The APOllO E.Cotector

This year, APOllO introduced a new customized detecting platform - The APOllO E.Cotector.

Our customers can spontaneously choose plasmids from our biobrick libraries and match various plasmids for co-transformation. This procedure helps tailor our product to the wishes of our customers and gives our company a major advantage in the manufacturing process. The simple process of co-transformation can create a dual display system that replaces the process of ligating several insertion genes into one single lengthy plasmid, which tremendously increases our manufacturing efficiency.

Our biobrick libraries consist of three kinds of plasmids: (1) scFv probes detect the target we want, (2) color signals are for observation, and (3) GBP can connect to gold. Customers can detect what they need just by randomly selecting up to two plasmids.

Single Chain Variable Fragment (scFv)

ScFv is a fusion protein of the variable regions of heavy chain (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. Even with addition of a linker, scFv can still retain the complete function of antigen-binding site and specificity of the original immunoglobulin.

Figure 1. ScFv is a fusion protein of the variable regions of heavy chain (VH) and light chain (VL) of a monoclonal antibody.

Moreover, scFv can be easily produced and displayed by bacteria as it is only 20 percent of the size of most normal antibodies^[1]. In addition, scFv can be envisaged to be applied in the non-pharmaceutical sector, such as in food, cosmetic or environmental industries. The unique and highly specific antigen-binding ability of biosensors might, for example, be exploited to block enzymes that cause food spoilage or to detect environmental factors present in very low concentrations^[2].

Figure 2. With scFv probes, E.Cotector could be applied to various fields. For example, in health & medicine, food, agriculture, and environment industries.

Targeted drugs

We focus on health and medicine, leading us to realize the significance of the targeted drug's market. According to statistics, the usage rate of targeted drug therapy in cancer treatment has increased in less than ten years. In Figure 3, it was shown that the targeted drug is not commonly used, accounting for only 11% usage in 2003. Over one decade, it is estimated that the usage of targeted drug will dramatically increase to 46%. However, targeted drugs have been shown to have a more accurate attack on cancer cells and cause less harmful damage to normal tissues via specific binding to target molecules^[3]. Targeted therapy is alleged to be a "personalized medicine" because health care professionals can use clinical test results from a patient to select a specific drug that has a higher likelihood of being effective for that particular person. This indicates that detection for targeted drugs is a growing field and will become a prevalent pre-diagnosis for cancer treatment in the near future.

Figure 3. The usage of targeted drugs in cancer treatment.

Pre-diagnosis of targeted drugs treatment

To create the new era of tailored targeted drugs, doctors must aim at appropriate target molecules for patients with particular diseases. In 2014, the U.S. Food and Drug Administration (FDA) issued a guideline to facilitate the development and review of diagnostic tests. The diagnostic tests are a crucial step to identifying the abnormal cancer biomarkers as they can help medical practitioners determine which patients could benefit from the certain drugs as opposed to those who should not receive the medication. Treatment decisions must be optimal as to not cause bodily damage and waste time, money, and medical resources. FDA encourages the joint of targeted drugs therapies and precise diagnostics tests as they are essential in the safety and effectiveness of targeted drugs^[4].

The concept of combination therapy

Although targeted drugs treatments can lead to the dramatic regressions of solid tumors, the responses are often short-lived as the resistant cancer cells will arise after the period of treatment. The major strategy proposed for overcoming the resistance is combination therapy^[5]. The clinical and preclinical researches further indicates that targeted drug therapy combined with other types of therapies to treat cancers may attain greater effects than using only one therapy. With the concept of combination therapy, the doctor can improve the treating effect and reduce the occurrence of cancer cell resistance toward the targeted drugs. This lessens the probability that a single mutation will cause cross-resistance to both drugs^[6].

Contribution to existing Biobrick: Lpp-OmpA BBa_K103006

Figure 4. (Left) NCTU_Formosa's Lpp-OmpA sequence

Figure 5. (Right) Warsaw 2008 iGEM team's Lpp-OmpA sequence

The original Lpp-OmpA sequence contains: a Ndel restriction site, one to twenty-nine amino acids of LPP_ECOLI P69776 from UniProtKB, forty-six to one hundred fifty-nine amino acids of outer membrane protein A fragment, Gly-Ser-Gly unstructuralized linker BBa_K103007, and SacI restriction site. We successfully exerted the function of Lpp-OmpA by the following steps. We removed the Ndel and SacI restriction .Furthermore, Gly-Ser-Gly unstructuralized linker BBa_K103007 is said to allow proper folding of both fusion partners[7]. However, Lpp-OmpA fusion protein can still retain its function without GGGSGGGS sequence[8]. Therefore, we are left with one to twenty-nine amino acids of LPP_ECOLI and forty-six to one hundred fifty-nine amino acids of outer membrane protein A fragment. To shorten the distance of scFv and the outer membrane, we adjusted the amino acids of OmpA from forty-six to twenty-five and one hundred fifty-nine to one hundred thirty-eight. Then, we added a NcoI restriction site to allow convenient fusions. By this improvement, we perfectly utilized Lpp-OmpA to bring scFv onto the outer membrane E. coli and are completely proved by our experiments.