For higher resolution: Download Graphical Abstract
- We are able to predict functional mammalian promoter sequences (go there)
- We have created a functional biochemical synthesis method for the generation of promoters libraries (go there)
- We have developed novel standards for measurement of promoters in mammalian cells (go there)
- 4 RFCs, well characterized parts (Parts) , (RFCs)
- First attempts to create a eukaryotic standard chassis (Stable Cell Line)
- Multicolor and multi-functional output devices for promoter characterization (Output)
- Isolation and characterization of natural promoters (Natural Promoters)
Synthetic biology in mammalian systems will accelerate the pace of medical and fundamental research. Despite its huge potential, this field of synthetic mammalian biology is still in its infancy. Therefore, we want to lay foundations for the methodical usage of mammalian cells as chassis systems. For this purpose, two premises must be met: first, a mature cloning standard must be defined and standardized measurement protocols must be developed to ensure modularity and comparability of BioBrick constructs. Second, a comprehensive collection of biological parts and devices must be manufactured.
A cloning standard for mammalian BioBrick constructs has not yet been established as there are virtually no mammalian parts in the Registry up to now. We therefore analyzed all standards postulated so far and propose the BioBrick BB_2 proposal (Tom Knight) for future work with mammalian parts.
The standardized characterization of eukaryotic parts and devices is very challenging. We have developed standardized procedures for comparable measurements of promoter strength by transient transfection in mammalian cell lines. However, since mammalian cells, unlike bacteria and yeast, do not propagate plasmids, they will need to be stably transfected for an optimized characterization. To meet this requirement we created a preliminary cell line which includes FRT sites in its genome enabling stable transfections at defined sites.
We have manufactured a library of promoters, since they are a basic element of every biological construction kit. Promoters are crucial for the regulation of differential gene expression which is the fundamental principle of both natural and synthetic biological systems. However, natural promoters often underlie highly complex regulation mechanisms, which complicate the construction of stable synthetic networks.
Therefore, we have developed and successfully applied a synthesis method for synthetic promoters, and a strategy for their rational design. Our promoters can only be induced by predefined transcription factors. We claim that any synthetic promoter can be constructed by our methods.
Rational design of promoters relies on in silico tools: based on an elaborate evaluation of over 4000 promoter sequences throughout the human genome, we can predict functional sequences for promoters containing only transcription factor binding sites of interest. The necessary information is stored in HEARTBEAT (Heidelberg Artificial Transcription Factor Binding Site Engineering and Assembly Tool). HEARTBEAT is equipped with a GUI enabling the users to design synthetic promoters suitable for their own purposes. Furthermore, relying on a computer model based upon fuzzy logic, the outcome of the designed promoter can be simulated. In a reverse process considering the output, the model helps in optimizing the input sequence. We show that HEARTBEAT predicted sequences work in vivo.
Synthetic promoters offer a huge potential to fundamental research. For instance, they allow the construction of an assay monitoring several user-defined pathways at the same time. Also, controllable gene expression is very interesting for medical applications where it might enable selective targeting of cancer cells in virotherapy.Go to Top