Part:BBa_K5154002

EPG - Nanoluc

EPG - split Nanoluciferase fusion protein.

NanoLuc is a luciferase mutated from renilla luciferase, developed by Promega, with enhanced brightness (150 fold compared to native renilla luciferase) and enhanced stability. It is split into two parts and connected to EPG and act as a reporter for the EPG activation under the external magnetic field.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 487
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 487
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 537
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 487
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 487
  Illegal NgoMIV site found at 47
  
• 1000
  COMPATIBLE WITH RFC[1000]

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.