Team:Heidelberg/Project SaO
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:-Furhtermore, we have been able to generate a [[Team:Heidelberg/Project_Synthetic_promoters#Generation of a library of constitutive promoters|library of constitutive promoters]] of varying strengths as well as a [[Team:Heidelberg/Project_Synthetic_promoters#Generation and screening of a library of promoters putatively responsive to NF-κB|library of NFκB-responsive promoters]]. | :-Furhtermore, we have been able to generate a [[Team:Heidelberg/Project_Synthetic_promoters#Generation of a library of constitutive promoters|library of constitutive promoters]] of varying strengths as well as a [[Team:Heidelberg/Project_Synthetic_promoters#Generation and screening of a library of promoters putatively responsive to NF-κB|library of NFκB-responsive promoters]]. | ||
:-We also established [[Team:Heidelberg/Project_Measurement|methods for promoter characterization in eukaryotes]] utilizing microscopy, flow cytometry (FACS) and qRT-PCR to reproducibly 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. | :-We also established [[Team:Heidelberg/Project_Measurement|methods for promoter characterization in eukaryotes]] utilizing microscopy, flow cytometry (FACS) and qRT-PCR to reproducibly 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 | + | :-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. |
- | :-Besides the previous, we were able to provide | + | :-Besides the previous, we were able to provide two FPs (''GFP'' and ''mCherry'') as well as four localization sequences ('''''1x''''' ''Endoplasmic reticulum''; '''''1x''''' ''Nucleus''; '''''2x''''' ''Plasma membrane'') '''(link to them in Eukaryopedia)''', all in BBb format and proved that they could be used when fused together based on the protein fusion principle exploited in BBb format providing future users with the possibility to visualize at least 6 different promoters simultaneously. |
:At the end, we are proud to say that we have introduced many of the concepts, methods and tools that could serve as the basis for all other attempts in the study of eukaryotic gene regulatory systems. Not only have we allowed the chance for many of the researchers in many biological and medical fields to enhance the selectivity of their promoters, but also helped develop the devices necessary for further characterization with the technologies available for those working in the life- and biosciences today. Not neglecting the need for further improvement, with such a collection of tools available the ideas of selective protein and gene therapy, metabolic engineering, stem cell manipulation and better intracellular network modeling do not seem too far away. | :At the end, we are proud to say that we have introduced many of the concepts, methods and tools that could serve as the basis for all other attempts in the study of eukaryotic gene regulatory systems. Not only have we allowed the chance for many of the researchers in many biological and medical fields to enhance the selectivity of their promoters, but also helped develop the devices necessary for further characterization with the technologies available for those working in the life- and biosciences today. Not neglecting the need for further improvement, with such a collection of tools available the ideas of selective protein and gene therapy, metabolic engineering, stem cell manipulation and better intracellular network modeling do not seem too far away. |
Revision as of 13:17, 19 October 2009
Outlook and summary
References
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