Biosensors in microspheres

The hard-coated hydrogel prevented bacteria from escaping from the biosensor into the environment

Vera Sysoeva, N+1

The scientists packed the bacteria into a hydrogel with a hard shell that allows only small molecules to pass through, but does not allow the bacteria themselves or their genetic material to leave the capsule. Based on such a design, it is possible to create biological elements that perform simple computational operations and use them as biosensors – and not be afraid to release modified organisms into the environment.

The developers showed that the sensitivity of the system makes it possible to determine the excess of permissible concentrations of heavy metals in the water and tested the system on a sample from a local river. The work is published in Nature Chemical Biology (Tang et al., Hydrogel-based biocontainment of bacteria for continuous sensing and computation).

Genetically modified microorganisms are a promising tool for environmental purification, agricultural applications and the creation of living devices – for example, biosensors based on bacteria. However, their use is very limited: there is a possibility that bacteria or their genetic material will end up in the environment, and this is undesirable, even if the modified bacteria themselves are not dangerous.

Often, for safety reasons, synthetic biology uses chemical methods to contain microorganisms. For example, they equip bacteria with genes that encode substances that are toxic to the bacterium itself. Such a cell can survive only while it is in a nutrient medium with a specific antidote. However, such strategies are not ideal: like any living creatures, altered bacteria mutate and eventually acquire new properties. There is a non-zero probability that some of them will be able to learn how to survive without the help of developers. A reliable physical barrier, in addition to chemical containment methods, would expand the scope of safe use of biological devices.

Capsules for bacteria can be created on the basis of hydrogels – materials that can provide bacteria with an aqueous environment with nutrients that is comfortable enough for cell growth. Alginate gels (insoluble salts of alginate acid) – non-toxic and inexpensive – already used in medicine, for example, for the delivery of medicines. However, as physical barriers preventing the escape of bacteria, they are poorly suited: the same change in the acidity of the medium can destroy the gel.

Biologists from The Massachusetts Institute of Technology under the leadership of Timothy Lu (Timothy K. Lu) offered a solution to the problem: they packed alginate hydrogel with bacteria in a strong shell. As a material for a strong outer layer, the scientists chose an elastic polymer network made of polyacrylamide in combination with an alginate gel. The researchers note that it is possible to apply several outer layers for greater strength.

To prepare one such capsule, the researchers first mixed a liquid culture of Escherichia coli coli coli with 50 or 100 microliters of alginate gel with nutrients and formed spheres. Then the cores were coated with a strong layer of polyacrylamide and alginate.

The pores in the solid layer of such a capsule are small enough (5-50 nanometers) to prevent E.coli from passing through, and scientists have confirmed the effectiveness of the barrier experimentally. Spheres with a hard coating and without it were incubated in a warm environment, and checked whether bacteria had grown outside the capsule. Soft spheres without a shell could not hold the bacteria, and a day later there was a high density of E. coli in the medium. At the same time, during 72 hours of the experiment, solid capsules did not allow E.coli to escape. The authors also provided the bacteria with genes that made the cells dependent on a synthetic amino acid that does not exist in nature. As soon as the capsule ran out of this substance (after about 48 hours), the bacteria lost their viability. Together with the physical barrier, this approach reduces the likelihood of bacterial escape. In addition, the authors showed that the genetic material of cells (DNA macromolecules) cannot penetrate the barrier.

Capsules in a hard shell did not release bacteria into the nutrient medium; spheres of ordinary hydrogel with bacteria became sources of bacterial growth in the medium. Figures from the article by Tang et al.

The scientists also investigated the possibility of transferring information between bacteria from different capsules or with the environment using small molecules and ions. The authors placed bacteria in capsules that can produce a fluorescent protein in the presence of a small molecule of anhydrotetracycline. When the capsules were incubated in anhydrotetracycline solution (200 nanograms per milliliter), the fluorescence in them was 35 times stronger compared to capsules in a normal medium.

In another experiment, the authors of the work taught bacteria from different capsules to transmit information among themselves. Some bacteria, in response to the presence of anhydrotetracycline in the environment, produced a signaling molecule and isolated it outside the capsule, while others produced a fluorescent protein as soon as they felt this signaling molecule.

The interaction of capsules with different bacteria: some receive information from the environment and secrete a signaling molecule, others produce a fluorescent protein in response to the signaling molecule.

Finally, the researchers tested capsules with bacteria as biosensors. The authors tested whether E. coli could detect the presence of cadmium ions in water samples from the Charles River. Cadmium ions are common water pollutants that negatively affect human health. The scientists placed bacteria in the capsules that could produce the same fluorescent protein in response to the presence of zinc, cadmium or lead ions. It turned out that bacteria can detect the presence of cadmium in water at a concentration of 5 micromoles, which is lower than the concentration allowed by local regulations (8.9 micromoles).

The principle of operation of a capsule-based biosensor.

Previously, biologists taught bacteria to perform logical operations and even perform analog calculations. Another team of scientists has shown that not only chemicals, but also electricity can be used as gene switches in bacteria.

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