Phocl variant
DNA encoding the NES of MAPKK (NLVDLQKKLEELELDEQQ)26 and the NLS of SV40 (PKKKRKV)27were separately appended by PCR to the 5′ end of the gene encoding PhoCl. The gene encoding mCherry was inserted between the KpnI and XhoI sites of pcDNA3.1(+) (Thermo Fisher Scientific). Genes encoding NES … Visa mer Plasmid construction was performed through standard restriction-enzyme cloning. Pfu (Thermo Fisher Scientific), Taq (New England BioLabs), and Q5 (New England BioLabs) DNA polymerases were used for polymerase … Visa mer Overlap PCR was used to circularly permute the gene encoding mMaple, such that the original termini were joined with a GGSGG linker, and new … Visa mer All cell lines were grown from original frozen stocks without authentication or mycoplasma detection. The HEK cell line is listed in the International Cell Line Authentication … Visa mer The genes for mMaple, PhoCl, and PhoCl-MBP in pBAD/His B were used to transform E. coli strain DH10B or Top10. LB medium supplemented with 100 μg/mL ampicillin was inoculated with these transformants, and the … Visa mer Webb20 mars 2024 · We developed a fusion protein containing a solubilizing maltose-binding protein domain, PhoCl, and two copies of the RGG domain. Several seconds of illumination at 405 nm is sufficient to cleave PhoCl, removing the solubilization domain and enabling RGG-driven coacervation within minutes in cellular-sized water-in-oil emulsions.
Phocl variant
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Webb9 dec. 2024 · Here, we report the X-ray crystal structures of the PhoCl1 green state, red state, and cleaved empty barrel. Molecular dynamics (MD) simulations were performed to provide insight into the precise dissociation mechanism. Using structure-guided engineering and directed evolution, we have developed PhoCl2c with higher contrast … WebbThe finalized PhoCl system has a broad absorbance from 400 to 500 nm, and the dissociation rate is 500 s. Many proteins, ≈ such as Cre recombinase, Gal4 transcription factor, Pannexin-1 ion channel, could be controlled by PhoCl.[10]
Webb25 maj 2024 · The integrin-binding motif RGD was fused to the PhoCl and was decorated on the hydrogel. After being exposed to light at 405 nm, the PhoCl was cleaved and the RGD motif was released, resulting in detachment of the binding cells. The regulatory effect of the light illumination showed a time-dependent and cell density-dependent manner. Webb31 maj 2024 · PhoCl variants: PhoCl2c with higher dissociation contrast ratio, and PhoCl2f with faster dissociation rate. Optogenetic control of protein localization by PhoCl2 variants
WebbThe improved performance of PhoCl2c is also made apparent by the fact that this variant induced a greater degree of capase-3 activation compared to PhoCl1 (Fig. 5F). Cell … WebbPhoCl protein was engineered by Campbell group in 2013 ( Zhang et al., 2024 ). Upon the illumination of violet light (~400 nm), the chromophore undergoes a β-elimination reaction, leading to the cleavage of the polypeptide backbone. The two fragments of the protein then spontaneously dissociate and become unstructured.
Webb7 sep. 2024 · A topological variant of mMaple, named “PhoCl”, was reported by Zhang et al. in 2024 . To achieve the light-induced dissociation, PhoCl was engineered via circular permutation, followed with screening for variants with loss of …
Webb9 feb. 2024 · These proteins contain the chromophore retinal, or a variant, which in response to light isomerizes between an 11- or 13-cis and all-trans retinal. ... Fluorescent proteins such as PhoCl, PYP or Dronpa, which were also used to implement light control are not discussed further. port number tableport number to at\\u0026tWebbPhoCl is a new member of this class that works via a different mechanism - the PhoCl protein is irreversibly cleaved when exposed to light. Robert Campbell’s lab illustrates the … port number sql serverWebbmechanisms by engineering a photocleavable protein (PhoCl) that splits into two fragments upon violet light (~400 nm) illumination. PhoCl is a circularly permuted version of a green-to-red photoconvertible fluorescent protein (FP) in which the main chain break that accompanies photoconversion produces a short iron chef memesWebb10 dec. 2024 · The photocleavable protein (PhoCl) is a green-to-red photoconvertible fluorescent protein that, when illuminated with violet light, undergoes main chain … iron chef marion marionWebbFig. S5 Flow chart of PhoCl evolution. The PhoCl variants discovered during the screening process are represented in blue rectangles. The final PhoCl2 variants are represented in green rectangles. The library generation method, specific mutations discovered, and other key points, are represented in white rectangles. 7 iron chef menu aberfoyle parkWebbPhotocleavage leads to redistribution of a red FP (mCherry) between the cytoplasm and nucleus. Relative to PhoCl1-mCherry, PhoCl2c-mCherry fusion exhibited a more even red … port number straight talk