Team:Aachen/Project/2D Biosensor

From 2014.igem.org

Revision as of 14:46, 15 October 2014 by PatrickO (Talk | contribs)

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. To overcome the limitation of our REACh construct to bacteria that secrete autoinducers, we developed an alternative detection method (Galectin-3 construct), which is based on tagging cells with a fluorescent reporter.

[graph quorum senising]

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 are ready to use. Storage of the readily usable sensor chips is possible for 2 days at 4 °C when using LB medium or for 5 days if TB-medium is used. If you want to know exactly how our chips are manufactures, you can read up on more details in our Protocols section.

Aachen 14-10-14 Flowsheet OD-device part1 ipo.png
Application of WatsOn for investigation of solid surfaces.

The application of Cellock Holmes for detection of P. aeruginosa cells is simple: fist, a sampling chip is placed on a hard surface that is potentially contaminated with the pathogen. Second, the chip is put onto one of our sensor chips. Subsequently, the two layered chip-stack is put into a petri dish, which is inserted into our measurement device WatsOn for evalutation.

Aachen 14-10-14 Flowsheet OD-device part2 ipo.png
Mode of action inside WatsOn.

Inside WatsOn the chips are incubated at 37 °C and populations of microorganisms on the sampling-chips start to multiply. P. aeruginosa secrets an increasing number of 3-oxo-C12 homoserine lactones (HSLs) when multiplying. The chips can be illuminated with blue light at any time, while WatsOn takes a picture of the chip. The software Measurarty then 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.




Achievements

What was done so far

Testing our sensor Chips with a Platereader

300px
Testing K1319042 in our sensor chips
K1319042 in our sensorchip induced with 2 µl iPTG and measured with a Platereader. Blue color indicates no fluorescence, Red color indicates fluorescence.
300px
Testing K131026 in our sensor chips
K131026 in our sensorchip induced with 0.2 µl 3-oxo-C12 HSL and measured with a Platereader. Blue color indicates no fluorescence, Red color indicates fluorescence.

Detecting the 3-oxo-C12 HSL with K131026 in our sensor chip with WatsOn.

480px
Detection of 3-oxo-CC12 HSL with K131026
0,2 µl of 3-oxo-C12 HSL were put in the middle of the chip and then incubated at 37 °C in our WatsOn device.

Detecting Pseudomonas aeruginosa with K131026 in our sensor chip with WatsOn.

480px
Detection of Pseudomonas aeruginosa with K131026
Direct detection of Pseudomonas aeruginosa on our sensor chips. Sensor cell used were K131026.