Difference between revisions of "Part:BBa K4016005"

Revision as of 05:14, 21 October 2021

zdk

ZDark, a ~ 6 kDa z-affibody developed by the Hahn group, an engineered small protein that binds selectively only to the dark state of LOV2, the photosensory domain from Avena sativa phototropin

Usage and Biology

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[1].

LOVTRAP consists of the blue light-absorbing LOV2 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 LOV2 globular domain. This adduct interaction initiates the unraveling of the C-terminal Jα helix, characteristic of LOV2’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 LOV2 to its dark state. Zdk binds LOV2’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 LOV2 upon blue light irradiation (Kd> 4 uM)[2].

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.

Experimental validation

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

PCR

The PCR is performed with Premix EX Taq.

Upper-Prime: 5’-AAGAGAAAGGTGGAGGCCAGTgtggataacaaattcaataaa-3’

Lower-Prime: 5’- TCTCTTCTTCTTGGGACTGGCgctaccaccagaaccaccttt-3’

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.

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.

Sequence

This part is sequenced as correct after construction.

Sequence and Features

Functional test

SEAP assay

We construct tetR-asLOV2-zdk-Vp64 and TCE-SEAP to test the expression of this part. VP64 is a transcriptional activator. When fused to another protein domain that can bind near the promoter of a gene, VP64 acts as a strong transcriptional activator. TetR can recognize and combine with TCE then inhibit its downstream transcription. The interaction of asLOV2 and zdk would restrain the TCE’s inhibition and start the transcription of SEAP.

In order to verify the hypothesis that asLov2-zdk is controlled by blue light, and more specifically, asLov2-zdk aggregates in the dark and depolymerizes under blue light, we conducted simplified version of tet-off and SEAP assay related experiments. Once aslov2 and zdk can bind to each other, the tetR and Vp64 connected to them will also bind, and tetR combined with tetO, so as to open the downstream gene transcription. With the tTA, we can open the transcription of downstream genes, which is the transcription of SEAP gene. The result showed that, compared with dark condition, after 24 hours and 48 hours, the increase trend of enzyme activity under blue light irradiation condition is significantly reduced. Specifically, it can be proved that the blue light irradiation did act on the asLov2-zdk module, which separated the asLov2-zdk, stopped or reduced the start of SEAP gene transcription, and the detected enzyme activity is greatly reduced.

Result

Reference

[1] Joshua A. Hammer,Anna Ruta,Jennifer L. West. Using Tools from Optogenetics to Create Light-Responsive Biomaterials: LOVTRAP-PEG Hydrogels for Dynamic Peptide Immobilization[J]. Annals of Biomedical Engineering,2020,48(prepublish):

[2] Hui Wang,Marco Vilela,Andreas Winkler,Miroslaw Tarnawski,Ilme Schlichting,Hayretin Yumerefendi,Brian Kuhlman,Rihe Liu,Gaudenz Danuser,Klaus M Hahn. LOVTRAP: an optogenetic system for photoinduced protein dissociation[J]. Nature Methods: Techniques for life scientists and chemists,2016,13(9):