Team:Aachen/Project/2D Biosensor

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For our biosensor, our team genetically modified ''E. coli'' cells to be able to elecit a fluorescent response to autoinducers produced by the pathogen ''Pseudomonas aeruginosa'' during quorum sensing. In the case of ''P. aeruginosa'', these autoinducers are  N-3-oxo-dodecanoyl-L-homoserine lactone, or 3-oxo-C-12-HSL for short. The quorum sensing system of this pathogen contains the LasR activator which binds 3-oxo-C-12-HSL, and the LasI promoter, which is activated by the LasR-HSL complex. Both LasR activator and LasI promoter are available as BioBricks [http://parts.igem.org/Part:BBa_C0179 C0179] and [http://parts.igem.org/Part:BBa_J64010 J64010].
For our biosensor, our team genetically modified ''E. coli'' cells to be able to elecit a fluorescent response to autoinducers produced by the pathogen ''Pseudomonas aeruginosa'' during quorum sensing. In the case of ''P. aeruginosa'', these autoinducers are  N-3-oxo-dodecanoyl-L-homoserine lactone, or 3-oxo-C-12-HSL for short. The quorum sensing system of this pathogen contains the LasR activator which binds 3-oxo-C-12-HSL, and the LasI promoter, which is activated by the LasR-HSL complex. Both LasR activator and LasI promoter are available as BioBricks [http://parts.igem.org/Part:BBa_C0179 C0179] and [http://parts.igem.org/Part:BBa_J64010 J64010].
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As a reporter gene, we use GFP. However, expression of GFP is not simply controlled through the LasI promoter activity in our approach. Instead, our sensor cells contain a constitutively expressed fusion protein consisting of GFP and a dark quencher, and an HSL-inducible protease.  
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As a reporter gene, we use GFP. However, expression of GFP is not simply controlled through the LasI promoter activity in our approach. Instead, our sensor cells contain genes for a constitutively expressed fusion protein consisting of GFP and a dark quencher, and an HSL-inducible protease. We use the REACh protein as dark quencher for GFP and the TEV protease to cleave the complex.  
''Add: [Fig. 1: Scheme of our molecular approach]''
''Add: [Fig. 1: Scheme of our molecular approach]''
    
    
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When ''P. aeruginosa cells'' are stuck on our agar chip and come close to our sensor cells, latter will take up the HSL molecules secreted by the pathogens. Inside the sensor cells, the autoinducer will bind to the LasR gene product and activate the expression of the TEV protease. The protease will then cleave the GFP-REACh construct. When illuminated with light of 480 nm, the excitation wavelenght of GFP, our sensor cells in the vicinity of ''P. aeruginosa'' will fluoresce. On the other hand, sensor cells that were not anywhere close to the pathogens will not express the protease. Therefore, the GFP will still be attached to the dark quencher in these cells, and no fluorescent signal is produced.
{{Team:Aachen/Footer}}
{{Team:Aachen/Footer}}

Revision as of 15:14, 6 October 2014

2D Biosensor

Principle of Operation

Cellock Holmes is devised based upon a SynBio approach comprised of a two-dimensional biosensor and a measurement device. The two-dimensional biosensor is designed to recognize quorum sensing molecules secreted by the pathogen cells and generate a distinct fluorescence signal, while the measurement device is designed to recognize and analyse the produced signal.

A Novel Molecular Approach

For our biosensor, our team genetically modified E. coli cells to be able to elecit a fluorescent response to autoinducers produced by the pathogen Pseudomonas aeruginosa during quorum sensing. In the case of P. aeruginosa, these autoinducers are N-3-oxo-dodecanoyl-L-homoserine lactone, or 3-oxo-C-12-HSL for short. The quorum sensing system of this pathogen contains the LasR activator which binds 3-oxo-C-12-HSL, and the LasI promoter, which is activated by the LasR-HSL complex. Both LasR activator and LasI promoter are available as BioBricks [http://parts.igem.org/Part:BBa_C0179 C0179] and [http://parts.igem.org/Part:BBa_J64010 J64010].

As a reporter gene, we use GFP. However, expression of GFP is not simply controlled through the LasI promoter activity in our approach. Instead, our sensor cells contain genes for a constitutively expressed fusion protein consisting of GFP and a dark quencher, and an HSL-inducible protease. We use the REACh protein as dark quencher for GFP and the TEV protease to cleave the complex.

Add: [Fig. 1: Scheme of our molecular approach]

When P. aeruginosa cells are stuck on our agar chip and come close to our sensor cells, latter will take up the HSL molecules secreted by the pathogens. Inside the sensor cells, the autoinducer will bind to the LasR gene product and activate the expression of the TEV protease. The protease will then cleave the GFP-REACh construct. When illuminated with light of 480 nm, the excitation wavelenght of GFP, our sensor cells in the vicinity of P. aeruginosa will fluoresce. On the other hand, sensor cells that were not anywhere close to the pathogens will not express the protease. Therefore, the GFP will still be attached to the dark quencher in these cells, and no fluorescent signal is produced.


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