Team:Heidelberg/Project SaO
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:<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 characterize 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 TFs 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 systematized research and attempts for modeling 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 characterize 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 TFs 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 systematized research and attempts for modeling 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: | ||
- | [[Image:HD09_GFPmchr_PM.jpg|thumb|left|350px|Figure 1: GFP and mcherry localizing to the plasma membrane might serve as output in an assay cell line]] | + | :[[Image:HD09_GFPmchr_PM.jpg|thumb|left|350px|Figure 1: GFP and mcherry localizing to the plasma membrane might serve as output in an assay cell line]] |
:*Understanding disease within a network context. | :*Understanding disease within a network context. | ||
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:* Over the last three months we have been able to devise two independent methods to <span style="font-size:5mm;">design eukaryotic promoters</span> of desired selectivity and strength. The two methods referred to are based on different principles, one being a biochemical method [[Team:Heidelberg/Project_Synthetic_promoters#Results|(RA-PCR)]] and the other an ''in silico'' method [[Team:Heidelberg/HEARTBEAT|(HEARTBEAT)]]. Noteworthy is that the ''in silico'' method resulted in a tool that not only helps design promoters of required selectivity, but also helps evaluate the quality of promoters as well as provide online users (of our wiki) to use the same principle to design their own through an elegant Graphical User Interface (GUI). Also, we propose ways to [[Team:Heidelberg/Project_Synthetic_promoters#M-RA-PCR|combine]] the two methods. By applying these methods, 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 selection of specific promoters. | :* Over the last three months we have been able to devise two independent methods to <span style="font-size:5mm;">design eukaryotic promoters</span> of desired selectivity and strength. The two methods referred to are based on different principles, one being a biochemical method [[Team:Heidelberg/Project_Synthetic_promoters#Results|(RA-PCR)]] and the other an ''in silico'' method [[Team:Heidelberg/HEARTBEAT|(HEARTBEAT)]]. Noteworthy is that the ''in silico'' method resulted in a tool that not only helps design promoters of required selectivity, but also helps evaluate the quality of promoters as well as provide online users (of our wiki) to use the same principle to design their own through an elegant Graphical User Interface (GUI). Also, we propose ways to [[Team:Heidelberg/Project_Synthetic_promoters#M-RA-PCR|combine]] the two methods. By applying these methods, 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 selection of specific promoters. | ||
- | [[Image:HD09_ER2.jpg|thumb|left|200px|Figure 2: GFP and localizing to the ER might serve as output in an assay cell line]] | + | :[[Image:HD09_ER2.jpg|thumb|left|200px|Figure 2: GFP and localizing to the ER might serve as output in an assay cell line]] |
:* 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. | ||
Revision as of 09:02, 20 October 2009
Outlook and summary
References
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