Team:Heidelberg/Project SaO
From 2009.igem.org
(Difference between revisions)
(→Outlook and summary) |
(→Outlook and summary) |
||
Line 24: | Line 24: | ||
:*Building fine-tuned logic gates in cells. | :*Building fine-tuned logic gates in cells. | ||
- | :<!--A step further, but within context of our attempt to develop an array of promoters that could help in studying eukaryotic intracellular networks, the thought to provide a tool to help visualize such interactions was of importance.--> A high-level application of | + | :[[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]]<!--A step further, but within context of our attempt to develop an array of promoters that could help in studying eukaryotic intracellular networks, the thought to provide a tool to help visualize such interactions was of importance.--> A high-level application of synthetic promoters that lies close at hand is the development of a <span style="font-size:5mm;">"cell-based drug screening assay"</span>. Such an assay is based upon a cell line which is stably transfected with a multitude of promoters responsive to a variety of pathways. Each promoter would be linked to a unique output signal (see below). This cell line could be stimulated with a variety of drug candidates, and the molecular effect of each drug would directly be visualized. The availability of such a cell line would greatly accelerate the pace of drug discovery and pharmacology alike. We have created all the parts and knowledge required for such an assay, and would only need to assemble it. |
:<!--Advances in microscopy and the availability of a myriad of fluorescent proteins (FP) have made their utilization a very attractive option. However, because of the overlap of the spectra of many fluorescent proteins, the use of several FPs together in the same sample while avoiding leakage from one channel to another in many microscopes is a limiting factor in the total number of promoters, and thus pathways, that can be visualized with such an approach . To circumvent such a limitation, the idea to use a small number of fluorescent proteins together with a family of intracellular targeting sequences to which they could be linked was proposed. By conjugating each of the fluorescent proteins to be used to more than one different intracellular sorting signal, not only is the variability dependent on color alone, but both colour and localization. For example with two fluorescent proteins alone, one could be able to study only two promoters concurrently, however with the same two proteins and three localization signal to which each could be targeted, six different promoters could be studied together, where a green plasma membrane and a green nucleus would reflect the activity of two different promoters without interfering with the visualization of each other.--> | :<!--Advances in microscopy and the availability of a myriad of fluorescent proteins (FP) have made their utilization a very attractive option. However, because of the overlap of the spectra of many fluorescent proteins, the use of several FPs together in the same sample while avoiding leakage from one channel to another in many microscopes is a limiting factor in the total number of promoters, and thus pathways, that can be visualized with such an approach . To circumvent such a limitation, the idea to use a small number of fluorescent proteins together with a family of intracellular targeting sequences to which they could be linked was proposed. By conjugating each of the fluorescent proteins to be used to more than one different intracellular sorting signal, not only is the variability dependent on color alone, but both colour and localization. For example with two fluorescent proteins alone, one could be able to study only two promoters concurrently, however with the same two proteins and three localization signal to which each could be targeted, six different promoters could be studied together, where a green plasma membrane and a green nucleus would reflect the activity of two different promoters without interfering with the visualization of each other.--> | ||
- | :* 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]] 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. |
:* 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 09:01, 20 October 2009
Outlook and summary
References
|