Difference between revisions of "Part:BBa K4016002"
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<partinfo>BBa_K4016002 short</partinfo>
 
<partinfo>BBa_K4016002 short</partinfo>
  
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The ~ 15 kDa aslov2 domain consists of a main body that associates with a host-incorporated flavin cofactor, and a C-terminal Jα helix.
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This part interacts with LD3([[Part:BBa_K4016003]]) through the induction of small molecule. Technically, we use A1331852 and A1155463 to induce the dimerization of this part and LD3. Functionally, we use this pair to fuse with other proteins respectively, bringing our system together in a controllable way.
  
 
==Usage and Biology==
 
==Usage and Biology==
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Light-sensing proteins (LSPs) have been used extensively in optogenetics and other fields to trigger specific subcellular events with light. Upon irradiation with light, the LSP domain undergoes a conformational shift, often revealing a cryptic site or inducing dimerization with a binding partner, thereby triggering downstream effects. LSPs typically absorb light in the visible to infrared wavelengths, and, critically, can undergo their lighttriggered conformational change repeatedly without functional degradation of the protein.
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B-cell lymphoma-extra large (Bcl-xL) is an anti-apoptotic Bcl-2 protein found in the mitochondrial membrane.
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MotifGraft performs extensive structural searches in protein databases to identify scaffolds with backbone simi-larity to a binding motif, as well as structural compatibility to a given binding partner. Finally human apolipoprotein E4 (LD3) shows to have a good bond with Bcl-XL.  
  
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The aslov2 trap and release of protein (LOVTRAP) system in particular holds promise for biomaterial functionalization studies because the constituents are relatively small in size and bind to each other both specifically and tightly. LOVTRAP consists of the blue light-absorbing aslov2 domain of Avena sativa10 and its binding partner ZDark (Zdk). Upon irradiation with light (400– 500 nm), the flavin cofactor becomes excited, allowing it to form a covalent adduct with a cysteine residue in the aslov2 globular domain. This adduct interaction initiates the unraveling of the C-terminal Jα helix, characteristic of aslov2’s excited state. Thermal relaxation allows the cysteine adduct to unbind the flavin cofactor and the Ja helix to re-coil and dock with the main protein body, thereby reverting aslov2 to its dark state. Zdk binds aslov2’s dark state with a dissociation constant (Kd) of 26.2 nM, the highest affinity of any LSP system currently in use, and then unbinds aslov2 upon blue light irradiation (Kd> 4 uM).
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Two known small molecules, A1331852 and A1155463, have been reported to bind to Bcl-XL at less than 10 pM and were shown by SPR and size-exclusion chromatogra-phy coupled to a multi-angle light scatter (SEC-MALS) to dissoci-ate Bcl-XL from LD3, with an  apparent half-maximum inhibitory concentration (IC50) of 242 nM and 101 nM , respectively.
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LOVTRAP is particularly useful because it can be readily applied to a broad range of proteins and protein activities, and because it enhances the dynamic range, or lit-dark activity difference, for the targeted proteins.
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==Characterization==
 
==Characterization==
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This part BBa_K4016024 was cloned in pXQ109 plasmid and transfected into HEK293T cell lines using Invitrogen LipofectamineTM 3000.This part is validated through four ways: PCR, Sequence, and functional testing.
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===PCR===
 
===PCR===
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Methods
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The PCR is performed with Premix EX Taq.
  
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Upper-Prime: 5’-CAGCTAAAGTGCGAAAGCGGCGGCGAGTTCCTGGCCACCACC-3’
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F-Prime：5’CTAGCGTTTAAACTTAAGCTTGCCACCATGgagtctgggggag 3’（oXQ218 forward prime）
  
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Lower-Prime: 5’-CAGGTTGTTAATCTGttaCAGCTCCTTGGCGGCCTC-3’
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R-Prime：5’gctgtagtccaggatTCCGTACAGTTCCACGAAGGT3’（oXQ169 reverse prime）
  
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===Enzyme cutting===
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The PCR protocol is selected based on the Users Manuel. The Electrophoresis was performed on a 1％ Agarose glu. The result of the agarose electrophoresis was shown on the picture below.
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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 EcoRI and SpeI restriction endonuclease bought from TAKARA.
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The plasmid was cutted in a 20μL system at 37 ℃ for 2 hours. The Electrophoresis was performed on a 1％ Agarose glu.
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===Sequence===
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This part is sequenced as correct after construction.
  
  
 
==Experimental Validation==
 
==Experimental Validation==
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For this part of functional testing, we use two methods at the same time: tet-on and SEAP.
 
  
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===Tet-on===
 
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  In the pXQ162 plasmid, we can use two promoters to transcribe two parts, tetR-asaslov2 and Vp64-zdk. Once asaslov2 and zdk can bind to each other, the tetR and Vp64 connected to them will also bind, then this It’s time to meet the initial conditions of the tet-off system.
 
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  TetR combined with tetO, so as to open the downstream gene transcription. In the presence of effector, tetR changes its spatial conformation after being bound to the effector, and cannot bind to tetO, thus weakening the transcription of downstream genes.HSV VP16 is an important activator in the early transcription process of human herpetic virus, and the transcription intensity of downstream genes can be greatly improved by fusing its C-terminal with different transcription factors. Minimum basic promoter element miniPromoter when working alone transcription efficiency is extremely low, will its role and tetO combination become cis element, Ptet, can be recognized by tTA, also called tetR - VP16. Compared with the original TET-OFF system, the switching performance of the target gene can be better controlled. The most primitive activating factor is Vp16, and Vp64 is its tetramer. With the tTA, we can open the Transcription of downstream genes, which is the transcription of SEAP gene.
 
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It began the process of SEAP with chemiluminescence detection.
 
  
  
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===SEAP assay===
 
  
  
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<partinfo>BBa_K4016002 parameters</partinfo>
 
<partinfo>BBa_K4016002 parameters</partinfo>
 
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===Reference===
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[1] Giordano-Attianese, G. et al. A computationally designed chimeric antigen receptor provides a small-molecule safety switch for T-cell therapy. Nat Biotechnol 38, 426–432 (2020).

Latest revision as of 15:27, 21 October 2021


Bcl-xl

This part interacts with LD3(Part:BBa_K4016003) through the induction of small molecule. Technically, we use A1331852 and A1155463 to induce the dimerization of this part and LD3. Functionally, we use this pair to fuse with other proteins respectively, bringing our system together in a controllable way.

Usage and Biology

B-cell lymphoma-extra large (Bcl-xL) is an anti-apoptotic Bcl-2 protein found in the mitochondrial membrane. MotifGraft performs extensive structural searches in protein databases to identify scaffolds with backbone simi-larity to a binding motif, as well as structural compatibility to a given binding partner. Finally human apolipoprotein E4 (LD3) shows to have a good bond with Bcl-XL.

Two known small molecules, A1331852 and A1155463, have been reported to bind to Bcl-XL at less than 10 pM and were shown by SPR and size-exclusion chromatogra-phy coupled to a multi-angle light scatter (SEC-MALS) to dissoci-ate Bcl-XL from LD3, with an apparent half-maximum inhibitory concentration (IC50) of 242 nM and 101 nM , respectively.


Characterization

This part BBa_K4016024 was cloned in pXQ109 plasmid and transfected into HEK293T cell lines using Invitrogen LipofectamineTM 3000.This part is validated through four ways: PCR, Sequence, and functional testing.

PCR

The PCR is performed with Premix EX Taq.

F-Prime：5’CTAGCGTTTAAACTTAAGCTTGCCACCATGgagtctgggggag 3’（oXQ218 forward prime）

R-Prime：5’gctgtagtccaggatTCCGTACAGTTCCACGAAGGT3’（oXQ169 reverse prime）

The PCR protocol is selected based on the Users Manuel. The Electrophoresis was performed on a 1％ Agarose glu. The result of the agarose electrophoresis was shown on the picture below.


Sequence

This part is sequenced as correct after construction.


Experimental Validation

Result

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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]



Reference

[1] Giordano-Attianese, G. et al. A computationally designed chimeric antigen receptor provides a small-molecule safety switch for T-cell therapy. Nat Biotechnol 38, 426–432 (2020).