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Team:KULeuven/Project
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Revision as of 15:49, 2 October 2009
Blue Light Receptor
The receptor senses the blue light to which the bacteria are exposed. By choosing the light intensity, the wanted vanillin concentration is set. Upon photo-excitation the receptorprotein YcgF dimerizes and interacts directly with the repressor YcgE. This protein is bound to the promoter-region of the key-gene and inhibits its transcription. The dimerized YcgF acts as an anti-repressor and releases YcgE from the DNA. Thereupon, the transcribed key activates vanillin production, which is proportional to the entered intensity of the blue light.
Vanillin Receptor
The vanillin receptor senses the vanillin concentration outside the cell. This information is needed to create the feedback loop that controls vanillin synthesis. The receptor consists of two proteins: virA and virG. In the presence of vanillin, virA binds a phosphor and transfers it to virG. In turn, the phosphorylated VirG binds to a so-called vir box sequence, triggering transcription of antikey. The more antikey is produced, the more it can anneal to the key and the less vanillin is produced. This process goes on until there’s an equilibrium between measured and wanted concentration.
Key Lock Antikey
The key/antikey system compares the signal from the blue light and vanillin receptor in order to control vanillin production. The more the measured amount of vanillin exceeds the wanted amount (set by the blue light intensity), the less vanillin is produced. After blue light irradiation, the key is transcribed and ‘unlocks’ the vanillin synthesis pathway. In response to the produced vanillin, the vanillin receptor then activates transcription of the antikey. Key and antikey are complementary RNA-strands: their annealing is favoured over the reaction between key en lock leading to vanillin synthesis.
Vanillin Production
A vanilla odour is created by synthesizing the molecule Vanillin. The starting point is tyrosine, an amino acid produced endogenously in E.coli. The subsequent pathway involves a combination of five enzymes. By locking both the transcription of the first and the third enzyme we prevent vanillin synthesis without the presence of the key.
‘Essencia coli’ is a vanillin producing bacterium equipped with a control system that keeps the concentration of vanillin at a constant level. The showpiece of the project is the feedback mechanism. Vanillin synthesis is initiated by irradiation with blue light. The preferred concentration can be modulated using the intensity of that light. At the same time the bacterium measures the amount of vanillin outside the cell and controls its production to maintain the set point. The designed system is universal in nature and has therefore potential benefits in different areas. The concept can easily be applied to other flavours and odours. In fact, any application that requires a constant concentration of a molecular substance is possible.
How it works...
Feedback control system
The novelty of this project is a unique control system that functions through a feedback mechanism. The desired value of an odour like Vanilla can be adjusted using the intensity of a blue light beam. Essencia coli then senses the concentration of the odour molecule and activates the inhibition mechanism of an anti-key. In this way the concentration can be kept constant at any initial set point.
This concept can also be applied to other odours or even flavours. In theory, any molecule that acquires a constant value is a possible candidate.
Vanillin synthesis
To produce the odour of Vanilla, Essencia coli synthesises Vanillin. This molecule is derived from the amino acid tyrosine, in a process involving five enzymes. The successive transcription and translation of Sam8, Sam5, COMT, Fcs, Ech leads to Vanillin production, which then diffuses out of the cell.
Blue Light
A key/lock mechanism opens the door to synthesis. The transcription of the 'key' gene commences when the blue light receptor absorbs a photon of blue light, thereby activating the promotor. The intensity of the light can be adjusted at will. The 'key' mRNA ("A") then interacts with the 'lock' and clears the road for RNA Polymerase to transcribe the locked genes.
VirA/VirG Vanillin Receptor
Essencia coli is equipped with the two component regulatory system VirA/VirG, derived from Agrobacterium tumefaciens. The VirA protein senses phenolic compounds -in this case Vanillin- and then transduces the signal through phosphate transfer to the VirG protein. VirG acts as a response regulator by binding to the upstream region of the anti-key gene and activating its transcription. The anti-key ("B") is the anti-sense mRNA of the key and will therefore block it after binding.
Key and antikey
When the antikey is present, it will bind the key with such great affinity that a complex antikey+key occurs. So, the more Vanillin is produced, the more of the anti-key will be transcribed and will bind to key. The bound complex of key and antikey puts a stop to the synthesis of vanillin.
