design

The genetic fence combines a chemically-activated gene switch with inhibition of a fatal gene pathway. In the presence of the fence compound, methoxyfenozide, the genetic switch activates transcription of an inhibitory mechanism, which halts an otherwise fatal gene pathway.

Genetic Fence gene map   click to enlarge

Genetic Fence Figure 2   click to enlarge

Fatal Gene
A protein called Barnase prevents plants from growing outside the fence. Barnase, a non-specific RNAse, cleaves all pieces of RNA it encounters in the cell, thus derailing the cell’s metabolism and causing it to die. Barnase is expressed constitutively in all iGarden plants, so in the absence of Methoxyfenozide none will grow.

Inhibitor
A protein called Barstar inhibits Barnase. When expressed in cells, Barstar prevents Barnase from destroying the iGarden plants. By putting Barstar under control of the switch-activated promoter (5xGal4 Upstream Activating Sequence plus 35s minimal promoter), barstar is expressed in the presence of methoxyfenozide.

Switch
The switch component of the fence activates gene transcription in the presence of methoxyfenozide. Two receptor proteins, Ecdysone (EcR) and Retinoic Acid (RXR), form a dimer in the presense of methoxyfenozide. We created two fusion proteins, one combining Ecdysone with VP16 activating domain (an activating domain recruits the machinery to start transcription), and one combining Retinoic Acid with Gal4 DNA binding domain (which binds to a specific stretch of DNA where transcription will begin). When methoxyfenozide dimerizes Ecdysone and Retinoic Acid, VP16 activating domain and Gal4 DNA binding domain act together to initiate transcription of Barstar.

Thus, Methoxyfenozide turns on the switch, which results in Barstar expression, which inhibits Barnase, allowing the plant to grow.

Genetic Fence Flowchart: Full   click to enlarge

Genetic Fence Flowchart: Simplified   click to enlarge