Team:Aachen/Project/2D Biosensor

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=== Choose the medium ===
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=== Medium ===
At the beginning we had no own device and had to work with the equipment of the lab: a Molecular Imager&reg; Gel Doc<sup>TM</sup> XR+ from BIO-RAD. The Gel Doc<sup>TM</sup> uses uv light and has only two filter. Because of this it was not ideal for our project.  
At the beginning we had no own device and had to work with the equipment of the lab: a Molecular Imager&reg; Gel Doc<sup>TM</sup> XR+ from BIO-RAD. The Gel Doc<sup>TM</sup> uses uv light and has only two filter. Because of this it was not ideal for our project.  
LB medium has under uv light a hight fluorescence. The background fluorescence was to high to see a signal of our cells so we used minimal media (NA, M9, Hartman) to minimize the background. This worked fine for the first tries but the cells don't grow well in the minimal media.
LB medium has under uv light a hight fluorescence. The background fluorescence was to high to see a signal of our cells so we used minimal media (NA, M9, Hartman) to minimize the background. This worked fine for the first tries but the cells don't grow well in the minimal media.
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For longer storage up to 5 days in the fridge (+ 4°C) TB medium can be used. It has a smal background flourescence in WatsOn.
For longer storage up to 5 days in the fridge (+ 4°C) TB medium can be used. It has a smal background flourescence in WatsOn.
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=== Choose the agar concentration ===
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=== Agar concentration ===
For the sensor chip an agarose concentration of 1.5% is used.
For the sensor chip an agarose concentration of 1.5% is used.
We first tryed agar. When the agar conzentration is lower than 1.5% the chip is not stable and brakes fast. When the agar conzentration is higher than 1.5% the medium gets solid befor pooring it into chip form.
We first tryed agar. When the agar conzentration is lower than 1.5% the chip is not stable and brakes fast. When the agar conzentration is higher than 1.5% the medium gets solid befor pooring it into chip form.
In the end we choose agarose instead of agar because the agarose is better linked and so the difusion is not so high.
In the end we choose agarose instead of agar because the agarose is better linked and so the difusion is not so high.
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=== Induction ===
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For the induction of the used constructs we use IPTG or 3-oxo-C12 HSL.
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The sensor cells with K1319042 can ditect a IPTG concentration of 1 mM.
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The sensor cells with K131026 can ditect a HSL concentration of 500 µg/ml
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{{Team:Aachen/Footer}}
{{Team:Aachen/Footer}}

Revision as of 10:01, 14 October 2014

2D Biosensor

With our 2D biosensor technology we are able to detect the pathogen Pseudomonas aeruginosa on solid surfaces. The sensor system is comprised of two distinct but inseparable modules, a biological part and a technical part:

  • Sensing chips containing Cellocks, our engineered detective cells that fluoresce in the presence of the pathogen, make up the biological part of Cellock Holmes.
  • Our measurement device WatsOn and the complementary softare Measurarty complete our sensing technology on the technical side.


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 to generate a distinct fluorescence signal, while the measurement device is designed to recognize and analyse the produced signal.

Our sensor cells are immobilized in agar chips. To make the chips, we mix the sensing cells, also known as Cellocks, with liquid LB agar. In the course of our project, we designed a casting mold specifically for the production of our agar chips. When the agar has cooled down, the chips are cut out of the mold and ready to use . If you want to know exactly how our chips are manufactures, you can read up on more details in our Protocols.

Add: Flow chart of chip measurement -> Patrick is working on it


Aachen Flowsheet OD-device test ipo.png
How to use our 2D sensor device
NOT FINISHED!


To detect P. aeruginosa cells, the chip is placed on a hard surface that is potentially contaminated with the pathogen. Subsequently, the chip is introduced into our measurement device WatsOn where it is incubated at 37 °C. After a short time, the chip is illuminated with blue light. WatsOn takes a picture of the chip and the software Measurarty analyzes any fluorescent signal. Depending on the intensity of the signal and the size of the spot, Cellock Holmes can calculate concentration and distribution of P. aeruginosa on the sampled surface.


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; here you can read more about the REACh construct and the TEV protease.

Aachen REACh approach.png
Our novel biosensor approach
Expression of the TEV protease is induced by HSL. The protease cleaves the GFP-REACh fusion protein to elecit a fluorescence response.

When P. aeruginosa cells are stuck on our agar chip and come close to our sensor cells, the latter take up the HSL molecules secreted by the pathogens. Inside the sensor cells, the autoinducer binds to the LasR gene product and activate the expression of the TEV protease. The protease then cleaves 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 give a fluorescence signal. On the other hand, sensor cells that were not anywhere close to the pathogens do not express the protease. Therefore, the GFP will still be attached to the dark quencher in these cells, and no fluorescence is produced.


Achievements

What was done so far

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