Part:BBa_K5154001
EPG ( Electromagnetic Perceptive Gene)
EPG is a promising protein identified from Kryptopterus bicirrhis by Prof. Assaf A. Gilad's research group (Krishnan et al,2018). It has been named Electromagnetic Perceptive Gene (EPG), and demonstrated exceptional in vivo responsiveness to magnetic fields. This protein can generate action potentials when exposed to external magnetic fields.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 282
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 282
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 332
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 282
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 282
- 1000COMPATIBLE WITH RFC[1000]
Introduction
Many animals possess the ability to detect magnetism for navigation purposes. The Kryptopterus bicirrhis, commonly known as the glass catfish, is one such organism that responds accurately to external magnetic fields. For effective navigation under Earth's magnetic field, which ranges between 20 and 65 microteslas [1], the magnetic field-sensing protein must be highly sensitive, making it a strong candidate for magnetogenetic applications. A promising protein has been identified from K.bicirrhis, by Prof. Assaf A. Gilad's research group [2]. It has been named Electromagnetic Perceptive Gene (EPG), and demonstrated exceptional in vivo responsiveness to magnetic fields. This protein can generate action potentials when exposed to external magnetic fields.
Figure 1 | Magnetic stimulation of EPG in HEK293T cells induced significant increases in [Ca2+]. Magnetic stimulation was applied between 10s and 20s. Data showing kinetics of fluorescence intensity response for individual cells under different conditions. Decreased fluorescence intensity changes were observed in 0 Ca2+ and 0 Ca2+ + EGTA after magnetic stimulation. No increase in signal was observed for control (non-transfected) and thapsigargin treated cells [2].
Structure
Figure 2 | Structure of EPG predicted by Alphafold2. Clear three finger domain structure typically found in Ly6/uPAR protein family.
Presence of flexible N-terminal and C-terminal residues originally allows the EPG to be anchored on the cell surface membrane and interact with various membrane receptors, providing it with magnetism sensing capabilities [3]. The core of EPG consists of a rigid “Head” and three flexible “Fingers”. The head is connected up by internal disulfide bond therefore maintaining its structural integrity, while the fingers are significantly more flexible and was suspected to be the main contributor of magnetosensitivity of EPG [3].
Figure 3 | The core of EPG consists of a rigid “Head” and three flexible “Fingers”.
References
1. Finlay, Christopher Charles, et al. "International geomagnetic reference field: the eleventh generation." Geophysical Journal International 183.3 (2010): 1216-1230.
2. Krishnan, Vijai, et al. "Wireless control of cellular function by activation of a novel protein responsive to electromagnetic fields." Scientific reports 8.1 (2018): 8764.
3. Ricker, Brianna, et al. "Proposed three-phenylalanine motif involved in magnetoreception signalling of an Actinopterygii protein expressed in mammalian cells." Open Biology 13.11 (2023): 230019.
EPG-Nanoluc test
Design
In order to characterise EPG, it is necessary to first test EPG on a reporter system that allow us to monitor the status of EPG under different magnetic conditions. The reporter genes should have characteristics including: being able to function as a split protein system, fast response time, signal amplification and reversibility. We have looked at various potential systems to compare their feasibility, benefits and drawbacks:
Figure 1 | Consideration on potential reporter system for EPG
Eventually we chose to first test the system on the reporter protein - Nanoluc. NanoLuc is a luciferase mutated from renilla luciferase, developed by Promega, with enhanced brightness (150 fold compared to native renilla luciferase) and enhanced stability. We planned to fuse the N-term Nluc fragment (1-65) to the N terminal of EPG, and C-term Nluc fragment (66-171) to C terminal of EPG. We took reference to this paper [1] which suggest adding a padding linker between Nluc and EPG which provide flexibility, preventing false positive signal when EPG is not active. We retrieved the sequence and split site from [2] this paper, and fused to the N - term, C - term of EPG.
Figure 2 | EPG Nanoluc protein, Mode of Action
Optimisation of linker design was not done in this step, as it is only meant to be a demonstrative and characterisation step of EPG
Construction
Plasmid Construction
We have designed two different plasmids for expressing EPG-NLuc Fusion protein, having different promoters, therefore can provide convenience in protein expression and characterization. We chose to assemble the plasmid from scratch using the existing FreeGene distribution containing the required promoter, RBS, Terminator and backbones, and also secondary level assembly parts.
Figure 3 | EPG-Nanoluc plasmid construction
We have successfully constructed the listed plasmids, and they have been sequence verified.
Figure 4 | Gel electrophoresis results of constructed plasmids
Figure 5 | Sequencing result for constructed plasmid
Expression verification
SDS-PAGE is used for protein expression verification
Figure 6 | SDS-PAGE result
Characterisation: test with plate reader
In order to verify the activity of EPG-NLuc construct, we proposed an initial protocol for measuring luminescence signal from the plate reader kindly lended by BMG labtech.
The EPG will be activated by a specially made magnetic plate device, allowing accurately control the time of activation, strength of activation and environmental control.
Knowing from prior literature review, the scale of reaction of EPG should be ~ second range. Therefore, we decide to prioritise the measurement speed to get an more accurate result.
The are innate drawback of this method, that it cannot measure the luminescence activity during the magnetic activation, which limits our ability to accurately measure the exact response profile. Instead, we are going to use modelling to model the decay of the activation, therefore determine the peak activation of EPG.
Figure 7 | Result for EPG-Nanoluc activated by external magnetic field. Data have been collected through plate reader. Non adjusted graph has been zeroed by blank with test solution plus LB. Adjusted graph has normalised the curve using the peak value detected before the activation. The box chart illustrates the datapoint right after the measurement, compared to the maximum value.
Other limitations have also been identified, including the delay in measurement, zeroing issue, luminescence cross talk. But the greatest one is still the inability to measure and activate at the same time. In order to overcome such limitation, we have designed another procedure to characterise EPG-Nluc construct with higher precision and better temporal resolution.
Characterisation: test with custom hardware
A luminometer embedded Electromagnet have been designed to carry out the test. The luminometer consists of a sample carrier, a photodiode, a gain controller and a signal processing unit. It has the capability to apply different magnetic field waveforms, at a resolution of 0.4% max magnetism, the highest magnetic field can reach 400mT.
Eventually, the device can measure luminescent signal, but it turns out to be not sensitive enough to detect our EPG signal, since the NLuc is still not bright enough to be detectable. Therefore this approach have been unsuccessful.
We have proposed two possible solutions to test EPG under similar setup, but with a different readout system：
- Fluorescent reporter - see EPG-TEV
- More sensitive Read out system - SiPAD
Conclusion
We have successfully done preliminary characterisation of EPG-NanoLuc construct, and retrieved data on how EPG behaves under different activation conditions.
References
1. Grady, Connor J., et al. "A putative design for the electromagnetic activation of split proteins for molecular and cellular manipulation." Frontiers in Bioengineering and Biotechnology 12 (2024): 1355915
2. Zhao, Jia, et al. "Self-assembling NanoLuc luciferase fragments as probes for protein aggregation in living cells." ACS chemical biology 11.1 (2016): 132-138.
TEV-EPG testing
Design
<p>As described in the description, EPG is capable to generate action potential through calcium influx in higher animals. However, it is impossible to replicate the same mode of action in lower eukaryotes and prokaryotes. In order to overcome this issue, a separate, independent pathway needs to be constructed.
We have two options to relay the signal from EPG, either through transcription level control or through protein level control. For transcription level control, it is necessary to engineer transcription factor, allowing it to be regulated by EPG, then control the target gene expression. This setup provides flexibility, and we have proposed a design fusing EPG with LacI adapting the design from [1]. However, We discovered that the insertion site for lacI is difficult to predict, and the position of linker, type of linker will have a profound effect on the activity of protein. Considering the timescale of the project, we decide to give up this approach but switch to the protein level control.
Figure 1 | EPG-TEV mode of action
We chose TEV as the actuator.
TEV is a protease found in Tobacco Etch Virus, has high specificity, and has been shown to retain high activity even when splitted into two parts.
We retrieved sequence of TEV, and constructed fusion protein by placing linker between N-TEV , EPG and C-TEV.
Reference
1. Liu, Meizi, et al. "OptoLacI: optogenetically engineered lactose operon repressor LacI responsive to light instead of IPTG." Nucleic Acids Research (2024): gkae479.
| biology | Kryptopterus bicirrhis |