Team:Fatih-Medical/Sherlocoli

Overview

Today Cancer is one of the most serious health problems and there is still no common effective cure for this disease. It is a group of diseases characterized by unregulated growth and spread of abnormal cells. If the spread is not controlled, it can result in death.

Cancer is the second most common cause of death in the US, exceeded by only heart disease. In 2012, about 577,190 Americans are expected to die of cancer, more than 1,500 people a day. 
In most cases, cells derived from the primary tumor permeate through the bloodstream or lymphatic system and pose a greater death risk than the expansion of the original neoplasm. These cells are particularly called Circulating Tumor Cells (CTC) and are essential for establishing metastasis.

Detection of CTCs in the blood in pre-metastatic phase of cancer offers a new opportunity for the early diagnosis and prognosis of the disease.
Nowadays, there are only two major strategies for detection of Circulating Tumor Cells. 
These are reverse transcription PCR (RT-PCR) and immunomagnetic or other immunoseparation techniques [1]. However, these methods are costly and time consuming.

Our project is focused on CTC/Free DNA detection and the early diagnosis of cancer by the means of SynBio. We want to establish a CTC detection and additionally a Free DNA detection device in E.coli which will operate in a short period of time and reduce the expanses. As a result, our system could become a simple routine blood test for cancer diagnosis in the future.

EpCAM is a transmembrane protein, which is overexpressed on almost all CTCs. For our recognition system, we will use C215 Mouse Monoclonal Antibody which is very suitable for the EpCAM antigen[2].
We will install two protein complexes (TEVp N Terminal-OmpA-C215 and TEVp C Terminal-OmpA-C215) into the bacterial cell wall. As a result of the interaction between these two protein complexes, the split TEVp parts will fuse to form an active enzyme and trigger a signal pathway. [see details]

Apart from CTCs as cancer biomarkers which we can find in a patient’s blood, there is also free DNA containing oncogenes which is released after lysis of tumor cells. This DNA also indicates the existence of a tumor. One of our goals is to find these free DNA molecules in the blood using TAL effectors – customizable proteins which are able to target specified DNA sequences [3].

Our construct for this part of the project is very similar to the CTC detection part: First, a special DNA sequence containing a point mutation which is the cancer causing component will be determined. After that, C215 antibodies from the protein complex mentioned above will be replaced by two compatible customized TAL effector proteins. When our target DNA is attached by the neighboring TAL effectors, the merging of TEV N and TEV C Terminal will trigger the same signal pathway as above. [see details]

Regarding all of the difficulties which we may encounter, we decided to construct two different signaling pathways. One is based on the FRET effect and the other on the Fast Response module of Team Imperial College London 2010. [see details]



References:
[1] Hayes DF, Smerage J. (2008). "Is There a Role for Circulating Tumor Cells in the Management of Breast Cancer?". Clin Cancer Res 14 (12): 3646–50. doi:10.1158/1078-0432.CCR-07-4481. PMID 18559576

[2] Bjork P, Jonsson U, Svedberg H, Larsson K, Lind P, Dillner J, Hedlund G, Dohlsten M, Kalland T (1993) - Isolation, partial characterization, and molecular cloning of a human colon adenocarcinoma cell-surface glycoprotein recognized by the C215 mouse monoclonal antibody. J Biol Chem 268: 24232– 24241


[3] Mussolino C, Cathomen T. TALE nucleases: tailored genome engineering made easy, Curr Opin Biotechnol (2012), doi:10.1016/j.copbio.2012.01.013