Purposes

In-field detection of aromatic compounds in environments has always been desirable, and convenience has always been an important requirement for in-field detection. To meet this requirement, the detection process should be fast and the result should be easily read by naked eyes. Furthermore, the device we design to realize all these should be readily portable.
When designing such a portable user-friendly device, the most challenging part would be developing the preservation method. As we choose Escherichia coli, which is unable to germinate spores, as our host strain, special method should be developed to protect the bacteria from temperature changes and physical stress while keeping them alive. We used alginate encapsulation as a basic solution to this problem and built advanced device based on such preliminary design to measure the concentration of aromatics in samples.^[5]

Alginate Encapsulation

Alginate is a polysaccharide consisting of β-D-mannuronic acid (M) and α-L-guluronic acid (G) residues. When exposed to calcium ions, G residues will immediately cross-link to form a gel-like material that may serve as a matrix to safely contain our biosensors.
Alginate has been frequently used as the biological encapsulation material for various organisms such as Saccharomyces cerevisiae, Escherichia coli and mammalian cells^[1][2]. It stands out because of several distinct characteristics^[8]:
(1) Stable and inexpensive, even edible.
(2) Does not interfere with biosensors we constructed.
(3) Ease to shape and manipulate.
(4) Provide reliable protection against environmental stresses.
The alginate encapsulation successfully solve the problem of lethal dehydration and oxidation stress upon our biosensor strains, so no recovering process is required.

Experimental protocol:
1.5% Alginate solution was boiled and kept warm at 40°C.
E.coli was grown overnight in LB medium at 37 °C in 15 ml Falcon tube, then were harvested by centrifugation at 4000 r.p.m. for 10 minutes and then resuspended in 500 μl of fresh LB media. mixed with 3 ml alginate solution, and dropped into 0.2M calcium chloride in room temperature(20 to 24℃) to form bead-like alginate encapsulation.
Alginate beads were washed in PBS to eliminate calcium ions and then stored in the solution with protective agents or drilled-water.

Results

Demonstration of our convenient in-field detection device was performed by soaking the alginate capsules containing bacteria with specific biosensors into solutions with corresponding aromatics and incubate for a certain amount of time.

For NahR biosensor, after 3 hours of incubation, the fluorescence generated by capsules soaked in solution with 100μM 4-MeBzO can be easily discerned by naked eyes using a simple blue light source, while the capsules soaked in blank solution has no visible fluorescence. The concentration of 100μM was chosen according to previous tests because under such a concentration, high induction ratio can be obtained without noticable toxicity to bacteria. (fig. 5b)

Although such demonstration was not sufficiently rigorous, it verifies the possibility that our products could be used in field to achieve a rough detection.

Advanced Design

Based previous test results on the alginate encapsulation method, we reasoned that a hydrogel patterning and transferring method could serve multi-purposes, including quantitative detection, implementing adaptors through cell communication, and realization of bandpass filter by constructing a inducer concentration gradient.

PDMS Template Design PDMS (polydimethylsiloxane) is particularly known for its unusual rheological (or flow) properties. PDMS is optically clear and, in general, inert, non-toxic, and non-flammable. PDMS is a material with no marked harmful effects on organisms in the environment and is frequently used in the microfluridic chips.
Parallel square wells which are 500μm wide, 500μm long and 170μm deep were etched on to a PDMS template, with a distance of 500 μm between them. This design was aiming at preventing interaction of E.coli between different wells. The shape of our design could be easily adjusted according to customers' need.^[5].
Pattern Transferring The alginate solution and bacterial culture mixture (described in the protocol of alginate encapsulation) was transferred into the wells. After treatment with calcium ions, the mixture solidify. The PDMS with the mixture was then transferred to a agarose layer. (Its concentration is of 1.5% or 2%) In 5 minutes, the PDMS template was peeled off with the cell patterns left on the agarose layer.
Improvements For adaptors, the adaptor E.coli cells could be cultured in the agarose layer. If the substrate of adaptor exists, it would be converted into the compound which could be dected by corresponding biosensor.
For the bandpass filter, the agarose layer could be pre-treated so that the sample and water was aliquoted on two sides of the agarose layer. After 6 to 12 hours treatment, a concentration gradient would be constructed by diffusion. (The diffusion time could be calculated according to the mass of inducers and the concentration of agarose layer.)

Results

References
[1] Koch, S., Schwinger, C., Kressler, J., Heinzen, C. H., & Rainov, N. G. (2003). Alginate encapsulation of genetically engineered mammalian cells: comparison of production devices, methods and microcapsule characteristics. Journal of microencapsulation, 20(3), 303-316.
[2] Wang, N., Adams, G., Buttery, L., Falcone, F. H., & Stolnik, S. (2009). Alginate encapsulation technology supports embryonic stem cells differentiation into insulin-producing cells. Journal of biotechnology, 144(4), 304-312.
[3] Morgan, C. A., Herman, N., White, P. A., & Vesey, G. (2006). Preservation of micro-organisms by drying; a review. Journal of Microbiological Methods,66(2), 183-193.
[4] de las Heras, A., & de Lorenzo, V. (2011). In situ detection of aromatic compounds with biosensor Pseudomonas putida cells preserved and delivered to soil in water-soluble gelatin capsules. Analytical and bioanalytical chemistry, 400(4), 1093-1104.
[5] Choi, W. S., Kim, M., Park, S., Lee, S. K., & Kim, T. (2012). Patterning and transferring hydrogel-encapsulated bacterial cells for quantitative analysis of synthetically engineered genetic circuits. Biomaterials, 33(2), 624-633.
[6] Leslie, S. B., Israeli, E., Lighthart, B., Crowe, J. H., & Crowe, L. M. (1995). Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying. Applied and environmental microbiology, 61(10), 3592-3597.
[7] Peking iGEM 2010 wiki
[8] iGEM: Imperial Collage/Encapsulation, 2009
[9] van der Meer, J. R., & Belkin, S. (2010). Where microbiology meets microengineering: design and applications of reporter bacteria. Nature Reviews Microbiology, 8(7), 511-522.