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From 2010.igem.org

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Revision as of 23:40, 16 July 2010

Contents 1 Breakdown 1.1 Green Reception 1.2 Blue Emission 1.3 Transmission 1.4 Signaling Cascade 2 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 green input into chemical information is independent from the machinery that transforms chemical information into blue output, and both are quite different from what transmits the information. Therefore, we can work with & model these three sub-devices.

Green Reception

Green Reception is composed of a two-component system. Firstly, we have a sensing agent (CcaS). This protein shows two basal states, both with histidine kinase activities but each with an affinity for different substrates: a phenomenon known as photoconversion. We plan on using the Green phase regulator (CcaR) who happens to be a Transcription Factor.

When our regulator is in a phosphorilate state, it shows greater affinity for DNA. Thus it is active. The target promoter region has been recently identified. We thus plan on constructing our reporter genes under this promoter. Such a construction would be an <IF Light> logic gate. This system is quite similar to the EnvZ-OmpR system.

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

Blue Emission

Blue 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, LumP), two catalyze the oxidation step (LuxA, LuxB), one adjusts the emission spectrum (LumP), 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. Likewise, LumP does not actually shift the spectrum, but the enzyme generates a substrate that does.

As you may imagine, these genes (sauf LumP) 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 green light, the following cascade will ensure:

• CcaS switches to Green conformation
• Kinase activity starts
• Phosphorilated CcaR concentration builds up
• Transcription of genes downstream of target promoter: LuxA & LuxB
• Oxidation of substrate
• Light emission

Further Information

For Green Reception, see [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2474522/ this paper].

For Blue Emission, see the work of Edinburg 2009.