Difference between revisions of "Part:BBa K4016004"

Revision as of 18:39, 20 October 2021

asLOV2

The ~ 15 kDa aslov2 domain consists of a main body that associates with a host-incorporated flavin cofactor, and a C-terminal Jα helix.

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.

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).

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.

Figure 1. Schematic figure of asLOV2-zdk interaction

Experimwntal validation

This part was validated through 4 ways: PCR,enzyme digestion, sequencing and functional test.

PCR

Upper-Prime: 5’-CAGCTAAAGTGCGAAAGCGGCGGCGAGTTCCTGGCCACCACC-3’

Lower-Prime: 5’-CAGGTTGTTAATCTGttaCAGCTCCTTGGCGGCCTC-3’

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.

Sequencing

This part was sequenced as correct after construction.

Sequence and Features

Experimental Validation

We constructed two promoters to transcribe two parts,tetR-asaslov2 and Vp64-zdk, into one plasmid. Once aslov2 and zdk can bind to each other, the tetR and Vp64 connected to them will also bind.

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. It began the process of SEAP with chemiluminescence detection.

SEAP assay

Since protein-protein interactions occur in mammalian cells, it is expected that the protein will conform to a more natural state and undergo post-translational modifications. Therefore, the experimental results of this assay are likely to reflect biologically important interactions. The two proteins are expressed as a fusion protein of tetO and tTA, and the SEAP reporter gene is transcribed on the plasmid only when the proteins interact. The interaction between the two proteins can be detected by SEAP analysis.

Result

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.

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):