Customized Biobricks for creating Detection Platform

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

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

In 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 can still reserve the complete function of 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.

Figure 2. ScFv is a fusion protein of the variable regions of heavy(VH) and light chain(VL) of a monoclonal antibody

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(OmpA).

Figure 3. Figure 3. Transmembrane protein: Lpp-OmpA 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.^[1]

Figure 4. To change the scFv sequence easily, We designed the NcoI restriction site between Lpp-Omp-A and scFv. Due to the reason of mix site problem when designing EXSP restriction site between Lpp-OmpA and scFv, NCO1 restriction site rather than EXSP 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 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.

Gold Binding Polypeptide

nother 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 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].

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.

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.

Health and Medical Detection Platform:
Commitment to Targeted drugs Prescription

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 8. The main concept of the process for applying the targeted drug therapy.

Figure 9. We design the platform to detect multimarkers as the reference for applying the combination therapy.

Probe: scFv of targeted drug

Each distinct scFv of targeted drug is displayed on the surfaces of E. coli by using a transmembrane fusion protein, Lpp-OmpA^[5]. By displaying different scFv on different E. coli, we can detect various specific molecules, which are specific to these scFv of targeted drugs. Generally, while the anti-EGFR scFv (originate from Cetuximab) is displayed on the surface of E. coli, we can detect EGFR by this APOllO E.Cotector.

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 individually in direct by distinguishing those different colors of APOllO E.Cotectors. In simple words, since APOllO E.Cotector displays scFv of targeted drugs we can identify the corresponding specific molecules, hence offering an accurate prescription of monoclonal antibody targeted drug.

Figure 10. E.Cotector

Cell Staining Experiment

To prove that E. Cotectors have successfully displayed scFv of Bevacixumab, Cetuxicumab and Trastuzumab respectively, we designed cell staining experiment. The mainly concept is utilizing those color signal APOllO E.Cotectors, which respectively display scFv of targeted drugs to stain the cancer cells, they may specify each of the targeted molecules –EGFR, VEGFR or HER2 on the cell directly in same time.

In fact, in clinical situation, doctors may stain cancer tissue slides by specific antibody to judge whether to use targeted drugs therapy. Therefore, in the future, doctors may obtain results by E.Cotector’s cell staining to define whether these monoclonal-antibody-targeted drugs are optimal to use for the patients. That is, by this application, we will offer a multimarker diagnosis for doctors to judge whether to use combination targeted drugs in a more direct method.

Figure 11. Concept of Cell Staing Experiment

Transducer: Immobilizing on Gold with GBP

With the concept of biosensor, APOllO’s customers may deem the E.Cotector to play the role of biological recognition part and the gold chip to act as the 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.

Figure 12. Concept of Biosensor via E.Cotector