Team:Heidelberg/Project SaO
From 2009.igem.org
(Difference between revisions)
(→) |
(→) |
||
Line 11: | Line 11: | ||
__NOTOC__ | __NOTOC__ | ||
+ | =='''Outlook and summary'''== | ||
+ | |||
:<span style="font-size:10mm;">T</span>he emergence of interest in manipulatable eukaryotic systems has posed much pressure on the development of methods to help understand and characterise eukaryotic gene regulation. Those methods go beyond the already rather sophisticated methodology still being established in prokaryotes to investigate and thereafter engineer these cells as needed.[[Team:Heidelberg/Project_SaO#References|[1]]]. For one thing, the design of promoters exclusively responsive to one transcription factor (TF) within eukaryotic cells could certainly help improve our understanding of the key components of one pathway or the other, while eliminating the cross-talk often observed with many naturally occurring promoters. Such promoters have often posed a challenge to researchers studying signal transduction in eukaryotic systems because of the different types of TF a single regulatory element can bind, and a single TF having multiple target regulatory regions[[Team:Heidelberg/Project_SaO#References|[2]]]. With the emergence of systemized research and attempts for modelling biological systems, the availability of data with minimal experimental variability and highly accurate experimental conditions has also contributed to the need for such finely-tuned promoters. Once such exclusive promoters could be available and methods for their characterization established, it is not so hard to imagine the revolutionary effect they could have on eukaryotic research. Some of many applications could be: | :<span style="font-size:10mm;">T</span>he emergence of interest in manipulatable eukaryotic systems has posed much pressure on the development of methods to help understand and characterise eukaryotic gene regulation. Those methods go beyond the already rather sophisticated methodology still being established in prokaryotes to investigate and thereafter engineer these cells as needed.[[Team:Heidelberg/Project_SaO#References|[1]]]. For one thing, the design of promoters exclusively responsive to one transcription factor (TF) within eukaryotic cells could certainly help improve our understanding of the key components of one pathway or the other, while eliminating the cross-talk often observed with many naturally occurring promoters. Such promoters have often posed a challenge to researchers studying signal transduction in eukaryotic systems because of the different types of TF a single regulatory element can bind, and a single TF having multiple target regulatory regions[[Team:Heidelberg/Project_SaO#References|[2]]]. With the emergence of systemized research and attempts for modelling biological systems, the availability of data with minimal experimental variability and highly accurate experimental conditions has also contributed to the need for such finely-tuned promoters. Once such exclusive promoters could be available and methods for their characterization established, it is not so hard to imagine the revolutionary effect they could have on eukaryotic research. Some of many applications could be: | ||
Line 33: | Line 35: | ||
:-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 attempted, 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 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. | :-As a further attempted, 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 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 2 FPs (GFP and mCherry) as well as 4 localization sequences ( | + | :-Besides the previous, we were able to provide 2 FPs (''GFP'' and ''mCherry'') as well as 4 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 12:35, 19 October 2009
Outlook and summary
References
|