Ljubljana/finalsystem
From 2007.igem.org
Performance of the Final Functional Systems
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Subtytle
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System based on the activation by HIV protease activity
he process of system activation was observed using fluorescent confocal microscopy. Cells were transfected with CMV–CD4–HIVprotease cleavage site–mCherry–NLS and optionally cotransfected with HIV protease on pNL4.3 plasmid. In cells without HIV protease fluorescence was detected at the plasma membrane (Fig. 8A), whereas in HIV protease cotransfected cells, fluorescent protein mCherry was released from the membrane and translocated to the nucleus (Fig. 8B). Similar results were obtained when mCherry was substituted with T7 RNA polymerase and additional construct pT7–mCerulean was transfected to detect the T7 RNA polymerase activity. In cells with HIV protease cotransfection, mCerulean was detected in cytosol (Fig. 9B), but no fluorescence at all was observed in the control experiment without of the HIV protease activity (Fig. 9A).
The system, using myristoylation signal instead of CD4 to anchor the T7 RNA polymerase to the membrane was also tested and we obtained similar results (data not shown).
We can conclude that in the presence of HIV protease, T7 RNA polymerase is released from the membrane and translocates to the nuceleus where it transcribes genes under the T7 promoter.
Fig. 8. HIV protease cleaves the linker between the membrane anchor and mCherry. HEK293T cells were transfected with CMV-CD4-HIVprotease cleavage site-mCherry-NLS (A) and additionally with HIV protease plasmid pNL4.3 (B). In cells without HIV protease, membrane localization of mCherry reporter protein was observed (A), but in the presence of HIV protease mCherry-NLS was released to cytosol and translocated to nucleus (B).
Fig. 9. T7 RNA polymerase is activated by the cleavage of the linker on the membrane anchor by HIV protease. HEK293T cells were transfected with CMV-CD4-HIVprotease cleavage site-T7 RNA polymerase-NLS, reporter plasmid T7-mCerulean (A) and additionally with HIV protease plasmid pNL4.3 (B). HIV protease cut off T7 RNA polymerase from the membrane, thus activating it. Consequently mCerulean under the T7 promoter was transcribed and detected in cytosol (B). In the absence of protease the system remained inactive and no fluorescence was observed (A).
