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Bluetooth Bacteria

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Project Description

 

The Problem

 

The field of optogenetics uses light to manipulate of cell behavior. With this technique, light-sensitive proteins generally known as “opsins” can be engineered to activate target genes or cells with the input of light at variable wavelengths. It allows a wide range of applications such as activation of neural circuits or muscle cells (Duebel et al., 2015; Mahmoudi et al., 2017). Although optogenetics is a powerful tool, light delivery remains a major problem. Invasive surgery and tethered light sources are often required to reach deep tissue or hard to reach places, limiting its potential applications (Nimpf & Keays., 2017).

Inspiration

 

To address this problem, we looked into different methods of manipulating cell behavior non-invasively. During the beginning of our project, we found that wireless fidelity (Wi-Fi) radiofrequency radiation globally affected cell behavior, causing a change in gene expression and even affecting antibiotic resistance (Said-Salman et al., 2019; Taheri et al. 2017). Inspired by the idea of wireless connectivity (hence the name Bluetooth Bacteria), we asked ourselves: Can we wirelessly control gene expression in bacteria?

After further investigation, we found that the field magnetogenetics is developing new methods to manipulate cell behavior non-invasively. Magnetogenetics explores the effect of magnetic fields on cells through a variety of techniques.

The Solution

 

The Bluetooth Bacteria aims to wirelessly control gene expression in Escherichia coli (E. coli). Our design features three main parts. First, magnetic nanoparticles are attached to the surface of an E. coli chassis and stimulated with an alternating magnetic field (AMF). Second, upon AMF stimulation, magnetic nanoparticles dissipate heat and raise the cytoplasmic temperature of the bacterium. Third, the change in temperature activates heat sensitive dimers, acting as a thermal bio-switch to regulate gene expression.

Magnetic Nanoparticles

 

The key component of the Bluetooth Bacteria is magnetic nanoparticles. Iron oxide magnetic nanoparticles (MNP) dissipate heat when stimulated with AMF (Christiansen et al., 2017; Chen et al., 2016). Iron oxide MNP can generate heat in response to AMF through the Brownian mechanism of relaxation (rigid body rotation) and Néel relaxation (internal magnetic realignment) (Rosensweig, 2002; Davis et al., 2020). The ability of magnetic nanoparticles to dissipate heat has been applied to cancer hyperthermia, drug release, and neural stimulation. In cancer hypothermia, magnetic nanoparticles targeted to a tumor site can raise its temperature and induce apoptosis (Pankurst et al., 2003). Stimulation and heating of magnetic nanoparticles loaded onto thermal sensitive liposomes have also been used to trigger a chemical release to targeted neural circuits (Rao et al., 2019).

To utilize this property in the Bluetooth Bacteria, magnetic nanoparticles are localized to the surface of E. coli. We have proposed three methods of attachment. The first method of attachment is to use amine-functionalized magnetic nanoparticles that electrostatically attach to the surface of E. coli. The second method of attachment is to use biotinylated-coated magnetic nanoparticles that bind streptavidin expressing on the surface of E. coli. The third method of binding is to use antibodies that target proteins native to the E. coli outer membrane.

Figure 1. Amine-functionalized MNPs electrostatically interact with the surface of E. coli

Figure 2. Biotinylated MNPs can non-covalently bind to E. coli expressing streptaviin on its surface.

Figure 3. MNPs functionalized with antibodies can bind native proteins on the surface of the E. coli outermembrane

Temperature Control

 

The second main aspect of the Bluetooth Bacteria is temperature control. Once magnetic nanoparticles are attached to the surface of E. coli, MNPs can be stimulated with an alternating magnetic field (AMF) to dissipate heat and activate gene expression.

To accomplish this, the Bluetooth Bacteria requires AMF-generating electromagnetic device. Our device is based on a simple and cost-efficient design presented by Christiansen et al (2017). An AMF device functions as a low loss inductor that is coupled to a target sample through a circuit. It will produce an AMF input that will stimulate the MNPs to generate heat. Our device will be scaled to stimulate a small volume of sample, and it will produce an alternating magnetic field with amplitudes from 100-500 kHz at timed intervals. The interval system will allow the heating and cooling of our sample to ensure that the temperature of our sample remains constant (Christiansen et al., 2017).

Figure 4. an AMF-generating device will produce an alternating magnetic field (AMF) to stimulate and heat magnetic nanoparticles in a sample.

Heat-Sensitive Dimers

 

The final part of the Bluetooth Bacteria is to activate gene expression based on the change in temperature. For our project, we required a high-performance temperature-dependent thermal bioswitch that can be used to respond to a change in temperature in E. coli. In mammalian cells, temperature-sensitive TRVP1 channels are often used as a method to respond to MNPs, where a local temperature increase opens TRVP1 sensitive channels and causes an influx of calcium ions (Stanley et al., 2016; Huang et al., 2010). However, this would not be effectively expressed in E. coli due to the lack of post-translational modifications and double membrane in bacteria. Alternatively, temperature-sensitive transcription factors known as TlpA and TcI can respond to the change in temperature and control gene expression in bacteria.

TlpA is a temperature-dependent transcriptional autorepressor from the virulence plasmid of Salmonella typhimurium. The protein undergoes temperature-dependent uncoiling between 37 °C and 45 °C. During the low-temperature dimeric sate, it blocks transcription from a 52-bp TlpA operator promoter. TcI is a temperature-sensitive variant of the bacteriophage lambda repressor cI (cI857) that will act on a tandem pR-pL operator-promoter. At 30°C, TcI is repressed gene expression and allows gene expression at 42 °C (Piraner et al, 2017).

The Bluetooth Bacteria will express TlpA or TcI. At lower temperatures, gene expression will be turned off. But at higher temperatures, which is controlled by the MNPs and AMF-generating device, gene expression will be turned on. As a result, wireless gene expression is achieved through MNPs, AMF stimulation, and heat-sensitive dimers.

Figure 5. At low temperatures, TlpA (37 °C) or TcI (30°C) turns off gene expression, preventing RNA polymerase from binding.

Figure 6. At high temperatures, TlpA (45 °C) or TcI (42°C) undimerizes and allows gene expression.

References

 

• Duebel, J., Marazova, K., & Sahel, J. (2015). Optogenetics. Current Opinion in Opthalmology, 26(3). 226-232.


• Mahmoudi, P., Veladi, H., & Pakdel, F. G. (2017). Optogenetics, Tools and Applications in Neurobiology, 7(2): 71-79


• Nimpf, S., & Keays, D. (2017). Is magnetogenetics the new optogenetics? EMBO J, 36(12): 1643-1646


• Said-Salman, I. H., Jebaii, F. A., Yusef, H. H., & Moustafa, M. E. (2019). Global gene expression analysis of Escherichia coli K012 DH5 α after exposure to 2.4 GHz fidelity radiation. Scientific Reports, 9, 14425.


• Taheri, M., Mortazavi, S. M. J., Moradi, S., Mansouri, G. R. & Nouri, F. (2017). Evaluation of the Effect of Radiofrequency Radiation Emitted From Wi-Fi Router and Mobile Phone Simulator on the Antibacterial Susceptibility of Pathogenic Bacteria Listeria monocytogenes and Escherichia coli. Sage J, 1-8.


• Christiansen, M., Howe, C. M., Bono, D. C., Perreault, D. J., & Anikeeva, P. (2017). Practical methods for generating alternating magnetic fields for biomedical research. Review of Scientific Instruments, 88(8): 084301.


• Chen, R., Christiansen, M. G., Sourakov, A., Mohr, A., Matsumoto, Y., Okada, S., Jasanoff, A., & Anikeeva, P. (2016). High-Performance Ferrite Nanoparticles through Nonaqueous Redox Phase Tuning. Nano Letters, 16: 1345-1351


• Davis et al. (2020). Nanoscale Heat Transfer from Magnetic Nanoparticles and Ferritin in an Alternating Magnetic Field. Biophysical Journal, 118 (6): 1502-1510.


• Rosensweig, R. R. (2002). Heating magnetic fluid with alternating magnetic field. Journal of Magnetism and Magnetic Materials. 252: 370-374.


• Pankhurst, Q. A., Connolly, J. S., Jones, K., & Dobson, J. (2003). Journal of Physics D: Applied Physics 36 (13): R167.


• Rao, S., Chen, R., LaRocca, A. A., Christiansen, M. G., Senko, A. W., Shi, C. H., Chian, P., Varnavides, G., Xue, J., Zhou, Y., Park, S., Ding, R., Moon, J., Feng, G., & Anikeeva, P. Remotely controlled chemomagnetic modulation of targeted neural circuits. Nature Nanotechnology, 14: 967-973.


• Stanley, S., Sauer, J., Kane, R. S., Dordick, J. S., Friedman, J. M. (2016). Remote regulation of glucose homeostasis in mice genetically encoded nanoparticles. Nat Med, 21 (1): 92-98.


• Huang, H., Kelikanli, S., Zeng, H., Ferkey, D. M., & Pralle, A. (2010). Remote control of ion channels and neurons through magnetic-field heating of nanoparticles. Nature Nanotechnology, 5: 602-606.


• Piraner, D. I., Abedi, M. H., Moser, B. A., Lee-Gosselin, A., & Shapiro, M. G. (2017). Tunable thermal bioswitches for in vivo control of microbial therapeutics. Nature Chemical Biology, 13: 75-80.