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 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.

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.

In addition to protein inhibition, a second switch-activated promoter regulates expression of a Lac Inhibitor Protein modified with a nuclear localization signal(LacIN). LacIN halts the transcription of the otherwise constitutive Actin2 promoter modified to include LacO sites (Act2LacO promoter). Barnase is placed under control of Act2LacO, so that it is expressed unless LacIN is present as well. Through LacIN and Barstar, Barnase is inhibited at both the transcriptional and the protein levels in the cell.

Sensor

The sensor component of the fence activates gene transcription in the presence of the fence compound 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 (which 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 and LacIN.

Thus, Methoxyfenozide activates the sensor, which results in Barstar and LacIN expression, which inhibits Barnase, allowing the plant to grow.

Genetic Fence Design   click to enlarge

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Genetic Fence Flowchart: Full   click to enlarge