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From 2010.igem.org

Contents 1 Breakdown 1.1 Blue Reception 1.2 Red 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 blue input into chemical information is independent from the machinery that transforms chemical information into red output, and both are quite different from what transmits the information. Therefore, we can work with & model these three sub-devices.

Blue Reception

Blue Reception is composed of a slightly complicated system. Firstly, we have a quimeric sensing protein (LovTAP). This protein is composed of a sensing domain (a Light-Oxygen-Voltage domain) as well as the TAP protein from the Triptophan pathway. This protein will dimerize when struck by blue light. As a dimer, it shows greater affinity for DNA at the TrpO promoter. However, it acts as an inhibitor to transcription. Occupied TrpO is repressed while free TrpO is active.

Our construction is consequently based on an "inhibit the inhibitor" logic. By constructing our reporter genes under a repressed promoter (TetO), and having LovTAP repress the inhibitor of said promoter (TetR), we establish a direct <IF Light> logic gate.

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

Red Emission

Red Emission is composed of mainly two enzymes. Our first enzyme (Luciferase) is a mutated form of the wild type enzyme found in Photinus pyralis. Our mutant is expected to glow red instead of the wild type blue-green. This enzyme catalyzes the oxidation reaction that yields light. The substrate for this reaction (Luciferin) is a most complicated molecule, and to our knowledge no one has ever managed to produce it within an E. coli chassis. Therefore, we need to inoculate the medium with luciferin to enable the reactions. However, recently a new enzyme was discovered (LRE) that recycles luciferin. We thus need only an initial inoculation with luciferin and from there on, the system is sufficiently autonomous.

We plan on using Luciferase as a reporter gene, while having LRE expressed constitutively. Again, 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 blue light, the following cascade will ensure:

• Lov-domain conformational change
• LovTAP dimerization
• TrpO promoter is repressed
• Concentration of TetR collapses
• TetO promoter is free
• Luciferase downstream of TetO is transcribed
• Oxidation of substrate
• Light emission

Further Information

For Blue Reception, see [http://partsregistry.org/Part:BBa_K191003 LovTAP].

For the Red Emission mutation, see [http://dx.doi.org/10.1016/j.ab.2005.07.015 this paper].

For the Red Emission protein, see [http://partsregistry.org/Part:BBa_I712019:Design this BioBrick part].