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 and a technical part:

• Sensor 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 software Measurarty complete our sensing technology on the technical side.

Principle of Operation

Cellock Holmes is designed upon a SynBio approach comprising a two-dimensional biosensor and a measurement unit. The two-dimensional biosensor is devised 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 transform regular E. coli cells into Cellocks.

The principle of quorum sensing Microorganisms can sense the presence of their own kind based on quorum sensing which is a form of chemical communication. Depending on their cell density, quorum sensing allows these cells to activate or deactivate certain gene expression cascades (Waters and Bassler, 2005) for a specific function.

Our sensor cells, Cellocks, 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.

Assay to detect P. aeruginosa using Cellock Holmes This flow sheet shows the procedure to sample and detect P. aeruginosa: A sampling chip is briefly put onto the potentially contaminated surface, added onto one of our sensor chips and inserted into WatsOn.

Using Cellock Holmes, we developed a simple assay to detect P. aeruginosa.
Initially, a so called sampling chip is placed on a solid surface that is potentially contaminated with the pathogen. Subsequently, 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 genetically modified organisms (GMOs) into the environment. The two layered chip-stack is then put into a petri dish which is inserted into our measurement device WatsOn for evalutation.

Mode of action inside WatsOn Chips are incubated at 37°C to stimulate cell growth and then illuminated with blue light to excite fluorescence. A picture is taken and analyzed for fluorescence signals using the software Measurarty.

Inside WatsOn, the chips are incubated at 37°C and the sampled populations of microorganisms attached on the sampling chip start to grow and multiply. 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.
P. aeruginosa secrets an increasing number of quorum sensing molecules that are recognized by Cellocks, thereby producing a fluorescence signal. For detection of P. aeruginosa, we focused on a quorum sensing molecule called N-3-oxo-dodecanoyl-L-homoserine lactone (for short: 3-oxo-C₁₂-HSL), which is involved in virulence regulation of P. aeruginosa (Jimenez, Koch, Thompson et al., 2012). The incorporation of the 3-oxo-C₁₂-HSL detection system into the Cellocks is explained in detail in the REACh Construct section.

Development & Optimization

iLOV and GFP in the Gel DocTM Sensor cells producing iLOV (A) and GFP (B) 1.5 h after induction.

Equipment and medium selection

Our first approach (before developing our own device) was to use the Molecular Imager® Gel Doc™ XR+ from BIO-RAD in our lab to detect fluorescence, which uses UV and white light illuminators. However, only two different filters were available for the excitation light wavelength, which resulted in very limited possibilities for the excitation of fluorescent molecules. For example, it was possible to detect the expression of iLOV in our sensor chips, but in contrast the detection of GFP was not possible. Hence, the Gel Doc™ was not suitable for our project.

Differend medium in the Gel Doc™ complex media exhibited high background fluorescence while less back- ground fluorescence was observed with the minimal media (HM, M9, NA).

Testing our chips' shelf-life Chips of [http://parts.igem.org/Part:BBa_K131026 K131026] in NEB were stored 5 days at 4°C. The right chip was induced with 0.2 µL of 500 µg/mL HSL and an image was taken after 1.5 h.

We tested different media (i.e. LB, M9, NA, HM and TB) for the preparation of our sensor chips. The details of media composition can be found in the Protocols section. We screened for an optimized media composition that results in minimal background fluorescence and supports cell growth. The resuts of the analysis are presented in the table below. Because of the reduced fluorescence compared to TB medium when using WatsOn for sensor chip evaluation and because of sufficient cultivation conditions for our Cellocks LB medium was chosen over TB medium for sensor hip manufacturing.

Experiments were conducted to test long-time storage of the sensor chips at low temperature and by the addition of glycerol. 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. Hence, we concluded that long time storage of the sensor chips is not possible under the tested conditions. However, it is 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.

Sensor chip manufacturing using the closed mold When injecting the liquid agar into a closed mold we encounter problems due to frequent bubble formation.

Optimal agarose concentration for sensor chip manufacturing

For the sensor chip manufacturing, agarose was preferred over agar, because of a uniform linkage that resulted in a better chip homogeneity. In addition, agarose reduced diffusion of the inducer molecules through the chip. A reduced diffusion is vital to observe distinct fluorescent spots on the sensor chips and thus further optimzation of our 2D biosensor was done using agarose-based chips.

The finalized chip mold An open casting mold was found to be optimal for sensor chip manufacturing, because this approach was fast, easy to handle and generated a reproducible chip quality.

Optimal chip configuration

Several approaches were tested for the production of agarose-based sensor chips with reproducible quality. The first approach was to cast every sensor chip individually. To achieve a plain chip surface (a requisite for high quality images), we casted the sensor chips between two microscope slides in an initial attempt. However, this approach was rejected, because the agar was too liquid and leaked from the microscope slides. In the second approach, we prepared a closed mold into which liquid agar was injected with a pipette, but we encountered a high number of bubbles in the resulting chips. Bubbles in the sensor chips interfered with fluorescence evaluation. Finally, we used an open mold into which the agar was poured right after mixing with the sensor cells. Chips were cut out along precast indentations in the casting mold after the agar solidified. 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.

Induction of the sensor chips

For initial testing of our molecular constructs, we simulated the presence of P. aeruginosa using IPTG or 3-oxo-C₁₂-HSL. However, for induction a minimal volume is required as our initial experiments showed that diffusion of the inducers through the chip hindered the formation of distinct spots on the chips. An optimal and low volume of 0.2 µl was chosen for induction. Sensor cells based on E. coli BL21, which incorporated the K1319042 construct were able to detect IPTG concentrations down to 1 mM (0.2 µl), and as well for the sensor cells based on E. coli BL21 which incorporated the REACh constructs. Sensor cells based on E. coli BL21, which incorporated the [http://parts.igem.org/Part:BBa_K131026 K131026] construct were able to detect HSL concentrations down to 500 µg/ml (0.2 µl). Furthermore, detection of growing P. aeruginosa cells based on secreted HSLs was possible using the [http://parts.igem.org/Part:BBa_K131026 K131026] construct. The experiments conducted during induction of our sensor chips are described in more detail in the Achievements section.

Achievements

We developed and optimized a 2D biosensor, which was able to detect IPTG and 3-oxo-C₁₂-HSL during artificial induction and we demonstrated the ability to detect living cells of the pathogen Pseudomonas aeruginosa using the same 2D biosensor. To ensure that the fluorescence signal resulted from the sensor constructs build in the sensor chips and not from the medium or E. coli itself [http://parts.igem.org/Part:BBa_B0015 B0015] in NEB was used as negative control during sensor chip induction with IPTG, HSL and P. aeruginosa (Negativ control, displayed below).

Negativ control B0015 in NEB as negativ control induced with A) 0.2 µl of 100 mM IPTG, image taken after 2.5 h; B) 0.2 µl of 500 µg/ml HSL (3-oxo-C12), image after 2.5 h; C) with 5 spots of Pseudomonas aeruginosa on the left and one big spot on the right, image taken after 2 h

Testing our Sensor Chips in a Plate Reader

To establish a prove of principle for our sensor chip design we used our construct [http://parts.igem.org/Part:BBa_K1319042 K1319042], which incorporates an IPTG inducible iLOV. E. coli cells containing the latter construct were introduced into sensor chips and fluorescence was measured every 15 minutes subsequently to induction with 2 µl 100 mM IPTG (Testing K1319042 in our sensor chips, displayed on the left). We observed a distinct difference in fluorescence between the non-induced chip (top) and the induced chip (bottom). The middle of the bottom chip started to exhibit a clear and visible fluorescence and the fluorescence increased over time and spread outward. The top chip, however, also showed increase in the measured fluorescence over time which was primarily due to a leaky promoter and background fluorescence.

Detecting 3-oxo-C₁₂-HSL with Sensor Chips

In an initially attempt to detect 3-oxo-C₁₂-HSL, we incorporated the [http://parts.igem.org/Part:BBa_K131026 K131026] construct generated by the 2008 iGEM Team Calgary in our sensor chips. The latter construct generates a fluorescent signal based on GFP in presence of 3-oxo-C₁₂-HSL molecules produced by P. aeruginosa during quorum sensing (Jimenez, Koch, Thompson et al., 2012). First, we tested the construct by direct induction of 3-oxo-C₁₂-HSL (0.2 µl, 500 µg/ml) whereby fluorescence measurement was taken every 15 minutes with an excitation wavelength of 496 nm and an emission wavelength of 516 nm (Testing K131026 in our sensor chips, displayed on the rigth). A distinct fluorescence signal was observed on the induced chip (bottom) compared to the non-induced chip (top). Fluorescence started in the middle of the chip (point of induction) and then extended outwards, still showing an increasing fluorescence signal. The basal level of fluorescence was attributed to leakiness of the promoter and general background fluorescence of growing E. coli cells. In the induced chip (bottom), the background fluorescence was lower than in the non-induced chip (top), because the signal masked the noise. The difference between the induced and non-induced chips indicated a clear response to the HSL and proofed the ability of our 2D sensor chip design to detect HSLs produced by Pseudomonas  aeruginosa.

Detecting IPTG with sensor chips

The video displayed on the left side (Detecting IPTG with sensor chips) shows the construct [http://parts.igem.org/Part:BBa_I746909 I746909] from the 2007 iGEM Team Cambridge, which expresses super folder GFP under the control of a T7 promoter in combination with our 2D sensor chip technology. The [http://parts.igem.org/Part:BBa_I746909 I746909] construct was introduced into BL21(DE3) cells making the expression IPTG inducible due to the T7 RNA Polymerase encoded in the genome of BL21(DE3) under the control of a lacI promoter. While the left chip does not show visible fluorescence, the right chip exhibits a strong fluorescence signal. This elucidates the ability of the sensor chip technology to detect IPTG. The fluorescence response is also high enough to be detected and analyzed by our measurement device WatsOn.

Detecting the 3-oxo-C₁₂-HSL with K131026 in our Sensor Chips with WatsOn

The next step towards the final goal of detecting living cells of Pseudomonas aeruginosa was to reproduce the detection of 3-oxo-C₁₂-HSL, which was established in the plate reader, in our own WatsOn device. Therefore, we again used E. coli BL21(DE3) cells containing [http://parts.igem.org/Part:BBa_K131026 K131026] and induced with 0.2 µL 3-oxo-C₁₂-HSL in a concentration of 500 µg/mL. The right chip was induced and - as a negative control - the left chip was not induced. Pictures were taken every four minutes (Detection of 3-oxo-C12 HSL with K131026, displayed below).
The result was a clear replication of the success of the plate reader experiment. The induced chip showed a clear fluorescence response eminating from the center where the induction with HSL took place, thus demonstrating the ability of not only our sensor chip technology, but also our measurement device WatsOn to successfully detect 3-oxo-C₁₂-HSL.

Detecting Pseudomonas aeruginosa with K131026 in our Sensor Chip with WatsOn

After establishing the successful detection of 3-oxo-C₁₂-HSLs with our sensor chips the next step was to detect living cells of Pseudomonas aeruginosa with our measurement device WatsOn. Therefore sensor chips based on [http://parts.igem.org/Part:BBa_K131026 K131026] were prepared and the right chip was induced with 0.2 µl of a Pseudomonas aeruginosa culture while the left chip was not induced (Detection of 3-oxo-C12 HSL with K131026, displayed below). On the induced chip, a clear fluorescence response was visible in response to P. aeruginosa. The fluorescence signal emerged outward from the induction point and showed a significant difference to the non-induced chip. The results clearly demonstrate the ability of our sensor chip technology and our measurement device WatsOn to detect P. aeruginosa!

Outlook

After successfully detecting P. aeruginosa, the next step in developing our sensor chip platform further is an improvement of the sampling chip. The current technique of using a simple agarose chip is not sufficient to collect all microorganisms from the sampled surface. Therefore, the aim is to improve the sampling chip by trying different, more adhesive material.

Furthermore, diffusion in the sensor chips will be reduced to limit the spread of the fluorescence signal. Currently, the fluorescence spot grows at lot beyond the point of detection and makes it difficult to successfully differentitate between multiple points of induction. By introducing different diffusion barriers into our chips, the growth of the fluorescence spots might be limited, thus enabling the detection of multiple sources of fluorescence lying close together.

Additionally, the application of our sensor chips in combination with our WatsOn device is currently being evaluated for the detection of signals other than fluorescence. Detecting bio- and chemiluminescence has been identified as an area of potential future application.

References

• Waters, C. M., & Bassler, B. L. (2005). QUORUM SENSING: Cell-to-Cell Communication In Bacteria. Annual Review of Cell and Developmental Biology, 21(1), 319-346. Available online at http://www.annualreviews.org/doi/full/10.1146/annurev.cellbio.21.012704.131001.

• Jimenez, P. N., Koch, G., Thompson, J. A., Xavier, K. B., Cool, R. H., & Quax, W. J. (2012). The Multiple Signaling Systems Regulating Virulence in Pseudomonas aeruginosa. Microbiology and Molecular Biology Reviews, 76(1), 46-65. Available online at http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3294424/#B63.