Team:Heidelberg/Project SaO
From 2009.igem.org
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:* For <span style="font-size:6mm;">output</span>, we suggest using a variety of fluorescent proteins (with non-overlapping spectra) coupled to localization tags. We were able to provide two FPs (GFP and mCherry) as well as four localization sequences (1x Endoplasmic reticulum; 1x Nucleus; 2x Plasma membrane). We show that combining our FPs with out lcalization sequences works, and thus provide future users with the possibility to visualize at least 6 different promoters simultaneously. | :* For <span style="font-size:6mm;">output</span>, we suggest using a variety of fluorescent proteins (with non-overlapping spectra) coupled to localization tags. We were able to provide two FPs (GFP and mCherry) as well as four localization sequences (1x Endoplasmic reticulum; 1x Nucleus; 2x Plasma membrane). We show that combining our FPs with out lcalization sequences works, and thus provide future users with the possibility to visualize at least 6 different promoters simultaneously. | ||
- | :* We also established methods for promoter [[Team:Heidelberg/Project_Measurement|<span style="font-size:5mm;">measurement</span>]] in eukaryotes utilizing microscopy, flow cytometry (FACS) and qRT- | + | :* We also established methods for promoter [[Team:Heidelberg/Project_Measurement|<span style="font-size:5mm;">measurement</span>]] in eukaryotes utilizing microscopy, flow cytometry (FACS) and qRT-PCR. Promoter activity was not measured by microscopy before; we established this technique in order to be able to measure protein levels in different compartments and thus make the output legible. <!--determine the activity of the promoters developed utilizing both approaches in more than one mammalian cell line, besides providing [[Team:Heidelberg/Project_Measurement#A promoter measurement kit for use in mammalian systems|measurement and normalization devices]], as well as a device that allows the integration of any Biobrick-β (BBb)-compatible block in a plasmid and its transfection into eukaryotic cells in a working form. All together, a standard method to characterize any promoter using such parts was established and [[Team:Heidelberg/Project_Measurement#Two units for promoter activity in mammalian cells|units]] describing promoter strength (as measured using these methods) were also defined.--> |
:* <!--As a further attempt, we tried to establish a [[Team:Heidelberg/stables|cell line]] that overcomes the variability in measurements caused by drawback of transient transfection by allowing the stable integration of inserts at a predetermined integration site in the genome of the cell line in use. Such an approach helps eliminating epigenetic variability in gene expression control. Although, this part was never realized in its final form, we are proud to have introduced the value of such a concept to the emerging field of eukaryotic promoter research and our own experimental observations have further strengthened our belief in the need of such a cell line in the future.--> Not only to overcome challenges for promoter measurement, but also to lay the foundations for the proposed drug-screening assay as well as any other synthetic biology idea in mammalian cells, we emphasize the importance of [[Team:Heidelberg/stables|stable cell line creation]]. | :* <!--As a further attempt, we tried to establish a [[Team:Heidelberg/stables|cell line]] that overcomes the variability in measurements caused by drawback of transient transfection by allowing the stable integration of inserts at a predetermined integration site in the genome of the cell line in use. Such an approach helps eliminating epigenetic variability in gene expression control. Although, this part was never realized in its final form, we are proud to have introduced the value of such a concept to the emerging field of eukaryotic promoter research and our own experimental observations have further strengthened our belief in the need of such a cell line in the future.--> Not only to overcome challenges for promoter measurement, but also to lay the foundations for the proposed drug-screening assay as well as any other synthetic biology idea in mammalian cells, we emphasize the importance of [[Team:Heidelberg/stables|stable cell line creation]]. |
Revision as of 08:35, 20 October 2009
Outlook and summary
References
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