From 2012.igem.org

Revision as of 09:14, 12 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

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Last year's project: Synchronized Oscillatory System

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week 12: 16 july - 22 july

Office work

Lab work

Biobrick BBa_J04450 was cloned and miniprepped.

Hepatitis B

Tuesday:

Digestion of

• Hepatitis B PCR product (with pre- and suffix) with Spe1 and Pst1
• BBa_J04500 (an IPTG inducible promoter with RBS) with Xba1 and Pst1
• BBa_PSB1K3.ml (linearized plasmid backbone) with EcoR1 and Pst1

PCR purification on the digest as a replacement for the heat inactivation

Wednesday:

Ligation of

• Hepatitis B PCR product with BBa_J04500
• the Hepatitis B PCR product with BBa_PSB1K3.ml

The ligations where done in duplo – once following the standard iGEM protocol and once using equimolar amounts of Hep B and the vector (DNA concentrations where measured by using NanoDrop)

Electrotransformation of the 2 different constructs with DH5α

• Grow transformed E.coli on plates containing kanamycin
• A plasmid with a GFP coding device was used as a positive control (growing on a plate containing ampicillin)

Thursday:

Colony PCR

• of 20 colonies transformed with the Hep B + BBa_J04500 construct
• of 10 colonies transformed with the Hep B + BBa_PSB1K3.ml construct

out of 20 samples taken from the transformation with the HepB + BBa_J04500 construct – 7 turned out to have the right insert. The colonies transformed with the Hep B + BBa_PSB1K3.ml construct did not have any Hep B inserts

TuYV

• Mach1 electrocompetent E. coli cells were transformed with last week's ligation mixtures. Results were unsatisfying.

• 'Coat Protein + readthrough' gene amplicons were digested, ligated into pSB1K3 backbone and transformed into our electrocompetent Mach1 E. coli cells. After two attempts and a colony PCR, results were still unsatisfying.

PLRV

After a two week quest for PLRV infected plant material, we obtained infected potato leafs from the Dutch General Inspection Service for Agricultural seed and seed potatoes.

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