From 2012.igem.org

Revision as of 07:13, 24 September 2012

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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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• Safety
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  • 1. General safety
  • 2. Virus-related safety
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  • 5. Safety of applications
• Modeling
  • Human Body Model
  • Tertiary folding prediction
  • VLP assembly model
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Last year's project: Synchronized Oscillatory System

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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 straight forward method to detect VLPs is Electron Microscopy (EM). We prepared and investigated multiple samples ourselves. This work was done at the Virology department of the Wageningen University.

Electron microscopy versus Light Microscopy

Electron microscopy works similar as light microscopy, but instead of visible light being used to illuminate the sample, it is done by electrons. The EM has multiple lenses to focus the beam to magnify and light the sample. The lenses are electromagnetic instead of glass and therefore allow a much higher resolution than conventional light microscopy. In theory it is possible to reach a resolution of around 0.005 nm, but in practice it is mostly around 1-2 nm. Possible reasons are certain errors of the lenses, inexperience of the operator (for example our teammembers) or the sample is not thin enough. All these factors can reduce the resolution of the electron microscope. We investigated various samples obtained in our experiments with the help of the EM. The wild types of the Cowpea chlorotic mottle virus (CCMV) and Hepatitis B (HepB) were detected. Multiple variations of CCMV have been tested too, unfortunately with mixed results. [add pictures]

</p>

EM Course

The goal of the course was to get familiar with the EM and to handle it safely. Dr. Jan van Lent of the Virology department of Wageningen UR gave us an introduction to EM with a small presentation. During this instruction he explained detailed the electron miscroscope itself, the preparation of good samples and how to operate the EM properly. Afterwards he showed the sample preparation in the wetlab. Preparation of the sample is critical to have a good resolution, during the course we learned how to do it. The sample must be thin, between 2 - 300nm, and stable in the electron microscope. This can be done by drying or cryo-freezing the sample. After drying or freezing, the sample is stained with a coating containing a heavy metal salt. This coating reflects the electron waves of the microscope, whereas the spots with the VLPs have no coating at all. The missing coating allows the electron waves to go through unhindered. The information of reflection and permeability is processed to an full image which shows the VLPs.

After the preparation of the samples Dr. van Lent explained the whole procedures of visualising a sample. After this last introduction we were able to analyse and investigate multiple samples ourselves.

The EM

Learning how to prepare the samples

A copper grid on which the sample has to be fixated

Preparing a grid with Virus-Like Particles

The copper grids are very delicate, therefore they should be handled with great care

After the VLPs have been applied to the grid, they have to be fixed with a heavy metal salt solution.

Jan van Lent explaining how to operate the EM itself

Operating the EM ourselves.

The result, a screen full of VLPs

At the end of the day we were able to use EM for our own experiments starting with sample preparation, handling of the microscope and proper visualization. After the course, the attending team members, are allowed and trained to use the EM without supervision.

Dynamic Light Scattering (DLS)

Another method to detect VLPs is 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 an indirect way to detect particles. We applied this technique as a complimentary method next to electron microscopy.

When light travels through the sample and hits a particle, it will scatter in all directions. The DLS machine detects 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 its 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 investigated various samples with the help of DLS. The main issue was that the sample was not pure or not concentrated enough. After optimization of 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. </p>

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