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 unit. 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 recognizes and analyzes the produced signal. On the molecular side, we use the REACh construct to turn regular E. coli cells into Cellocks. We also developed another molecular approach as an alternative to our REACh approach: The Galectin-3 construct overcomes the limitation of the REACh method to bacteria that secrete quorum sensing molecules because it 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 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 up to 2 days at 4 °C when using LB medium or up to 5 days if TB medium is used. A detailed description of the sensor chip manufacturing can be found in our Protocols section.

Application of WatsOn for investigation of solid surfaces.

The application of Cellock Holmes for detection of P. aeruginosa cells is straightforward: Fist, a sampling chip is placed on a solid surface that is potentially contaminated with the pathogen. Second, the sampling chip is removed from the surface and put onto one of our sensor chips. Theorectically, the sensor chips could be directly used for sampling, however, this was avoided in our project to match biosafety regulations and to prevent the spread of GMOs into the environment. Third, the two layered chip-stack is put into a petri dish which is inserted into our measurement device WatsOn for evalutation.

Mode of action inside WatsOn.

Inside WatsOn, the chips are incubated at 37 °C and populations of microorganisms stuck on the sampling chip start to multiply. P. aeruginosa secrets an increasing number of quorum sensing molecules in growing cell colonies which are taken up by our sensor cells where they induce a fluorescence signal. In case of P. aeruginosa, we focused on a quorum sensing molecule called N-3-oxo-dodecanoyl-L-homoserine lactone, or 3-oxo-C-12-HSL for short, while our sensor cells comprise of genetically modified E. coli cells which are able to elecit a fluorescent response to the autoinducers. How we incorporate this 3-oxo-C-12-HSL detection system into the sensor cells is explained in more detail in the REACh Construct section.

During incubation the chips can be illuminated with blue light at any time, and a photo of the chips is taken. The software Measurarty then analyzes any fluorescent signal.

Development & Optimization

Medium

Prior to using our own device for detection of fluorescence emitted by the sensor chips we used equipment readily available in the lab. A Molecular Imager® Gel Doc^TM XR+ from BIO-RAD was available which uses UV and white light illuminators. However, only two different filters were available for the excitation ligth wavelength, which resulted in very limitted possibilities for the excitation of fluorescent molecules. For example, it was possible to detect the expression of iLOV in our sensor chips but the detection of GFP was not possible. It was thus determined that the Gel Doc^TM XR+ was not suitable for our project. (iLOV_GFP_HM_1,5h.png)

Regarding the medium used for our sensor chips, LB medium showed a high background fluorescence when exposed to UV light. Surprinsingly, the background fluorescence resulting from the LB medium was to high to detect a signal emitted by our sensor cells. Instead we tried using minimal media (NA, M9, Hartman) in order to minimize background fluorescence. The appliction of minimal media was sufficient to minimize the background fluorescence, but this approach came with the drawback of minimal to zero growth of our sensor cells. (Chip_medium_geldoc.png)

Further experiments were conducted to test long-time storage of the sensor chips. Storage at -20 °C resulted in the loss of our sensor cells. Adding 5-10% (v/v) glycerol ensured survival of the sensor cells, but resulted in an expression stop of fluorescence proteins. Thus, the idea of long time storage of the sensor chips had to be passed on. However, it was possible to store ready-to-use sensor chips for 2 days at 4 °C when using LB medium and storage for 5 days was possible with chips made from TB medium.

In our device WatsOn, optimized wavelengths of 450 nm and 480 nm were used for excitation of iLOV and GFP, respectively. When exposed to either excitation wavelength the LB medium showed minimal background fluorescence and no difficulties were observed in cultivation of our Cellocks. Furthermore, a reduction of background fluorescence compared to LB medium was observed when using TB medium for sensor chip manufacturing in combination with fluorescence evaluation using WatsOn. (5Tage_K131026_neb_tb_1,5h)

Agar Concentration

For the sensor chip manufacturing, a concentration of 1.5% agarose was found to be optimal. When agarose concentrations below 1.5% (w/v) were used the sensor chips were easily damaged and were not transportable. Agar concentrations above 1.5% (w/v) had to be avoided, because the agarose started to solidify before it could be poured into the chip casting mold. Agarose was chosen above agar, because of more even linkage resulting in a better chip homogenity. In addition, agarose reduced diffusion of inducer molecules through the chip. A reduced diffusion was desired in order to achieve distinct fluorescent spots on the sensor chips.

Chip Form

Various approaches were tried for production of sensor chips with reproducable quality. The first approach was to cast every sensor chip individually. In order to achieve a plain chip surface, which was required for high quality images, we tried to cast the sensor chips between two microscope slides. This approach had to be rejected, because the agar was too liquid. In a second try, we produced a closed mold into which liquid agar was injected using a pipette, but we encountered a high number of bubbles in the chips when using this method. Bubbles in the sensor chips resulted in problems during fluorescence evalutaion. (2_chipform.jpg)

Finally, we used an open mold into which the agar was poured right after mixing with the sensor cells. When the agar had solidified the chips were cut out along precast indentations in the casting mold. An advantage of the open mold was the ability to simultaneously produce nine sensor chips while the surface tension of the liquid agar ensured a plane chip surface. (final_chipform.jpg)

Induction

For the induction of the used molecular constructs we simluated the presence of P. aeruginosa by using IPTG or 3-oxo-C12 HSL. The sensor cells with K1319042 in BL21 cells can detect a IPTG concentration of 1 mM (0.2 µL). (2µl_IPTG_1mM_K1319042_1h.png) The sensor with the REACh constructs in BL21 cells can detect an IPTG concentration of ??? (Plate reader???) The sensor cells with K131026 in BL21 cells can detect an HSL concentration of 500 µg/mL (0.2 µL). It also can detect Pseudomonas aeruginosa. (Zeitaufnahmen bearbeitet von Arne)

Achievements

We are able to detect IPTG, 3-oxo-C₁₂ HSL and Pseudomonas aeruginosa. To proof that your sensor cells produce the flourescence signal and not something else we have B0015 in NEB as negativ control.

Testing our Sensor Chips with a Platereader

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

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