Team:Heidelberg/Project SaO

The 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.[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[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:

• Understanding disease within a network context.

• High-accuracy studying of signaling transduction pathways.

• Designing better experiments to understand noisy genetic control in eukaryotes.

• Selective protein expression in target cells.

• Combinatorial gene therapy.

• Metabolic engineering.

• 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. 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 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 dependant on colour 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 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)(link to GUI).

-Furhtermore, we have been able to generate a library of constitutive promoters of varying strengths as well as a library of NFκB-responsive promoters.

-We also established 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 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 units describing promoter strength (as measured using these methods) were also defined.

-As a further attempted, we tried to establish a 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 (1xEndoplasmic reticulum; 1xNucleus; 2xPlasma 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.

References

[1] Venter M., Synthetic promoters: genetic control through cis engineering,Trends in Plant Science,12;118-124

[2] Carey M. et al., Transcriptional Regulation in Eukaryotes: Concepts, Startegies and Techniques. New York:CSHL, 2000, p.18-25