Team:Freiburg Transfection

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Figure 1_Transfection shows the configuration of the construct. Lipocalin, the fluorescein binding Anticalin, exhibits the extracellular part of the construct. The transmembrane region is appropriate to that of the EGF-receptor erbb1. Split-beta-Lactamase the intracellular part is labeled to the yellow fluorescent protein to detect membrane localization.<br>
Figure 1_Transfection shows the configuration of the construct. Lipocalin, the fluorescein binding Anticalin, exhibits the extracellular part of the construct. The transmembrane region is appropriate to that of the EGF-receptor erbb1. Split-beta-Lactamase the intracellular part is labeled to the yellow fluorescent protein to detect membrane localization.<br>
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'''Figure 1_Transfection'''
'''Figure 1_Transfection'''
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Revision as of 14:49, 28 October 2008


Freiburg2008 small header.gif



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_Transfection


1) Localisation at the cell membrane
To show the localisation of the constructs at the cell membrane transfection of the construct signalpeptide-Lipocalin-transmembraneregion-betaLactamase1-YFP was performed.
Figure 1_Transfection shows the configuration of the construct. Lipocalin, the fluorescein binding Anticalin, exhibits the extracellular part of the construct. The transmembrane region is appropriate to that of the EGF-receptor erbb1. Split-beta-Lactamase the intracellular part is labeled to the yellow fluorescent protein to detect membrane localization.

Freiburg2008 Lipo bla1+YFP.jpg
Figure 1_Transfection

Membranelocalisation of the construct signalpeptide-Lipocalin-transmembraneregion-betaLactamase1-YFP is visible in transfected 293T cells (Figure 2_Transfection). The fluorescence of the cells is most likely restricted to the cellmembrane which confirms the assembly of the construct in the cell membrane.
In Comparison wuth wirds deutlicher
Freiburg2008 SP LIPO GGGS TM bla1 YFP 1.jpg
Figure 2_Transfection

Freiburg2008 TV CMV YFP CFP loeslich.jpg
Figure 3_Transfection


Freiburg2008 Lipo+Split CFP.jpg Freiburg2008 Lipo+Split YFP.jpg





Freiburg2008 SP LIPO GGGS TM bla1 YFP 2.jpg



METHODS
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Transfection of 293T cells
One day before transfection cells were counted in the Neubauer chamber and 6*10^4 cells/cm² were seeded in 6 well plates. Approximately 1 hour before transfection cells were washed with 1xPBS and fresh DMEM medium was added. For transfection 2µg of DNA were mixed with 25µl CaCl2 and ddH2O was filled up to 250µl. After an incubation on ice for 20 min 250µl BBS (2x) were added. This mixture was given to the cells and after 4-12 hours cells were washed and fresh medium was added.

Freiburg08 FT3.png