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From 2010.igem.org

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Revision as of 23:25, 16 July 2010

Contents 1 Breakdown 1.1 Red Reception 1.2 Green Emission 1.3 Transmission 1.4 Signaling Cascade 1.5 Further Information

Breakdown

We decided to break down our device into 3 sub-devices: Reception, Emission, and Transmission. The rationale is as follows: the machinery that transforms the red input into chemical information is independent from the machinery that transforms chemical information into green output, and both are quite different from what transmits the information. Therefore, we can work with & model these three sub-devices.

Red Reception

Red Reception is composed of a two-component system. Firstly, the chimeric protein Cph8 possess a light-sensing domain (PCB) and a histidine kinase domain (EnvZ). The chromophore has two states, and light triggers the passage of state. Thus, under dark conditions Cph8 shows kinase activity; under light conditions it does not.

Cph8's substrate is OmpR, a well studied Transcription Factor. When phosphorilated it shows greater affinity for DNA. OmpR regulates two promoters in an antagonistic way: in high concentrations of active OmpR, OmpC is active and OmpF is repressed. In low concentrations of active OmpR, OmpC is repressed and OmpF is active.

We plan on constructing our reporter genes under the OmpF promoter and starting our system in a <Dark> state (where active OmpR concentration is high). Thus we hope to achieve an <IF Light> logic gate by using Cph8 as a sensing mechanism, and OmpF as a response one.

The input for this sub-device is light, the output is Polymerases per Second.

Green Emission

Green Emission is composed of a series of enzymes that generate light by the oxidation of a substrate. Our sub-device has 6 enzymes (LuxA, LuxB, LuxC, LuxD, LuxE, LuxY), two catalyze the oxidation step (LuxA, LuxB), one adjusts the emission spectrum (LuxY), and three generate and recycle the substrate (LuxC, LuxD, LuxE). We plan on having the adjusting enzyme, as well as the 3 regenerating enzymes expressed constitutively. We would then only use the oxidation enzymes as reporters for whatever event we are observing.

While the oxidation per se does not generate light, it does generate an intermediate molecule in an electronically exited state. When said molecule returns to a basal energy state, a photon is released.

As you may imagine, these genes constitute an Operon. This is the Lux Operon from Vibrio fischeri. The input for this sub-device is Polymerases per Second, and the output is light.

Transmission

We are planning on using two methods for transmitting light across our devices. The first is through mono-directional fiber optic cables connecting the beakers. The second is through a micro-controller coupled device. We still have to construct such a device though...

Signaling Cascade

When our device is struck by red light, the following cascade will ensure:

• PCB conformation change
• EnvZ kinase activity abolished
• Phosphorilated OmpR concentration collapse
• Transcription of genes downstream of OmpF promoter: LuxA & LuxB
• Oxidation of substrate
• Light emission

Further Information

For Red Reception, see [http://partsregistry.org/Coliroid Coliroid].

For Green Emission, see the work of Edinburg 2009.