There are seven different composite parts existing in our first module. As the module possesses the detection mechanism of cancer cells related with NanoFactory parts and quorum sensing device, there are two groups of parts:
NanoFactory is a protein complex which is consisting of three different domains. Each domain has its unique ability and responsibility. These domains are Binding Zone, Connecting Zone and Enzyme Zone. This design is inspired by an attitude in an article which we confronted during our extense research. Nanofactories are actually used for extracellular and nanoscale functions of specific proteins or chemicals.
In our design, our NanoFactory is designed for efficient cancer detection via a specific cancer marker whose number elevates in some typical cancers and powerful stimulation of our modified bacteria in colon for the production of our anti-cancer peptides. These two completely different functionalities are fixed together with a connecting peptide called protein G.
EpCAM (Epithelial Cell Adhesion Molecule) is an antigen of normal epithelial cells in human body, whose number are highly elevated in some cancer types such as colon and breast. This typical increase of number is found significant for the detection, staging and predicting the aggressiveness of tumors. For this purpose, anti-EpCAM antibody is well studied in literature.
According to the articles, this antibody has three different binding domains for the antigen. The affinity profiles of these sites have been studied and the biggest value has been designated. That binding site is called C215; which we determined to use in our project for the detection of EpCAM antigen, therefore, cancer cells. Nanofactory Binding Zone includes C215 sequence. Between the connecting zone (protein G) and binding zone, an additional linker sequence is found necessary to attach; because we want to prevent possible misfolding problems of our peptides in the construct. After the protein G to the N-terminus, a second linker sequence again inserted, just before the enzyme zone begins.
Enzyme zone is responsible for the production of autoinducer-2 (AI-2) quorum sensing unit from its substrate S-adenosyl homocysteine (SAH). This reaction occurs in two steps, each step is under control of one enzyme: LuxS and Pfs. We designed our NanoFactory by combining these enzymes as an enzyme complex and attached it to the second linker that comes after the protein G.
Lsr system is consisting of three main components: pLsr promoter, LsrR repressor and LsrK enzyme. Lsr promoter is under control of LsrR repressor. This repression is inhibited by a phosphorylated AI-2 that this reaction is done by LsrK.
In our system, NanoFactory Enzyme Zone can convert SAH into AI-2 in the extracellular matrix. The accumulation of AI-2 ends up with the diffusion into bacteria in colon. In bacterial environment, AI-2 is phosphorylated by LsrK; this results the inhibition of repression of LsrR on the promoter. Thereby, the production of the desired protein can be regulated with this system. We want to take the production of our cancer killing peptides under our control by using Lsr system.
"
