Team:Freiburg/Project/pRIG15 17

• Home
• Project
• Team
• Results
• Human Practice
• Notebook

• Introduction
• Overview
• Diagnostics today
• The DiaCHIP
  • DNA Engineering
  • Cell-Free Expression
  • Protein Purification
  • Surface Chemistry
  • Optical Detection
• Outlook

• Members
• Acknowledgements
• Partners

• The DiaCHIP
  • Main Results
  • Diagnostics
  • Our Device
  • DNA Array
  • Cell-Free Expression
  • Binding on Surface
  • Modeling
• BioBricks
• Collaborations
• Interlab Study
• Achievements

• Science Fair
• Theater Project
• Public Opinion
• College Talk
• Ethics

• Labjournals
• Techniques
  • Gibson
  • E.coli Strains
• Protocols
• Materials
• Safety
• Wiki-Tricks
• Glossary

• Home
• Project
• Results
• Team
• Practice
• Notebook

Menu

<< Back

pRIG15_17

The Human Immunodeficiency Virus (HIV) causes acquired immunodeficiency syndrome, AIDS, which leads to major impairments of the immunsystem. ¹⁾ For our experiments we used a sequence coding for a recombinant protein that contains six epitopes from different proteins of the HI Virus (HIV-1-trans-activating (tat) encoding region, one epitope of the reverse transcriptase, one of the p24 protein, one of the envelope protein gp41, one of gp120).²⁾

Figure 1: pRIG15_17. BBa_K1621004 inserted into the submission backbone pSB1C3.

To insert the sequence for HIV tat/pol/gag/env into pSB1C3 we designed Gibson primers with compatible overhangs that also included the start codon ATG. This fragment was amplified via PCR and then assembled with the digested pSB1C3 backbone using Gibson assembly. To prove correct insertion of our fragment we did a test digest and sent the whole plasmid for sequencing.

We inserted the sequence coding for HIV tat/pol/gag/env into pET_22b+ for overexpression in E.coli. We could show interaction of the HIV tat/pol/gag/env antigen with a polyclonal anti-HIV-1 p24 antibody (figure 2).

Figure 2: Western Blot of HIV multi-epitopic antigen. HIV multi-epitopic antigen was analyzed by 12.5% SDS-PAGE. The anti-HIV-1 p24 polyclonal antibody was used in a dilution of 1:5000. The secondary antibody (anti-rabbit HRP) was diluted 1:5000. The expected molecular weigth is 20.5 kDa.

Link to GeneBank file: BBa_K1621004.gb.
Link to Registry: BBa_K1621004

¹⁾ Douek et al. 2009. Emerging concepts in the immunopathogenesis of AIDS. Annual review of medicine

²⁾ Rahimi et al. 2015. Optimization of multi-epitopic HIV-1 recombinant protein expression in prokaryote system and conjugation to mouse DEC-205 monoclonal antibody: implication for in-vivo targeted delivery of dendritic cells. Iranian journal of basic medical sciences