnano-CIB1 through CRY2-CIB1 interaction and forms a dimerized complex.

2.Inside the complex, GFPnano-CIB1 targets GFP.

3.GFP is degraded by ubiquitin-proteasome system recruited by Trim21

Characterization

This part was validated through four ways:PCR, enzyme digestion, sequencing and functional test.

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 preparation of the plasmid was performed with TIANprep Mini Plasmid Kit from TIANGEN. The cutting procedure was performed with XbaI and KpnI restriction endonuclease bought from TAKARA.

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 1195
  Illegal BglII site found at 1654
  Illegal BamHI site found at 2133
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 161
  Illegal AgeI site found at 1079
  Illegal AgeI site found at 1808
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1431
  Illegal BsaI.rc site found at 840
  Illegal SapI.rc site found at 948
  

Functional validation

In order to simplify the test and optimization procedure,, we used GFP as thewas used as the target protein, therefore a simple fluorescent imaging could be sufficient to evaluate the degradation efficiency. HEK-293T cells were co-transfected with GFP-Fluc-Rluc expressing plasmid and LiPrePro plasmid/empty vector.

Figure 2. Experimental validation approach.

Result

Fluorescent imaging showed slight decrease (~5%) of GFP fluorescence in LiPrePro expressing group comparing to the control group (Fig.3)under 48 h blue light illumination

Figure 3.Fluorescence images (A) and quantified fluorescent intensity(B) of HEK-293T cells co-transfection and blue light stimulation with pcDNA3.1(control group), LiPrePro(Trim21-CRY2 and GFPnano-CIB1 pairs).

Since the result wasn't satisfying enough, we adjusted the linker length between modules to help proteins folding correctly. Click here to see the improvement and results: Part:BBa_K4016040, Part:BBa_K4016042

Reference

[1] Taslimi, A. et al. Optimized second-generation CRY2–CIB dimerizers and photoactivatable Cre recombinase. Nat Chem Biol 12, 425–430 (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.

[3] Foss, S. et al. TRIM21—From Intracellular Immunity to Therapy. Front. Immunol. 10, 2049 (2019).

[4] Liu, C. et al. Predator: A novel method for targeted protein degradation. http://biorxiv.org/lookup/doi/10.1101/2020.07.31.231787 (2020) doi:10.1101/2020.07.31.231787.

[5]Kennedy, M. J. et al. Rapid blue-light–mediated induction of protein interactions in living cells. Nat Methods 7, 973–975 (2010).

[6]Bugaj, L. J., Choksi, A. T., Mesuda, C. K., Kane, R. S. & Schaffer, D. V. Optogenetic protein clustering and signaling activation in mammalian cells. Nat Methods 10, 249–252 (2013).