Purposes

To realize the idea of in-field detection, a device with remarkable convenience for monitoring environmental water pollution should be proposed. This device must be capable of determining if the specific kind of aromatic compound exists in a water sample, and perhaps more significantly, measuring specific aromatic compound’s concentration semi-quantitatively. To meet the requirement of convenience, the detection process must be fast and the result must be read with naked eyes or with user-friendly devices. As for measuring the concentration, a concentration gradient could be constructed by the pre-treating method of the device, so different response patterns may roughly reflect the concentration. All this requirements must be carefully designed with biosafety concerns.
The most challenging part of a biological detection device is the preservation method. The sensor strain we use, Escherichia coli, failed to germinate spores or gemma to resist general preservation conditions of dehydration, temperature changes and physical interference. Several approaches were designed to achieve valid maintainance benefits. Based on the fundamental designs, an advanced device with multifunctions was proposed to measure the concentration carrying potential for further improvements.

Alginate Encapsulation

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

Alginate is a polysaccharide consists of β-D-mannuronic acid (M) and α-L-guluronic acid (G) residues. If treated by calcium irons, G residues are cross-linked and a coat would be formed within a rather short period of time.
Alginate is frequently used as the biological encapsulation material for various organisms such as Saccharomyces cerevisiae, Escherichia coli and mammalian cells^[1][2]. It came into our consideration as the encapsulating material for its outstanding specifications, which include^[8]:
(1) Food grade materials which are stable and inexpensive
(2) Possibility to use every biosensor we constructed
(3) Ease to shape and manipulate
(4) Protection against environmental stresses
The alginate encapsulation successfully solve the problem of dehydration and oxidation stress upon our biosensor strains, so no reviving process is required. With the support of protective agents, the period of validity could be longer than a month in 4℃.

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. )

Figure 3. The results indicated that inositol was a protective agent of maintained the viability of bacterial cells. With the existance of maltodextrins(MD), the optimal validity maintaining could be achieved. (figure from: A. Heras and V. Lorenzo, 2011)
The link to the original paper: http://link.springer.com/article/10.1007/s00216-010-4558-y

Trehalose

Figure 4.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. It is also important to produce in cell trehalose by the bacteria spontaneously, which involves certain gene expression and an osmotic press induction. In E. coli, the otsA and otsB genes are responsible for trehalose biosynthesis from UDP glucose. These genes encode trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase, respectively. And 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 5.The tests of induction with the existance of different concentration of protective agents, inositol and trehalose. The NahR was induced by 100μM of inducers. As is illustrated, these two protective agents didn’t interfere the inducing process within a fairly high concentration.

Advanced Designs

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(Link:), 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(Link:), 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 6. The design and experiment protocol of hydrogel patterning and transferring method. This method is potential for conducting cell communication and semi-quantitative detection.

Results

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

Figure 7.The test results of alginate encapsulation beads with XylS and NahR biosensors in it. a and b: After 10h incubation, the fluorescence intensity is sufficiently high to be discriminated by blue LED and after 12 hours, by naked eyes. c: Same inducer(4-MeSaA,100μM) with 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. 7b) 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. 7c) (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. 7d)
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].
Although the evidence of this device was inadequately rigorous, the actual products could be used in field to achieve a rough detection.

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.

Reference

[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.