Overall Goal
Our project aims to enable precise spatiotemporal control of gene activation in response to magnetic fields. This is done by combining two cellular responses into an AND gate. By then using a hardware system involving orthogonally applied magnetic fields to activate the two responses along different axes, we can target expression at only the intersection between them.
In order to achieve this, we had to design:
- Cellular pathways that respond to magnetic fields
- AND gates that integrate these pathways to create a detectable output
- Hardware systems capable of
- Generating two orthogonal magnetic fields
- Localising these fields along a desired axis
- Imaging our output signal (fluorescence/chemiluminescence/colour change)
Pathways
In this project, we employ three methods to modulate gene expression based on magnetic fields. Two of these make use of magnetic nanoparticles, either to heat cells or to exert a mechanical force on their membrane.
Magnetic (direct)
Electromagnetic perceptive gene (EPG) undergoes a conformational change when placed in a magnetic field, bringing its termini closer together. Fusing each half of a protein of interest to the different termini of EPG, allows us to regulate it with magnetic fields.
Thermal (Indirect)
Temperature sensitive transcription factors, such as TlpA and TcI, only bind their promoters at certain temperatures. Placing these promoters upstream of a reporter, e.g., mRFP, allows us to control its expression through the heating of cells.
Mechanical (Indirect)
Mechanosensors on the cell surface activate certain transcription factors under cell wall stress. Placing the relevant promoters upstream of a reporter, e.g., LacZ, allows us to control its expression through activation of the mechanosensor.
| Magnetosensitive | Thermal | Mechanical | |
|---|---|---|---|
| Magnetic Nanoparticle | N/A | Small (10-20nm) | Large (250nm) |
| Field properties | DC ~35mT | High Frequency ~5mT | Low Frequency ~20mT |
| Biological Components | E. coli
|
E. coli
|
S. cerevisiae
|
EPG (Direct response to magnets)
Click here to see our engineering cyclesKryptopterus bicirrhis (glass catfish) have been shown to swim away from both static and alternating magnetic fields. By cloning cDNA libraries from K. bicirrhis into X. laevis oocytes and measuring membrane current alteration, Krishnan et al (2018) were able to show that the magnetosensitive response is controlled by a single open reading frame, termed the electromagnetic perceptive gene (EPG).
Expression in mammalian cells suggests that EPG is a cell membrane protein that acts by mediating calcium influx (Krishnan et al, 2018; Hwang et al, 2020; Ricker et al, 2023). While initial studies by Krishnan et al (2018) suggested that the mechanism did not involve a conformational change, more recent studies show that proteins split between the termini of EPG regain their function in a magnetic field (Grady et al, 2024). This suggests that a conformational change brings them together.
Inspired by optogenetic approaches using Lov2 (Liu et al, 2024), which undergoes conformational changes when it absorbs light, we designed a regulatory system based on fusion proteins. By inserting EPG into regulatory elements, enzymes or structural proteins, we can regulate their activity using direct magnetic fields. In order to demonstrate our ability to accurately control cell behaviour, we proposed two designs using EPG. One of these allows us to characterise the behaviour of EPG, while the other is to integrate it into the AND gate system. For characterisation, we designed an EPG-NanoLuciferase fusion protein that can monitor EPG activity under different field strengths. For integration into the AND gate system, we designed an EPG-TEV protease that can cleave effector proteins.
EPG-NanoLuc
Click here to see our engineering cyclesIn order to characterise the behaviour of EPG under different magnetic field strengths, we needed a reporter that has been shown to work as a split protein. We also needed it to have a fast response time, be amenable to signal amplification, and be reversible. Table 1 shows a comparison of the different systems we considered.
Of these options, we decided that the optimal system for characterisation was NanoLuc, a synthetic luciferase developed by Promega. It is derived from Renilla luciferase, with enhanced brightness (150X) and stability.
We split NanoLuc into two fragments (65 and 106 amino acids long), as done by Zhao et al (2015). We also added linkers between the NanoLuc fragments which provide flexibility and reduce background signal (Grady et al, 2024).
We designed four different plasmids expressing EPG-NanoLuc, differing in their promoter and backbone. We also designed control plasmids, with Renilla luciferase acting as our positive control.
EPG-TEV
Click here to see our engineering cyclesWe designed a fusion protein comprising EPG and split TEV protease, enabling the hinge-like motion of EPG to transmit a magnetic signal that activates the TEV protease. TEV protease is a widely used engineered protein capable of regulating multiple synthetic signaling pathways, including protease logic gates, transcription factors, and proto-proteins.
To validate the compatibility between split TEV and EPG, we developed a prototype expression system. This system features an EPG-TEV fusion protein with a swappable linker on one plasmid and a FRET-based TEV protease reporter on another. The reporter is composed of sfGFP linked to an EYFP mutant, REACh2, via a TEV-cleavable linker. In the absence of TEV activity, FRET occurs between the fluorescent proteins, rendering the fusion protein non-fluorescent since REACh2 itself is non-fluorescent. However, upon TEV-mediated cleavage of the linker, sfGFP is released and becomes fluorescent, which can be detected by a plate reader or photodiode.
Since the activity of the split proteins is highly dependent on the type of linker between EPG and split TEV, we have proposed a series of constructs with varying linker types. Flexible linkers promote interaction between the TEV subunits, enhancing overall activity, while rigid linkers control the distance between the split TEV proteins to reduce false-positive activity. Our future objective is to screen different linker combinations to identify those that maximize activity while minimizing noise.
Within the scope of this project, we plan to begin by screening TEV-EPG constructs using both a flexible 5-amino acid linker and a rigid 5-amino acid linker. Additionally, positive and negative controls, along with reporter plasmids, will be required for proper validation.
Thermal (Indirect repsonse to magnets)
Click here to see our engineering cyclesThe rapid vibration of small (10-20nm) magnetic nanoparticles (MNPs) under a magnetic field causes heat dissipation. By having these MNPs in the media with cells, temperature sensitive cellular pathways may be switched on/off by magnetic induction. MNPs can be attached to the cell surface via: (1) non-specific electrostatic interactions, if they are amine-functionalised; (2) biotin-streptavidin interactions, by biotin tagging membrane proteins and streptavidin coating the MNPs, (3) Histidine-Nickel interactions, by His-tagging membrane proteins and coating MNPs in NTA-Ni+ (University of Pittsburgh iGEM Team, 2020).
Many naturally occurring proteins take advantage of the conformation change caused by temperature shifts to act in a temperature-dependent manner. In particular, increases in temperature can disrupt multimerisation, which has a significant impact on transcription, as transcription factors often bind DNA as oligomers.
An example of this is TlpA in Salmonella typhimurium, which binds as a dimer to repress its targets at room temperature, but uncoils and unbinds under heat shock at 42C (Hurme et al, 1997; Piraner et al, 2016; Piraner, Wu and Shapiro, 2019). Similarly, TcI is a thermolabile mutant form of the cI protein of bacteriophage lambda, which unbinds its operon at temperatures above 37C (Valdez-Cruz et al, 2010). Both of these examples involve depression under heat shock, whereas the native yeast HSF actively upregulates its targets under heat shock by binding a cis-regulatory sequence in their promoters (Santoro, Johansson and Thiele, 1998).
These transcription factors have known consensus binding sequences, and placing these in the promoter region of a gene of interest will allow us to switch this gene on in the presence of small MNPs and an alternating magnetic field.
Mechanical (Indirect response to magnets)
Superparamagnetic nanoparticles are magnetised in the presence of magnetic fields, and thus a slowly alternating magnetic field results in the oscillation of the particles. If bound to proteins on the cell surface, these oscillations can pull/push on the proteins to alter the tension on the cell wall/membrane.
Wsc1 is a cell membrane protein in S. cerevisiae which acts as a sensor for the cell wall integrity pathway, by activating the Rom2 GEF and thus the downstream MAPK signalling cascade, regulating the expression of at least 25 genes implicated in cell wall biogenesis (Philip and Levin, 2000). An elongated form of Wsc1 was engineered by Dupres et al (2009), such that it extended out of the cell wall with a His tag that could interact with their NTA-Ni+ functionalised atomic force microscopy tips. Prof. Heinisch kindly sent us the Y1 strain expressing this form of Wsc1, which could then interact with our NTA-Ni+ magnetic nanoparticles.
Prof. Heinisch also sent us plasmid pHPS100H, as described in Straede, Corran and Heinisch (2009), which contains:
- a fusion of the bacterial LexA transcription factor and the Wsc1-activated Rlm1 transcription factor
- LacZ downstream of the LexA operator
By transforming Y1 with pHPS100H we aim to engineer a strain that expresses beta-galactosidase (and thus turns blue) after induction with Ni-NTA nanoparticles and the application of a magnetic field.
AND Gates
We also designed AND gates to combine the individual pathways within each of our model organisms in order to increase specificity of cellular targeting. When the output requires two different signals in order to activate, we can induce expression of these pathways using orthogonal magnetic waves.
Magnetic-Thermal (E. coli)
The thermal pathway controls the expression of the fluorescent protein linked with quencher whereas the magnetic pathway controls the activity of the EPG-TEV fusion protein. Only when both pathways are active does the cell fluoresce.
Therefore, we can regulate the fluorescence level of the speicific cells by controlling the direct frequency magnetic field and high frequency magnetic field.
In the future, the fluorescent protein linker with quencher can be change to any protein that can be regulated by TEV. This includes other protease, transcription factors and enzymes.
Mechanical-Thermal (S. cerevisiae)
GAL4 is a two-domain yeast transcription factor, where one domain is required to bind DNA while the other recruits the transcriptional machinery to activate expression. If each domain is placed under the control of a different pathway, GAL4-controlled genes depend on the activation of both involved pathways. For example, the GAL4 DNA binding domain (DBD) can be controlled by the Rlm1 transcription factor from the Wsc1 pathway, and activated by mechanical stimulus. The GAL4 activating domain (AD) can be controlled by the HSP26 promoter in the HSF pathway, and stimulated by an increase in temperature. When both these signals are received, they will feed into the AND gate and result in the mScarlet-I3 output.
Hardware
Please see our hardware page for details on our design process.
References
Dupres, V., Alsteens, D., Wilk, S. et al. The yeast Wsc1 cell surface sensor behaves like a nanospring in vivo. Nat Chem Biol 5, 857–862 (2009). https://doi.org/10.1038/nchembio.220
Grady C.J., Castellanos F.E.A, Schossau J., Ashbaugh R.C., Pelled G., Gilad A.A., A putative design for the electromagnetic activation of split proteins for molecular and cellular manipulation Frontiers in Bioengineering and Biotechnology 2024 12 https://doi.org/10.3389/fbioe.2024.1355915
Hurme, R., Berndt, K.D., Normark, S.J., Rhen, M., 1997. A Proteinaceous Gene Regulatory Thermometer in Salmonella. Cell 90, 55–64. https://doi.org/10.1016/S0092-8674(00)80313-X
Hwang, J.; Choi, Y.; Lee, K.; Krishnan, V.; Pelled, G.; Gilad, A.A.; Choi, J. Regulation of Electromagnetic Perceptive Gene Using Ferromagnetic Particles for the External Control of Calcium Ion Transport. Biomolecules 2020, 10, 308. https://doi.org/10.3390/biom10020308
Krishnan, V., Park, S.A., Shin, S.S. et al. Wireless control of cellular function by activation of a novel protein responsive to electromagnetic fields. Sci Rep 8, 8764 (2018). https://doi.org/10.1038/s41598-018-27087-9
Meizi Liu, Zuhui Li, Jianfeng Huang, Junjun Yan, Guoping Zhao, Yanfei Zhang, OptoLacI: optogenetically engineered lactose operon repressor LacI responsive to light instead of IPTG, Nucleic Acids Research, Volume 52, Issue 13, 22 July 2024, Pages 8003–8016, https://doi.org/10.1093/nar/gkae479
Philip, B. and Levin, D. E. (2001) ‘Wsc1 and Mid2 Are Cell Surface Sensors for Cell Wall Integrity Signaling That Act through Rom2, a Guanine Nucleotide Exchange Factor for Rho1’, Molecular and Cellular Biology, 21(1), pp. 271–280. doi: 10.1128/MCB.21.1.271-280.2001.
Piraner, D., Abedi, M., Moser, B. et al. Tunable thermal bioswitches for in vivo control of microbial therapeutics. Nat Chem Biol 13, 75–80 (2017). https://doi.org/10.1038/nchembio.2233
Piraner, D.I., Wu, Y., Shapiro, M.G., 2019. Modular Thermal Control of Protein Dimerization. ACS Synth. Biol. 8, 2256–2262. https://doi.org/10.1021/acssynbio.9b00275
Ricker B, Mitra S, Castellanos A.E., Grady C.J., Woldring D, Pelled G, Gilad A.A., 2023 Proposed three phenylalanine-motif involved in magnetoreception signalling of an Actinopterygii protein expressed in mammalian cells. Open Biol. 3: 230019 http://doi.org/10.1098/rsob.230019
Santoro, N., Johansson, N., & Thiele, D. J. (1998). Heat Shock Element Architecture Is an Important Determinant in the Temperature and Transactivation Domain Requirements for Heat Shock Transcription Factor. Molecular and Cellular Biology, 18(11), 6340–6352. https://doi.org/10.1128/MCB.18.11.6340
Straede, A., Corran, A., Bundy, J., Heinisch, J.J., 2007. The effect of tea tree oil and antifungal agents on a reporter for yeast cell integrity signalling. Yeast 24, 321–334. https://doi.org/10.1002/yea.1478
Valdez-Cruz, N.A., Caspeta, L., Pérez, N.O. et al. Production of recombinant proteins in E. coli by the heat inducible expression system based on the phage lambda pL and/or pR promoters. Microb Cell Fact 9, 18 (2010). https://doi.org/10.1186/1475-2859-9-18
Zhao, J., Nelson, T.J., Vu, Q., Truong, T., Stains, C.I., 2016. Self-Assembling NanoLuc Luciferase Fragments as Probes for Protein Aggregation in Living Cells. ACS Chem. Biol. 11, 132–138. https://doi.org/10.1021/acschembio.5b00758
https://2020.igem.org/Team:Pittsburgh/Team
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