From 2012.igem.org

Revision as of 12:01, 17 September 2012

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  • The Team
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• Project
  • 1. Introduction
  • 2. PnA System
  • 3. Virus-Like Particles
    • CCMV
    • HepB
    • Polerovirus Natural BioBrick
  • 4. Outside Modification
  • 5. Inside Modification
  • 6. Detection of VLPs
  • 7. Applications
  • 8. The Constructor
  • 9. Final Overview
• Human Practices
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  • 1. Mini Symposium
  • 2. Visiting Secondary Schools
  • 3. Discovery Festival
    • Communication Science
  • 4. Stakeholders
  • 5. Munich CAS Conference
• Safety
  • Introduction
  • 1. General safety
  • 2. Virus-related safety
  • 3. Regulations
  • 4. Safety suggestions
  • 5. Safety of applications
• Modeling
  • Human Body Model
  • Tertiary folding prediction
  • VLP assembly model
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Detection of VLPs

The most challenging piece of science is the detection. When producing a new VLP you don't know if it forms. So we put a lot of effort in it, to detect and visualize the Virus-Like-Particles

Electron Microscopy (EM)

One of the direct methods to detect our VLPs is with Electron Microscopy or EM. We prepared and viewed multiple samples, this all was done in the Virology department of the Wageningen UR.

Media:EM vs LM

Electron microscopy works similar as light microscopy, but instead of visible light being used to illuminate the sample, electrons are being used. The same as light microscopy, electron microscopy uses multiple lenses to focus the beam so that the sample is properly lighted. The only differences with lenses are that the lenses of electron microscopy are electromagnetic instead of glass. EM has also a much greater resolution than conventional light microscopy. It is theoretically possible of around 0.005 nm, but in practice it is around 1-2 nm, this is because the lenses has certain errors, the operator (us) is inexperienced and the sample must be as this as possible. All these factors reduces 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.

EM Course

We received personally a course about the EM from Jan van Lent at the Virology department of the WageningenUR. In this course he explained how the EM works, how you prepare the samples and how to operate the EM.

The goal of the course wa to get familiar with the EM and how to handle it. First there was a small presentation about how the EM works and how to prepare the samples. Next he showed us one time how to prepare the sample afterwards we can prepare 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. The places where there is no coating (VLPs), electron wave can go through unhindered, while the places where there are coating, electron waves break apart. This result in a picture where you can see the particles.

After the preperation of the samples we were shown where the EM is and how to operate it. Similar to the sample presentation, he showed us first one sample and afterwards we could try it ourselfs.

(SLIDE SHOW EM PICTURES)

At the end of the day we were able to prepare samples and visulize them in the EM.

Dynamic Light Scattering (DLS)

Another method to detect virus-like-particles is with dynamic light scattering, 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.

Media:(PICTURE DLS OVERVIEW)

When light travels through the sample and hits a particle, light will scatter in all directions. The dynamic light scattering machine detect 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.

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