From 2007.igem.org

Bio-diode in our project mainly consists of four different types of cells which are constructed by MIT: Generator cells, Reporter cells, Amplifier cells and Block cells.

Generator: Generator cells could produce AHL signal constantly which could be sensed by special gene sequence to activate particular function. In our experiment, the production of AHL is controlled by LuxI promoter and could be promoted by the addition of IPTG and the function of Generator could be verified by the detector cells.

Reporter cell: this cell is designed to test the concentration of AHL signal. In this cell, LuxR gene under the control of tet promoter could produce LuxR protein which could activite the following Lux pR promoter when binding to certain amount of AHL signal, thus GFP expressed to make this cell green within the view of a fluoresence microscope. The intensity of fluoresence could be measured by a fluoresence spectrophometer to decide the concentration of AHL signal roughly.

Amplifier cells: Also containing a LuxR protein producer together with an AHL producer controlled by a Lux pR promoter, the amplifier cells could generate more AHL signal when it combines with the environmental AHL signal.

Block cells: Block cells contain a gene coding for a special enzyme digesting the AHL signal. The concentration of AHL signals in liquid culture could dramatically decrease after passing through the block cell congeries.

Mechanism: The three types of cells, generator cell、amplifier cells and block cells are immobilized into small beads with a diameter of 0.5 millimeter, then different beads are settled into particular parts of our specially designed equipment sequentially and respectively. Firstly, the liquid culture flow through the generator cells and carry the AHL signal into the amplifier cells which are activated to make more AHL signals. Finally, the concentration of AHL accumulates to such a level that block cells could not eliminate them completely, and the rest could be detected by reporter cells, a condition called ‘posivive-biased pass’ On the contrary, if the positions of amplifier cells and block cells in the equipment are reversed, the AHL produced by generator cell would be degraded by block cells at the very beginning resulting in no amplification of the signal, thus, hardly AHL could be detected in the outflow culture, a condition called ‘negative-biased block’. By this way, we successfully simulate the two most important features of diode and our project could be connected with other devices to construct more complex circuit system in the future.