Team:Pittsburgh/Poster

Poster: Pittsburgh

Bluetooth Bacteria: Wireless control of gene expression in E. coli

IGEM Team Members
Sabrina Catalano, Dara Czernikowski, Lia Franco, Victor So, Angel Zheng

IGEM Team Mentors
W. Seth Childers¹, Alex Deiters¹, Jason Lohmueller³, Jason Shoemaker², Sanjeev Shroff<sup>4

1</sup>Department of Chemistry, Dietrich School of Arts and Sciences, University of Pittsburgh
²Department of Chemical Engineering, Swanson School of Engineering, University of Pittsburgh
³Departments of Surgery and Immunology, University of Pittsburgh School of Medicine, University of Pittsburgh
⁴Department of Bioengineering, Swanson School of Engineering, University of Pittsburgh

Abstract
Our ability to control the function of engineered microbes is limited within hard-to-reach places, such as deep within human tissue, the rhizosphere surrounding crops, or pipes in manufacturing facilities. The field of magnetogenetics has begun to consider how magnetic fields could affect biological molecules and cells. In our project, we aim to control the function of bacteria via the remote activation of an alternating magnetic field (AMF). E. coli will be engineered to respond to AMF stimuli by attaching magnetic nanoparticles to the surface of the bacterial membrane in conjunction with cytoplasmic temperature sensitive transcriptional repressors to regulate gene expression of the green fluorescent protein. Upon AMF stimulation, magnetic nanoparticles heat up and increase cytoplasmic temperature, thereby inducing a conformational change of the repressor dimers and allowing transcription of target genes. This technology critically couples alternating magnetic field stimulation with thermal sensitive parts to enable “wireless” control of bacteria.

Introduction and Inspiration

Currently, gene expression is controlled by scientists using chemicals or light. We sought to come up with a non-invasive method, similar to optogenetics, that would allow for deeper penetration for areas like tissue. We considered products like Bluetooth and Wi-Fi that can transmit information between electronics over long distances to investigate how we may apply such mechanisms to a biological system. The Bluetooth Bacteria aims to wirelessly control gene expression in Escherichia coli (E. coli).

Project Goals

1. Attach functionalized magnetic nanoparticles to the surface of E. coli in conjunction with cytoplasmic temperature sensitive transcriptional repressors to regulate gene expression of GFP

2. Upon stimulation with an alternating magnetic field (AMF), MNPs can dissipate heat, allowing control of cytoplasmic temperature

3. An increase in temperature induces a conformational change of the repressor dimers allowing transcription of our target genes, enabling “wireless control” of bacteria

Magnetic Nanoparticles

To localize the MNPs to the surface of the bacteria, we came up with three options for optimizing attachment:



Electrostatic

• Through functionalizing the MNPs with positively charged amine or chitosan, the particles would interact with the negatively charged bacterial surface (Huang, 2010; Le, 2020).
• PROS: This method is simple and efficient to implement.
• CONS: It is nonspecific and will bind to any negatively charged surfaces.

Antibodies

• The specific antibodies which bind to E. coli surface proteins would be fixed to the MNPs.
• PROS: Compared to using electrostatic interaction, antibodies would allow more specific attachment.
• CONS: The antibodies will still bind to other bacteria which also express the epitope.

Streptavidin/biotin

• The E. coli would be first transformed to express streptavidin with Lpp-OmpA, allowing streptavidin to localize at the surface.
• PROS: The MNPs would be biotinylated and form an almost covalent bond with the streptavidin, allowing for the most specific interaction.
• CONS: By the time it is expressed to the surface, streptavidin may already be saturated with biotin; it is more difficult and time-consuming than the other two methods.

Temperature Control

An alternating magnetic field (AMF) is induced by the change in direction of an alternating current. As seen in the figure below, when the current moves counter-clockwise through the coiled magnetic core, a magnetic field is produced pointing upwards out of the coil through the center. When the current moves in the opposite direction, the magnetic field is produced is produced downwards through the center. The direction of the magnetic field is constantly alternating due to the nature of the alternating current running through the coil.

The magnetic nanoparticles are highly responsive to this magnetic current. They resonate with frequencies in the 100-500 kHz range and orient themselves over time to align with the field. Due to the alternating nature of the magnetic field, the nanoparticles are excited by the field. This induces a steady increase in temperature during AMF exposure. We chose to use this characteristic paired with an intermittent exposure to the AMF as a method for controlling temperature within our cells without needing to expose the entire solution to heat directly.

Heat-Sensitive Response

In order to allow wireless gene expression, we need heat-sensitive parts to respond to the change in temperature.

We explored two temperature-sensitive parts: heat shock promoters and temperature-sensitive transcription factors. These will regulate the expression of target gene mWasabi, a green fluorescent protein similar to GFP:

Heat shock promoters:
The plasmids we used express heat shock promoters upstream of the mWasabi genes. An increase in temperature induces endogenous heat shock proteins to bind to the heat shock promoters and upregulate mWasabi.

Temperature-sensitive transcription factors:
TlpA and TcI are repressors that respond to 45°C and 40°C, respectively (Piraner, 2017). At lower temperatures, these transcriptional repressors are in a dimerized state, preventing RNA polymerase from initiating transcription. When the temperature rises, they both undimerize and allow expression.

Surface Attachment

We tested for surface attachment by incubating BL21 E. coli strain with amine-functionalized magnetic nanoparticles (AF-MNPs).


The initial absorbance of BL21 E. coli strain culture was measured before the addition AF-MNPs (OD600 ~1). AF-MNP were added to the culture and incubated at room temperature for 15 mins. After-incubation, a strong magnet was used to remove the bacteria-nanoparticle conjugate, and the absorbance of the final supernatant was measured.





Figure 1. OD600 decreased significantly after magnetic separation (p=0.038) after adjusting for AF-MNPs that were not separated by magnet. P-values were calculated with one-tailed and two-tailed t-test.

Based on the decrease in OD600, our results suggest that AF-MNP were able to form a bacteria-nanoparticle conjugate. However, further imaging must be done to confirm the surface attachment of AF-MNP to the cell.

AMF-Emitting Device

Our device is a simple circuit composed of an amplifier, oscilloscope, function generator, capacitors, transformer, shunt resistor, vector board, and wire. It also includes an electromagnetic core with a coil of approximately 2.5 cm diameter in which we would place our magnetic nanoparticles to be excited.

While our design works in theory and in the literature, we were unable to produce the full circuit due to some shipping delays and inability to access an amplifier of the appropriate power (at least 50 W but preferably 100 W).



Once the circuit is completed, we would then experiment with the MNPs to determine their exact rate of heating when exposed to the AMF. With this, we would then develope an interval system which will allow the heating and cooling of our sample to ensure that the temperature of our sample remains within a small range (Christiansen et al., 2017).

Heat-shock Promoters and Heat-sensitive Dimers

To test our heat-sensitive parts, we performed 7-hour thermal inductions of the constructs. We measured the results in triplicate and calculated standard deviation and standard error for the measurements. We performed these experiments with cells that expressed mWasabi through heat-sensitive promoters and a separate constitutive mTangerine, which allows comparison of mWasabi output.

Figure 2. Thermal Induction of heat sensitive constructs for 7-hours at varying time points. Blue: grown at 45 C in a larger culture while shaking. Orange, Grey, Yellow: grown at 37°C, 40°C, 45°C, respectively in separate 100 uL aliquots in a Thermocycler without shaking.



A B Figure 3. 7-hour thermal incubation at 40°C (Figure 3A) and 45°C (Figure 3B) of heat sensitive constructs (mWasabi) in cells that constitutively express mTangerine. Fluorescence measured by plate reader normalized using cell density (OD600) to give approximate fluorescence per cell. Error bars gives standard error.

Based on our results, the heat-sensitive constructs can respond to the change in temperature. In particular, TcI and DnaK produces significant mWasabi expression at 45°C. This suggests that these heat-sensitive parts can be used to facilitate the gene output after stimulation with AMF.

Education

In effort to stay tuned on current advances in Synthetic Biology, we decided to create a podcast. The Bluetooth Bacteria Podcast presents the Pittsburgh iGEM team discussing controversial topics in synthetic biology, and how those current advances are applied to our everyday lives.

• Over 235 plays!
• Avaliable on seven different platforms, including Spotify and Apple Podcast
• Listeners from all around the world
• Topics Discussed
  • Animal Organ Donation
  • Aging
  • Genetic Dating


• Collaborations with iGEM Teams
  • William & Mary
  • Baltimore BioCrew
  • Pune
  • Toulouse
  • BITS GOA
  • Kings College – London

Conclusion

Based on our results, we succeeded in attaching magnetic nanoparticles to the surface of the cell, designing a device to emit to AMF to stimulate MNPs, and inducing heat-sensitive output.

• Amine-functionalized MNPs were attached to the surface of E. coli
  • However, further imaging must be done to confirm attachment
• Designed circuit to build the AMF-emitting device
  • Major components of the device were not available
  • Futher construction must be done to measure AMF input and control cytoplasmic temperature
• Our heat-sensitive transcription factors and heat-shock promoters induced gene expression at higher temperature

Short term goals

• Improve upon the concept of localized heating to produce spatial control like optogenetics
• Design bio-circuits using expanded library of temperature-sensitive parts
• Identify and expand the types of temperature-sensitive parts that we could use
• Explore using ferritin

Long term goals

• Develop design for use as a therapeutic to treat chronic diseases
• Optimize our system for easy application in research

Acknowledgements

Thank you to all of our sponsors for providing financial support and resources.



We want to especially thank all the experts that have taken the time out of their schedules to provide their input on our project. Their advice has been a valuable resource in determining the direction and design of our project.

• Dr. Siyuan Rao, PhD. - Postdoctoral Fellow in Bioelectronics Group at Massachusetts Institute of Technology
• Dr. Michael Christiansen, PhD. - postodoctoral researcher in the Department of Health Sciences and Technology at ETH Zurich
• Dr. Warren Ruder, PhD. - Associate Professor of Bioengineering and a William Kepler Whiteford Faculty Fellow in the Swanson School of Engineering at the University of Pittsburgh
• Dr. Jungsang Moon - PhD. Candidate at Massachusetts Institute of Technology

Special thanks to Dr. Mikhail Shapiro, PhD. and Dr. Polina Anikeeva, PhD. for providing us guidance and contacts for our project via email.

References

• Alphandéry, Edouard. “Applications of Magnetosomes Synthesized by Magnetotactic Bacteria in Medicine.” Frontiers in Bioengineering and Biotechnology, vol. 2, 2014. Frontiers, doi:10.3389/fbioe.2014.00005.
• Christiansen, Michael G., et al. “Practical Methods for Generating Alternating Magnetic Fields for Biomedical Research.” Review of Scientific Instruments, vol. 88, no. 8, Aug. 2017, p. 084301. aip.scitation.org (Atypon), doi:10.1063/1.4999358.
• Giustini, Andrew J., et al. “MAGNETIC NANOPARTICLE HYPERTHERMIA IN CANCER TREATMENT.” Nano LIFE, vol. 1, no. 01n02, 2010. PubMed Central, doi:10.1142/S1793984410000067.
• Huang, Yan-Feng, et al. “Amine-Functionalized Magnetic Nanoparticles for Rapid Capture and Removal of Bacterial Pathogens.” Environmental Science & Technology, vol. 44, no. 20, Oct. 2010, pp. 7908–13. ACS Publications, doi:10.1021/es102285n.
• Le, Thao Nguyen, et al. “A Convenient Colorimetric Bacteria Detection Method Utilizing Chitosan-Coated Magnetic Nanoparticles.” Nanomaterials, vol. 10, no. 1, Jan. 2020. PubMed Central, doi:10.3390/nano10010092.
• Piraner, Dan I., et al. “Tunable Thermal Bioswitches for in Vivo Control of Microbial Therapeutics.” Nature Chemical Biology, vol. 13, no. 1, Jan. 2017, pp. 75–80. www.nature.com, doi:10.1038/nchembio.2233.