Difference between revisions of "Part:BBa K4016004"

Revision as of 15:40, 16 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.

Characterization

PCR

Upper-Prime: 5’-CAGCTAAAGTGCGAAAGCGGCGGCGAGTTCCTGGCCACCACC-3’

Lower-Prime: 5’-CAGGTTGTTAATCTGttaCAGCTCCTTGGCGGCCTC-3’

Enzyme cutting

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. The plasmid was cutted in a 20μL system at 37 ℃ for 2 hours. The Electrophoresis was performed on a 1％ Agarose glu.

Experimental Validation

For this part of functional testing, we use two methods at the same time: tet-on and SEAP.

Tet-on

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.

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

Result

Sequence and Features