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Overall project
Our Project is based on CTC (Circulating Tumour Cells) detection and early diagnosis of cancer; we designed a complex system that will recognize CTCs, amplify the signal and report about CTC detection. So, when our bacterium finds CTC it binds to the EpCAM molecules which is sufficiently expressed on the CTC surface. This interaction is ensured by appropriate monoclonal antibodies which are presented on the becterial cell membrane.
As Sherlocoli identifies a Circulating Tumor Cell, two protein complexes with anti-EpCAM antibodies will move towards each other for the secure attachment to the EpCAM molecules. These monoclonal antibodies are linked to the extracellular end of transmembrane protein, OmpA. When OmpAs approach to each other, α and β halfs of TEV protease (Tobacco Etch Virus protease) which are attached to the intracellular end of OmpA fuse to form an active enzyme. As TEV protease gains an active form, it initiates a signaling system which outcome will show up the validity of the whole CTC Detection Module.
In the event of CTC detection, activated TEV protease cuts TEV cleavage site that connects GFP and LasR. Then, released LasR joins to the AHL1 which is constitutively synthesized by LasI gene. Afterwards, LasR connected to the AHL1 proceeds to the inducible promoter to initiate AHL2 production. Here, by means of quorum sensing AHL2 goes to the receiver cell and joins to the LuxR that is constitutively synthesized by a system established in receiver cell. Finally, the AHL2 - LuxR complex activates the inducible promoter of a reporter gene. As a result, the initial signal will be amplified by the production of a reporter protein in the neighboring receiver cells which will ensure an easier conclusion.
Our Safety Module aims self-destruction of the bacteria via emitting light. Here, light-sensitive chlor channels are synthesized in the bacteria and placed into the cell wall by the means of the halorhodopsin gene. In the presence of light, these chlor channels open and allow the entrance of chlor ions to the cell. Inside the cell, chlor ions activate the chlor promoter. Subsequently, an active chlor promoter starts the synthesis of holin and endolysin. As holin disintegrates the inner cell membrane, endolysin passes through the holes made by holin and disrupts the outer cell membrane which results in death of the bacterium.
