Team:Freiburg Transfection and Synthetic Receptor
From 2008.igem.org
(Difference between revisions)
Line 33: | Line 33: | ||
<h4>'''Localization at the cell membrane'''</h4> | <h4>'''Localization at the cell membrane'''</h4> | ||
To show the localization of the constructs at the cell membrane transfection of the construct signalpeptide-Lipocalin-transmembraneregion-betaLactamase1-YFP was performed.<br> | To show the localization of the constructs at the cell membrane transfection of the construct signalpeptide-Lipocalin-transmembraneregion-betaLactamase1-YFP was performed.<br> | ||
- | Figure 2_Transfection shows the model of the protein structure based on PDB files. Anti-fluorescein Anticalin is the extracellular part of the construct. The transmembrane region is identical to that of the EGF-receptor erbb1 (BBa_K157002). Split-beta-Lactamase ( | + | Figure 2_Transfection shows the model of the protein structure based on PDB files. Anti-fluorescein Anticalin is the extracellular part of the construct. The transmembrane region is identical to that of the EGF-receptor erbb1 (BBa_K157002). Split-beta-Lactamase (BBa_I757011), the intracellular part is labeled to the yellow fluorescent protein to detect membrane localization.<br> |
<br> | <br> | ||
[[Image:Freiburg2008_Lipo_bla1+YFP.jpg|500px]] | [[Image:Freiburg2008_Lipo_bla1+YFP.jpg|500px]] | ||
Line 41: | Line 41: | ||
Membranelocalization of the construct signalpeptide-Lipocalin-transmembraneregion-betaLactamase1-YFP is visible in transfected 293T cells (Figure 3_Transfection). The fluorescence of the cells is most likely restricted to the cellmembrane as evident by the strong signal at the surface of the cell spheres confirming the assembly of the construct in the cytoplasmamembrane.<br> | Membranelocalization of the construct signalpeptide-Lipocalin-transmembraneregion-betaLactamase1-YFP is visible in transfected 293T cells (Figure 3_Transfection). The fluorescence of the cells is most likely restricted to the cellmembrane as evident by the strong signal at the surface of the cell spheres confirming the assembly of the construct in the cytoplasmamembrane.<br> | ||
In comparison, 293T cells transfected with the construct transfectionvector-CMV-YFP show a uniformly distributed fluorescence all-over the cell (Figure 4_Transfection A and B).<br> | In comparison, 293T cells transfected with the construct transfectionvector-CMV-YFP show a uniformly distributed fluorescence all-over the cell (Figure 4_Transfection A and B).<br> | ||
- | Transfection with the construct transfectionvector-CMV-CFP, i.e. membrane targeting as well results in completely fluorescent cells (Figure 4_Transfection C and D).<br> | + | Transfection with the construct transfectionvector-CMV-CFP, i.e. without membrane targeting as well results in completely fluorescent cells (Figure 4_Transfection C and D).<br> |
<br> | <br> | ||
[[Image:Freiburg2008_SP_LIPO_GGGS_TM_bla1_YFP_1.jpg|710px]]<br> | [[Image:Freiburg2008_SP_LIPO_GGGS_TM_bla1_YFP_1.jpg|710px]]<br> |
Revision as of 15:46, 29 October 2008