1• To detect cancer cells specifically using the nanofactory complex.
Firstly, we had to devise a system which would be able to detect the cancer cells. This challenge was solved by searching for a specific marker present on cancer cells and that was EpCaM[1]. EpCAM markers are present on both healthy cells and cancer cells. However according to an article[1] the number of EpCAM present on cancer cells are 100 times more compared to a healthy cell. Nanofactories were the complexes we aimed to detect cancer cells. This complex consists of an Anti-EpCAM antibody that would bind to EpCAM antigens present on the cancer cells.We designed our bacteria as an effective alternative to presenting antibody parts of our nanofactory on its surface. It was an obligation for us to use anti-EpCAM for binding. While we were researching on EpCAM, we found anti-EpCAM’S active domain called C215. C215 does the same job as Anti-Epcam though it is smaller than anti-EpCAM. Therefore, our choice was C215. With presenting C215 on the outer membrane, we designed a bacteria called “nanobacterium”. This bacteria will find the cancer cells and accumulate around them, which will call our bacteria towards the cancer cells.
2• Producing AI-2 by our nanofactory without cease
After detection a system was required to call the bacteria towards the cancer cells, therefore we used a quorum sensing material called Auto Inducer-2(AI-2). AI-2 was produced by our nanofactories which enabled our bacteria to move towards the cancer cells via taxis method.
3• Expression of cancer specific apoptotic protein by the bacteria due to AI-2 concentration
We had to trigger the expression of our cancer killer protein with a stimulant. This complicated challenge was solved by using intentive AI-2 concentration near by of cancer cells. The bacteria will sytnthesize our protein when AI-2 concentration reaches to a certain level. Once the bacteria enter the environment of intensive concentration of AI-2, a mechanism is triggered which enable our bacteria to produce the killer protein.
4• Secreting the killer protein out of the cell with signal peptides.
We used HLYA signal peptide to secrete our apoptotic proteins from the bacteria. We also wished to use TorA signal peptide as an alternative to HLYA [3]. While using TorA, our proteins were secreted to the periplasm and not to the extra cellular matrix. As a solution to this problem we used a chemical substance which pokes holes on the outer membrane proteins and release our proteins inside.
(1)EpCAM overexpression prolongs proliferative capacity of primary human breast epithelial cells and supports hyperplastic growth Agnieszka Martowicz1, Johannes Rainer2, Julien Lelong2, Gilbert Spizzo1, Guenther Gastl13 and Gerold Untergasser3* (2) Biological Nanofactories Target and Activate Epithelial Cell Surfaces for Modulating Bacterial Quorum Sensing and Interspecies Signaling Colin G. Hebert,†, Apoorv Gupta,§ Rohan Fernandes,†, Chen-Yu Tsao,†, James J. Valdes,‡ and William E. Bentley†,§
(3) Improved secretory production of recombinant proteins by random mutagenesis of hlyB, an alpha-hemolysin transporter from Escherichia coli.
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