Difference between revisions of "Part:BBa K4016035"

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==Usage and Biology==
 
==Usage and Biology==
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This part is composed of Cryptochrome 2(CRY2) photoreceptor linked to Trim21. When induced by blue light, CRY2 dimerizes with its binding partner CIB1, effectively bringing the target site defined antibody GFP-nano. While achieving the purpose of optical control, Trim21 bind with antibody GFP-nano to prove that the PREDATOR PRO really works.
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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]
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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.
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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 interaction 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]
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In iGEM 2021,to develop a light inducible PREDATOR (LiPrePro) system, we intended to replace the core module of PREDATOR PRO system into Cry2/CIB1 pairs.Therefore, this part can be used togther with [[Part:BBa_K4016036]] to prove whether light-mediated protein degradation can be achieved with our system.
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3.GFP is degraded by ubiquitin-proteasome system recruited by Trim21
 
3.GFP is degraded by ubiquitin-proteasome system recruited by Trim21
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==Characterization==
 
==Characterization==
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==Functional validation==
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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.
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<html>
  
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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
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" class="figure-img img-fluid rounded"  height="350px">
  
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</figure>
  
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==Functional test==
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</html>
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===Method===
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Figure 2. Experimental validation approach.
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*Double luciferase reporter gene detection
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1.Preparation: Abandon the supernatant. Use PBS to wash cells. Abandon the added PBS.[two lines after two lines; separate to two steps]
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2.Lytic cells: after fully mixing the reporter gene cell lysate, add the reporter gene cell lysate to fully lyse the cells. After absorbing the cell culture medium, add reporter gene cell lysate with 200uL / hole in 24 orifice plate. Place the culture plate on a rocking platform or orbital shaker with gentle motion .
 
  
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3.After full cracking, cryopreservation(N2;liquid).Then frozen samples need to be tested at room temperature.
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===Result===
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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
  
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4.14000g centrifuge for 10min. Then suck up the supernatant for the test later.
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<html>
  
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5.The firefly luciferase detection reagent and the sea kidney luciferase detection buffer were melted and reached room temperature. The sea kidney luciferase detection substrate (100x) is placed on an ice bath or ice box.
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<figure class="figure">
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<img src="https://2021.igem.org/wiki/images/e/ef/T--NUDT_CHINA--Part_Result_35-36.png
  
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6.According to the amount of 50 uL needed for each sample, take appropriate amount of sea kidney luciferase detection buffer and add sea kidney luciferase detection substrate (100x) to prepare sea kidney luciferase detection working solution(1:100).
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" class="figure-img img-fluid rounded"  height="350px">
  
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7.Turn on the chemiluminescence meter or the multi-function enzyme labeling instrument with the function of detecting chemiluminescence according to the operating instructions of the instrument. the determination interval is set to 2 seconds and the determination time is set to 10 seconds. (each 12 seconds plus reagent; test in 6min)
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</figure>
  
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8.When determining each sample, take 30uL.The usage of the same batch of samples should be consistent.
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</html>
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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).
  
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9.Add 50uL of firefly luciferase detection reagent, beat well with a gun or mix well in other appropriate ways, and then determine RLU (relativelightunit).
 
  
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10.After completing the above steps for the determination of firefly luciferase, add 50uL of sea kidney luciferase detection solution, beat it well with a gun or mix it in other appropriate ways, and then determine RLU (relativelightunit).
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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]]
  
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11.When the sea kidney luciferase was used as the internal reference, the LUR value determined by firefly luciferase was divided by that determined by sea kidney luciferase. According to the obtained ratio, the activation degree of reporter gene among different samples was compared. If firefly luciferase is used as the internal reference, similar calculation can also be carried out.
 
  
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===Result===
 
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Figure 2. The result of double luciferase reporter gene detection
 
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To prove PREDATOR PRO really works, we use the experiment--double luciferase reporter gene detection to verify its outstanding ability of protein degradation under blue light inducing. The result showed that on the condition of blue light inducing, the efficiency of protein degradation is significantly better than in the dark condition, which specifically shows the PREDATOR PRO is workable.
 
  
 
===Reference===
 
===Reference===
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1.Kennedy, M. J. et al. Rapid blue-light–mediated induction of protein interactions in living cells. Nat Methods 7, 973–975 (2010).
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[1] Taslimi, A. et al. Optimized second-generation CRY2–CIB dimerizers and photoactivatable Cre recombinase. Nat Chem Biol 12, 425–430 (2016).
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2.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).
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3.Taslimi, A. et al. Optimized second-generation CRY2–CIB dimerizers and photoactivatable Cre recombinase. Nat Chem Biol 12, 425–430 (2016).
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4.Foss, S. et al. TRIM21—From Intracellular Immunity to Therapy. Front. Immunol. 10, 2049 (2019).
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5.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.
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[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.

Latest revision as of 18:03, 21 October 2021


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 interaction 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]

In iGEM 2021,to develop a light inducible PREDATOR (LiPrePro) system, we intended to replace the core module of PREDATOR PRO system into Cry2/CIB1 pairs.Therefore, this part can be used togther with Part:BBa_K4016036 to prove whether light-mediated protein degradation can be achieved with our system.


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.