Difference between revisions of "Team:NCTU Formosa/Design"

Cotransforming our various plasmids: probe of scFv, color signal and GBP, we created a customized detecting platform-The APOllO E.Cotector!

Tn the core design of customized E.Cotector, APOllO displayed scFv on the surface of E. coli as probes for detection
ScFv is a fusion protein of the variable regions of heavy(VH) and light chain(VL) of immunoglobulins, and the heavy chain and light chain are connected with a short linker peptide of about 15 to 20 amino acids. The function of linker is to connect the heavy chain with light chain. With aid of a linker, scFv still reserve completely functional antigen-binding fragment and specificity of the original immunoglobulin; that is ,the property of specificity of antibody to antigen has being maintained. Moreover, scFv is only 20 percent the size as antibody [4], therefore unlike monoclonal antibodies, which are often produced in mammalian cell cultures, scFv can be simply produced or displayed by bacteria such as E. coli.

To display scFv on the surface of E. coli, we use a transmembrane protein. The transmembrane protein is composed of lipoprotein(Lpp) and outer membrane protein(OmpA).
Lpp-OmpA was designed as a fusion protein consisting of the signal sequence and first 9 amino acid of Lpp, residue 46~159 of OmpA. The Lpp of the N-termini of this fusion protein targets the protein on the membrane while the trans-membrane domain of OmpA serves as an anchor. scFv is on the externally exposed loops of OmpA, which can be anchored to the outer membrane. Between the OmpA and scFv, there is a cut side of restriction enzyme called NcoI. With this cut side, the linked scFv can be easily changed just as a cassette.

The color signals that we have selected are florescence proteins and chromoproteins. Comperating with iGEM, all of resources were from the Giant library of iGEM kit which is accessible to every iGEMer. APOllO utilized the red florescence protein BBa_E1010, the green fluorescent protein BBa_E0040, the blue fluorescent protein BBa_K592100 , and the blue chromoprotein BBa_K592009.

Another plasmid we provide for customization is gold binding polypeptide, abbreviated as GBP. APOllO may display GBP on the surface of E.coli as glue for binding on gold surface.
GBP was designed with the three-repeated of following 14 amino acid sequences: [MHGKTQATSGTIQS], which was developed in an E. coli cell-surface display system. The binding sequence of GBP does not contain cysteine which can form a covalent thiol linkage with gold, the linkage to the gold surface in Self-Assembled Monolayers (SAMs)[2].
The mechanism of the connection between GBP and gold metal plane remains unknown. By using Molecular Dynamics (MD), it indicates that GBP, with an antiparallel β-sheet structure, can recognize gold surface via OH-binding. It is likely that the hydroxyl, together with amine, ligands on GBP recognize the atomic lattice of gold, aligning the molecule along the variants of a six-fold axis on the Au (111) surface [3].

By utilizing the concept of Cotransformation , APOllO can offer various E. Cotectors with scFv ,color signal or GBP and even any desired combination. Therefore, APOllO could customize the E. Cotector to satisfy the need of various detection platforms.