Team:The Citadel-Charleston/ProjectPopControl

Overview    Peptide YY   Population Control

     Early in the planning stages of The Citadel team's project, it became apparent that, while production of PYY3-36 by E.coli would be a great accomplishment, for the peptide to be an effective candidate for microbial supplementation and use in a brain-gut appetite control system such as we envisioned, it would need to be under strict regulation.  Overproduction of the peptide ought to be prevented by a module secondary to the original peptide production component.  This new module would work to maintain a specific, desirable level of PYY3-36 production.  The team set out to create such a device.

     The system that the team chose to accomplish a limitation of PYY3-36 production is simple in principle, built off the concept that the most effective way to prevent a cell from producing a peptide is to eliminate the cell.  With this in mind, the team constructed a poison-generating module that becomes activated only once the colony of cells has reached such a density that additional PYY3-36 production is not required. 

     Three key features make this population limit possible.  The first is inter-cellular communication, or quorum sensing.  The AHL family of molecules is produced constituatively by all cells in the colony.  These molecules accumulate within the cell and diffuse into the extracellular space.  As the colony grows, the concentration gradient is reversed, and uptake of AHL into the cells dominates.  In this way, AHL acts as a broadcasting system for the density of the colony as a whole.

     The second feature required for the team's population control module is the sensitivity tuner.  Thanks to work done previously by the Cambridge iGEM teams from multiple years, the Registry now boasts a set of parts designed to fine-tune the level at which transcription of a given downstream sequence can begin.  When coupled with the AHL inter-cellular communication system described above, these parts allow the designer to construct a device which sets a precise threshold marker for population size, the attainment of which triggers the transcription of downstream sequences in the circuit.  Essentially, sensitivity tuners have allowed The Citadel team to set up a gateway, through which only sufficiently dense colonies can pass.

     The third and final feature of the population control circuit is the element which actually performs the limiting, a cell poison. The CcdB gene was selected for this purpose, as it is lethal when expressed in E.coli and has been shown to be effective at population control in existing scientific research.  The peptide operates by rendering DNA gyrase nonfunctional.  Once the translation of CcdB has begun, the colony size begins to decrease as cells die from exposure to the poison.  The cell population size continues to drop until it is below the density level which is capable of producing enough AHL to trigger transcription of the poison downstream of the sensitivity tuner.  The cycle then repeats itself, with the colony beginning to grow again until it passes the threshold, then decreasing in density until poison production is cut off.  This oscillatory period has been shown in previous research to dampen as the cycles continue, leveling out along the “switch point” where CcdB is expressed

References

1. Batterham RL, Cohen MA, Ellis SM, Le Roux CW, Withers DJ, Frost GS, Ghatei MA, Bloom SR (September 2003). "Inhibition of food intake in obese subjects by peptide YY3-36". The New England Journal of Medicine 349 (10): 941–8.
2. Nonaka N, Shioda S, Niehoff ML, Banks WA (September 2003). "Characterization of Blood-Brain Barrier Permeability to PYY3-36 in the Mouse." The American Society for Pharmacology and Experimental Therapeutics 306 (3):948-53.
3. Batterham RL, Cowley MA, Small CJ, Herzog H, Cohen MA, Dakin CL, Wren AM, Brynes AE, Low MJ, Ghatei MA, Cone RD, Bloom SR (August 2002) “Gut hormone PYY3-36 physiologically inhibits food intake.” Nature 418: 650-4.
4. Halatchev IG, Cone RD (March 2005) “Peripheral administration of PYY3–36 produces conditioned taste aversion in mice” Cell Metabolism 1 (3): 159-68.