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Team:Wageningen UR/MethodsDetection
From 2012.igem.org
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Detection of VLPs
A key part of our project is the detection of VLPs. We need sufficient visualization to get conclusive evidence of VLP formation. Besides, we will investigate alternative methods to detect the formation and stability of Virus-Like Particles.
Electron Microscopy (EM)
The most direct method to detect VLPs is with Electron Microscopy (EM). We prepared and investigated multiple samples. This was done at the Virology department of the Wageningen UR.
Electron microscopy works similar as light microscopy, but instead of visible light being used to illuminate the sample, it is done by electrons. Similarly to light microscopy, electron microscopy uses multiple lenses to focus the beam so that the sample is properly lighted and magnified. The only differences with lenses are that the lenses of electron microscopy are electromagnetic instead of glass. EM has also a much higher resolution than conventional light microscopy. It is theoretically possible to reach a resolution of around 0.005 nm, but in practice it is around 1-2 nm, because the lenses have certain errors, the operator (us) is inexperienced and the sample must be as thin as possible. All these factors reduce the resolution of the electron microscope.
We checked various samples in the electron microscope. The wild types of CCMV and HepB were detected. Multiple variations of CCMV were also tested with mixed results. [add pictures]
EM Course
We were instructed about the use of the EM by Jan van Lent at the Virology department of Wageningen UR. During the course he explained how the EM works, how you prepare the samples and how to operate the EM.
The goal of the course was to get familiar with the EM and to handle it safely. First there was a small presentation about how the EM works and how to prepare the samples. Next he showed us how to prepare a sample in practical after which we prepared our own samples.
Preparation of the sample is critical to have a good resolution, during the course we learned how to do it properly. The sample must be thin, between 2 - 300nm, and must be stable in the electron microscope. This can be done by drying or cryo-freezing the sample. After drying or freezing we stain the sample with a coating containing a heavy metal salt. The places where there is no coating (VLPs), electron wave can go through unhindered, while the places where there is coating, electron waves break apart. This result in an image which shows the VLPs.
After the preparation of the samples we were shown where the EM is and how to operate it. Again, he showed the procedures visualising one sample after which we were able to analyse the rest of the samples ourselves.
(SLIDE SHOW EM PICTURES)
At the end of the day we were able to prepare samples and visualize them in the EM. After the course, the attending team members were allowed and trained to use the EM without supervision.
Dynamic Light Scattering (DLS)
Another method to detect virus-like-particles is with dynamic light scattering (DLS), also known as photon correlation spectroscopy or quasi-elastic light scattering. This technique is used to measure the size of particles and is thus an indirect way to detect particles. We used this technique as a complimentary method next to the electron microscopy.
When light travels through the sample and hits a particle, light will scatter in all directions. The dynamic light scattering machine detects the scattered light under a fixed angle. The particles in the solution will move randomly due to Brownian motion. As the particles move, the intensity will change. This is because the scattered light can undergo constructive or deconstructive interference with the scattered light of other particles. The larger the particle, the more slower it is in Brownian motion, the slower the change of intensity is. The change in intensity over time relates to the diffusion coefficient and this diffusion coefficient is related to the particle radius using the Stokes-Einstein equation.
We tested multiple samples with DLS. The main issue was that the sample was not pure enough or not concentrated enough. After optimizing the production and purification protocol we succeeded to detect VLPs. The main improvement was the implementation of FPLC, which filters out all aggregates and subunits, yielding pure VLP solutions.
Sources:
EM
- Jan van Lent
- Quantitative characterization of virus-like particles by asymmetrical flow field flow fractionation, electrospray differential mobility analysis, and transmission electron microscopy - Leonard F. Pease, Daniel I. Lipin, De-Hao Tsai, Michael R. Zachariah, Linda H.L. Lua, Michael J. Tarlov, Anton P.J. Middelberg
DLS
- Remco Fokkink
- Light Scattering from Polymer Solutions and Nanoparticle Dispersions - Wolfgang Schartl
- Nanoparticle-Templated Assembly of Viral Protein Cages - Chao Chen, Marie-Christine Daniel, Zachary T. Quinkert, Mrinmoy De, Barry Stein, Valorie D. Bowman, Paul R. Chipman, Vincent M. Rotello, C. Cheng Kao, and Bogdan Dragnea
