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Team:Heidelberg/Project SaO
From 2009.igem.org
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*Building fine-tuned logic gates in cells. | *Building fine-tuned logic gates in cells. | ||
| - | + | 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.--> | ||
Revision as of 18:09, 20 October 2009
Outlook and summaryThe 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 [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 [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: 
A high-level application of synthetic promoters that lies close at hand is the development of a "cell-based drug screening assay". 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. Over the last three months we have been able to devise two independent methods to design eukaryotic promoters of desired selectivity and strength. The two methods referred to are based on different principles, one being a biochemical method (RA-PCR) and the other an in silico method (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 combine the two methods. By applying these methods, we have been able to generate a library of constitutive promoters of varying strengths as well as a selection of specific promoters.  For output, 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 measurement 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.
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 engineering of eukaryotic gene regulatory systems. 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. References[1] Venter M., Synthetic promoters: genetic control through cis engineering, Trends in Plant Science, 12:118-124 [2] Carey M., Smale S. T., Hughes H., Transcriptional Regulation in Eukaryotes: Concepts, Strategies and Techniques. New York:CSHL, p. 18-25 (2000) |
