PstI restriction site was mutated. Includes start and stop codon.
Part comes from Origene human cDNA clone (NM_002176.2).
Granulysin, a T Cell Product,Kills Bacteria by Altering Membrane Permeability
Granulysin, a protein located in the acidic granules of human NK cells and cytotoxic T cells, has microbial activity against a broad spectrum of microbial pathogens. The substance released by cytotoxic T cells (CD8) when attached to infected body cells. It creates holes in the target cell membrane and destroy it. That is,Granulysin induces apoptosis in target cells and have antimicrobial action. It is broadly antimicrobial, killing microbes that cause, for example, tuberculosis and malaria, and can destroy some tumors. A series of peptides generated from the amino acid sequence of granulysin are potential antibiotics. Granulysin increased the permeability of bacterial membranes, as judged by its ability to allow access of cytosolic ??-galactosidase to its impermeant substrate.Granulysin functions to create holes in the target cell membrane and destroy it. Granulysin is able to induce apoptosis in target cells and also has antimicrobial action.
Granulysin can be purified via nickel affinity chromatography.
Granulysin is a member of the saposin-like protein (SAPLIP) family. The gene was synthesized at GENEART in pMA vector
dnMyD88 is a dominant negative form of protein MyD88 (an important protein in signaling cascade through TLR4 receptor). dnMyD88 is linked to GFP (acts as a reporter) through six aminoacid long protein linker.
dnMyD88-linker-GFP was made by using PCR overlap extension technique. This part is cloned in BioBrick vector pSB1AK3. Original sequence has PstI restriction site so we have to introduce a mutation without affect on amino acid sequence.
dnMyD88 is a dominant negative form of protein MyD88 (an important protein in signaling cascade through TLR4 receptor), which was our main target for modifying the signaling path. dnMyD88 is linked to Rluc (which acts as a reporter) through six aminoacid long protein linker.
dnMyD88-linker-Rluc was made by using PCR ligation technique. This part is cloned in BioBrick vector pSB1AK3.
Chemokine (CXC motif) receptor 4, fused to N-terminal half of ubiquitin.
CXCR4 and NUb fragments were fused with PCR overlap extension method without any linker added. The part is cloned into BBa_J52017 (a vector with eukaryontic terminator).
CXCR4 gene obtained from Origene, NUb gene obtained from Dualsystems.
A putative neuraminyllactose-binding hemagglutinin from Helicobacter pylori.
Chemokine (C-C motif) receptor 5, a HIV-1 co-receptor.
The part is cloned into BBa_J52017, a vector with eukaryontic terminator. PstI and EcoRI restriction sites inside CCR5 gene were mutated.
This is a part that is composed of C-terminal end of chimeric flagellin (BBa_K133016). We used aminoacids from 215th aminoacid on. Chimeric flagellin is composed of C- and N-terminal part of E.coli flagellin and of variable region of H.pylori. The E.coli parts are responsible for binding and activation of cell membrane receptor TLR5 to initiate immune response, while H.pylori variable region was used to array the immune response against H.pylori. This part was used in combination with CF213 - chimeric flagellin (BBa_K133010). Between the CF213 and CF215 we have put H.pylori antigens in order to array the immune response against H.pylori.
It was important to split the chimeric flagellin in the variable region to assure that the antigen will be in the right place and not to interrupt the 3d structure of the protein.
We used polimerase chain reaction from chimeric flagellin (BBa_K133016).
dnMyD88-linker-Rluc-linker-PEST191 is a part where dnMyD88 (very important protein in signaling cascade through TLR4 receptor) is linked to Rluc (reporter) and PEST191 (degradation tag). Both linkers are made of six aminoacids.
dnMyD88-linker-Rluc-linker-PEST191 was made by using PCR ligation technique. It was difficult since we had some problems with primers. This part is cloned in BioBrick vector pSB1AK3.
This part codes anti-Norovirus GII.4 scFv (single-chain variable fragment) which derives from human 12A2 antibody. This scFv is suggested to prevent binding of human norovirus to Hist-Blood Group Antigens. [1] This coding sequence was fused to INPNC-His (part number: ) or BclA-His (part number: ) in our project. For more information, please visit our Wiki page.
silent mutation was made to remove PstI: G255C
We have been given given the scFv-encoding plasmid (NoV GII.4 strain-specific antibody 12A2 (vector: pSCCA5-E8d)) from Dr. Kyoko Moriguchi of the Fujita Health University Department of Virology Parasitology.
Part is composed of TIR domain of Traf6 and GFP linked with 6 aminoacids long linker. GFP serves as reporter. For usage in eukaryotes.
dnMyD88 is a dominant negative form of MyD88, which has an important role in TLR signalling pathway. dnMyD88 contains only TIR domain of MyD88.
Toll-like receptor 3 cytoplasmic domain.
Cloned into pSBAK3 vector with terminator. Start codon is NOT included.
Sequence for chemokine receptor CD4, including signal peptide.
It is an imortant protein in signaling cascade through TLr receptors (MyD88 dependent pathway).