Design

Core of APOllO E.Cotector: Customization

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.

Figure 1. With the co-transform technique, we can insert any scFv or signal-related genetic sequences we want into the E.coli, and create a customized platform ─ the APOllO E.Cotector.

Single Chain Variable Fragment

Probe: scFv from targeted drug

In order to provide doctors with new, direct, and innovative methods in determining the usage of monoclonal-antibody-targeted drugs ,we redesigned the FDA approved monoclonal antibody targeted drugs, such as Bevacizumab (Avastin® anti-VEGF)^[1], Cetuximab (Erbitux® anti-EGFR) ^[2] and Trastuzumab (Herceptin® anti-HER2)^[3] into scFv as probes.

Figure 3. We design the platform to detect multimarkers as the reference for applying the combination therapy.

Each distinct scFv of targeted drug is displayed on the surfaces of E. coli. ScFv from targeted drugs can specifically detect target molecules.

Furthermore, as APOllO E.Cotector expressed scFv and color signal at the same time, the specific molecules that correspond to those scFv can be identified by different colors, hence offering an accurate prescription of monoclonal antibody targeted drug.

Figure 4. E.Cotector

Transmembrane Protein of scFv

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 A(OmpA).

Figure 5. Figure 6. Transmembrane protein: Lpp-OmpA is composed of lipoprotein(Lpp) and outer membrane protein A(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-terminal of this fusion protein targets the protein on the membrane while the transmembrane domain of OmpA serves as an anchor. ScFv is on the externally exposed loops of C-terminal of OmpA, which can be anchored to the outer membrane. Between the OmpA and scFv, there is a cut site of restriction enzyme called NcoI. With this cut site, the linked scFv can be easily changed like a cassette.^[1]

Figure 7. To change the scFv sequence easily, we designed the NcoI restriction site between Lpp-Omp A and scFv. When designing XbaI-SpeI restriction site between Lpp-OmpA and scFv, it cause mixed site. Therefore, NcoI restriction site rather than EX-SP restriction site was designed.

Color Signal

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

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Figure 8. Different Color Signal

Gold Binding Polypeptide

Another plasmid is gold binding polypeptide, abbreviated as GBP. APOllO may display GBP on the surface of E. coli for binding on gold surface.

GBP was designed with the three-repeated following 14 amino acid sequences: [MHGKTQATSGTIQS]. 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].

Figure 9. GBP can recognize and bind on the gold surface.

Transmembrane Protein of GBP

To display the GBP to the outer membrane of E.Cotector, we use a transmembrane protein called Long-chain fatty acid short as FadL. We selected the first 384 amino acid sequence from the FadL for the transmembrane protein of GBP.

Transmembrane Protein FadL transports GBP out of the surface of E. coli

Application: Immobilize on Gold

With the concept of biosensor, E.Cotector play the role of biological recognition part and gold chip act as signal transducer part. Nowadays, gold is the best choice for biosensor substrate because of its advantages of stability to external environment, the excellent capability of transducing electronic signals, the sensitive physicochemical properties, and most important of all, the specific interaction with GBP. Therefore, E.Cotector, which is able to attach on gold chip, can be regarded as the platform for combining with multiple precise physicochemical nanoscale detectors in various field, such as optical, electrochemistry or microbalance, and so on.

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