Team:Fatih-Medical/Dracoli/Assembly

Unlike the other modules in our project, the parts taking place in this module are assembled from the remaining BioBricks in the Registry. We intend to establish a light tunable self-destruction device in E.coli. To do so, we want to base our device on the ion transfer channels and ion/pH dependent promoters.

Halorhodopsin is a light driven ion channel which accepts chloride ions into the cell depending on the gradient difference. To activate the channel, it is been told that especially the green light exposure works well. In 2011, Hong Kong-CHUK iGEM team has characterized BBa_K559010 and has won Best BioBrick, Natural in Asia Regional Jamboree. Also, they have added the intracellular chloride-sensing cassette which can be used as chloride ion inducible PoPS generator.

Because of all these reasons, we decided to assemble halorhodopsin complete system with chloride-sensing cassette in order to have a light inducible PoPS generator. In conclusion, our goal is to synthesize self-destructive cell killing mechanisms such as endolysin-holin complexes, which have synergic effect on the lysis of cell wall of bacteria, or Limulus anti-lipopolysaccharide factor (LALF) which is demonstrated last year by Fatih Turkey iGEM team as gram negative bacteria (e.g. E.coli) growth inhibitor.

To generate a composite part, we used 3A assemble method and BioBrick Standards. Our final objective is to have a composite part as follows: halorhodopsin complete system + Pgad chloride-sensing unit + T4 endolysin + T4 holin. Unfortunately, the unavailability of the chloride-sensing unit and some problems we confront about synthesizing that sequence, we were not able to assemble this composite part.