From 2012.igem.org

Revision as of 11:44, 24 September 2012

• Home
• Team
  • The Team
  • Team Registration
• 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
  • Human Practices Home
  • 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
• Attributions
• Parts
• Notebook
  • Journal
  • Protocols

---

Last year's project: Synchronized Oscillatory System

iGEM Home

Contact Us

Our Sponsors:

Inside Modifications

Introduction

Since virus-like particles (VLPs) lack genetic content, they enclose an empty space. This space can be filled with proteins such as antibiotics, hormones, and all sorts of farmaceuticals. Modifications on the inside of the VLPs can increase binding affinity to the loaded substance. One of the modifications we pursue is adding a coil to the protein subunits at any location that is exposed on the inside of the VLP. In our case, this is a fusion to either the N-terminal or the C-terminal. The coils will facilitate strong attachment to the substance which carries the opposite coil. Another way to open up more options for loading our VLPs, is to make a change in the interior's charge. Normally, the interior of a VLP is positively charged, facilitating interaction with negatively charged DNA/RNA molecules. By changing the amino acid sequence that is located on the interior of a VLP, the positive charge can be changed into a negative charge. A VLP with a negatively charged interior can be loaded with all sorts of positively charged molecules, such as polymeres, metals, etc.

Our aims are:

Construct a Virus-Like Particle that has coils exposed on the inside.

Construct a Virus-Like Particle that has a negatively charged interior.

CCMV

The Cow Chlorotic Mottle Virus (CCMV) VLP is one of the best candidates for drug delivery, since it is tolerant of high temperatures and stable in a wide pH range. Next to that, the wild-type CCMV VLP is not specific for mammalian cells in vivo. Because the CCMV VLP is such a good candidate, we decided to place coils on the interior. Since the N-terminus of the CCMV VLP subunits is folded to the inside, this was the logic place to attach the coil to.

To get the most out of the CCMV VLP, the interior was given a negative charge. While the wild-type CCMV VLP can not be loaded with positively charged substances, this modified version can.

Results:

This attachment of a coil to the N-terminus of the CCMV VLP subunits was carried out in 3 subsequent PCR steps. Unluckily, the primer of the first step was not designed correctly, resulting in a frameshift over the entire VLP, making this construct useless. We discovered this on the 18th of September, when receiving our sequencing results.

Constructing a CCMV VLP with a negative charge was facilitated by 4 subsequent PCR steps. Luckily, this construct was finished in august and we were able to place an IPTG promoter combined with an RBS site in front of it. The next step was to produce VLPs with this new construct, which succeeded. As a proof of concept, the CCMV VLPs with a negative interior were loaded with iron ions and monitored using electron microscopy.

Figure 1: Electron Microscopy image of a CCMV VLP with negative interior.

Hepatitis B

It is known that deletions as well as modifications to the C-terminal of the Hepatitis B core protein are possible since this part of the protein is not important for VLP formation [1]. Knowing this we can assume that the fusion of a K-coil to the C-terminal will lead to the formation of a VLP with a coil presented on the inside. To obtain the maximal amount of space in the VLP capsule we decided to delete a part of the C-terminal that is normally used for DNA binding and not required for VLP folding. After looking at protein folding predictions it was decided to add a short flexible linker between coil and core protein instead. This modification was carried out in 4 PCR steps extending our construct one step at a time.

Figure 2: legend

Figure 3: primers for the 4 PCR reactions

Figure 4: PCR steps to obtain the fusion of linker and coil

Results:

After some problems with the last step of the PCR reaction a product with the correct length was obtained. Unfortunately the ligation into a vector and transformation with E. coli did not succeed yet.

References

1. Beterams, G., Böttcher B., & Nassal M. (15. Sept 2000). Packaging of up to 240 subunits of a 17 kDa nuclease into the interior of recombinant hepatitis B virus capsids. FEBS Lett., S. 169-76.