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

Figure 1. The structure of alginate and the cross-link between encapsulation product.

Alginate is a polysaccharide consisting of β-D-mannuronic acid (M) and α-L-guluronic acid (G) residues. When exposed to calcium irons, 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.
If additional protective agents were applied, the biosensor strains may be preserved at 4℃ for more than a month.

Experimental protocol:
1.5% Alginate solution was boiled and kept warm in 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 irons and then stored in the solution with protective agents or drilled-water.

Protective Agents

Inositol

Figure 2. The structure of myo-inositol, which is the active steroisomer, having crucial function in eukaryotic cells.

Bacterial under dehydration may face an increasing osmotic stress as the water activity decrease. A possible way to counteract the osmotic stress is to accumulate compatible solutes to maintain a high osmotic pressure in cytoplasm to stablize proteins and balance the dehydration in the environment^[4]. Inositol is highly compatible for this usage for its stability and hydrophilicity.
Previous work done by Aitor. et al. showed that inositol is highly efficient for preserving bacterial cells. (In that case, P. putida, which is the host of the transcriptional factors we used in our biosensors. )
The link to the original paper: http://link.springer.com/article/10.1007/s00216-010-4558-y

Trehalose

Figure 3. The structure of trehalose, which is a disarrcharide that is a major constituent of insects surving as energy storage compound. It has also been found efficient in preventing dehydration.

Trehalose has an advantage over other kinds of sugars for it has a relatively high glass transition, so the bacteria may maintain a glassy form with the temperature and humidity change.Additionally, high concentration of NaCl solution induces the expression of intracellular trehalose. ^[7]

To obtain a high validity maintainance, the protective agents’ we use were of ralatively high concentration, so we should test that the induction process was not interfered by the protecting agents. A series inositol and trehalose solutions of different concentration were tested.

Figure 4. The tests of induction with the existance of different concentration of protective agents, inositol and trehalose. The NahR was induced by 100μM of inducers. The basal line of none protective agent is shown. As is illustrated, these two protective agents didn’t interfere the inducing process within a fairly high concentration.

Results

There is visible difference between the beads with and without induction. and it would be clearer under blue light.

Figure 5.The test results of alginate encapsulation beads with XylS and NahR biosensors in it. a: After 10h incubation, the fluorescence intensity of XylS is sufficiently high to be discriminated by blue LED and after 12 hours, by naked eyes. b:The induction experiment conducted after 12 days preservation. Left: blank. Right: response of XylS with 100μM 3-MeBzO. c: Test for NahR beads with same inducer(4-MeSaA,100μM) for different incubation time. (from left to right: 0h, 3h, 6h.) showed that the fluorenscent intensity increase during the 3 to 6 hours after induction, but 3 hour is sufficient to create visible difference. d: results for different inducer’s concentration, incubated for 4 hours. (from left to right, 100μM, 10μM and 0μM of 4-MeSaA.)

For NahR, 3 hour induction was sufficient to discriminate the 100μM 4-MeBzO’s induction with the blank by naked eyes. The concentration of 100μM was chosen according to previous tests, indicating this concentration was to obtain highest induction ratio without noticable toxicity. (fig. 5b) 3 hours treatment would be possible to obtain noticable difference of induction, and 6 hours would be appropriate to detect aromatics to determine if the water sample can meet the Chinese government’s requirements for water sample in the environment.(fig. 5c) (Link to the Chinese Government’s Requirements)
After 4 hours incubation, the fluoresence of NahR biosensor worked as expected, and 10μM of 4-MeSaA can be detected by this device. (fig. 5d)
Additionally, This experiment was done after 10-day-storage in the 4℃, so the bacteria were likely to survive for more than 10 days. According to the paper’s report, the maintenance of validity of the bacteria could be sure to meet the detection requirements in more than a month^[4].

Figure 6. The response of NahR to the sample with 4-MeSaD treated by NahF. From left to right: 1,2: not been treated by the adaptor, NahF; 3.4: treated by NahF for 4 hours. 5,6: treated by NahF for 12 hours. 1,3,5 contain 10μM 4-MeSaD and 2,4,6 contain 100μM 4-MeSaD.

Although the evidence of this device was inadequately rigorous, the actual products could be used in field to achieve a rough detection.

Advanced Design

Based on the alginate encapsulation method and previous test results, a hydrogel patterning and transferring method could surve our purposes. This method would be multi-purposes including aromatic detection in a quantitative view, possibility of the cell communication serving the idea of adaptor, and potential for the application 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, which is frequently used in the microfluridic chips.
The parallel wells were etched on PDMS, using a PDMS template in which 500 μm×500 μm square patterns are microfabricated and equidistant from each other by 500 μm. The depth of the wells was 170μm. This design was aiming at prevent interaction of E.coli between different suqares, because the interaction may influence the diffusion process for constucting a concentration gradient. Addtionally, the shape could be altered according to the basal level and detection convenience^[5].
Pattern Transferring The alginate solution and bacterial culture mixture(described in the protocol of alginate encapsulation) was transferred in the wells. After treated with Calcium irons, the PDMS with the mixture was 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 substrate.
Improvements For adaptors, the adaptor E.coli cells could be encapsulated in the agarose layer. Then if the substrate of adaptor exist, it would be tranferred into the inducer which could be dected by corresponding biosensor.
For the bandpass filter, the agarose layer could be pre-treated, that sample and water was drilled on each side 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.)

Figure 7. The design and experiment protocol of hydrogel patterning and transferring method. This method is potential for conducting cell communication and semi-quantitative detection.

Results

Figure 8. The primary attempt to construct the advanced device. The agarose plate was pre-treated with 100μM 4-MeSaA for 12 hours, and the Photograph was took 4 hours after the induction.The lecuna in the left was designed for the inducer’s loading. For the transferred patterning, some alginate out of wells wasn’t removed. The one on the bottom indicating that even with the PMDS adhered, the biosensor could be induced within a relative short time.

Potentials

The advanced device design has potentials for the entire aroamtic toolkit’s application. Various biosensors could be encapsulated by the same method, and the agarose layer may also encapsulate another kind of biosensor, or the adaptor bacterial cells. As the fact that the communication between the agarose layer and alginate is fast, it’s possible to conduct multi-sensor assay by this device. It’s also applicable for the adaptors.

Different concentration gradient could be constructed on the agarose layers. More tests are required to obtain the diffusion constant and this parameter could be used to calculate the diffusion time. After determine the pre-treating time, the alginate encapsulation of bandpass filter would be applied to this device. If the concentration of each square pattern could be calculated, then the bandpass filter would show response in different position. Then we may roughly read out the concentration by the response position. This approach would provide an opportunity for in field aromatic detection.

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.