"
Team:Newcastle University/ProofOfConceptBrick
From 2008.igem.org
Newcastle University
GOLD MEDAL WINNER 2008
| Home | Team | Original Aims | Software | Modelling | Proof of Concept Brick | Wet Lab | Conclusions |
|---|
Home >> Proof of Concept Brick
The designs for regulatory networks produced by our software are too large and complicated to be implemented in the time available. We needed a smaller system to try out our ideas.
To demonstrate that the sensory and regulatory systems will work in isolation from the genes for subtilin production, we generated a proof-of-concept brick. This contains the two-component system that senses extracellular subtilin and in response activates genes with response elements in their promoters. The brick also contains one such promoter, at the far right-hand-side. The rational was that by simply placing this brick directly up-stream of any promoterless gene, that gene can be driven by extracellular subtilin, allowing us to rapidly produce positive controls for the two-component system.
In the original strains, a single operon contains all of the genes responsible for subtilin production and export, sensing, and down-stream gene regulation. For the BugBuster application, we needed to be able to pick up subtilin secreted by the unmodified strain, within our modified bacterium. This meant that our bacterium needed the genes for sensing subtilin, and for activating genes in response to it, but must not themselves produce subtilin. We re-designed the system in a bottom up fashion from virtual parts encoded in CellML.
Figure 1: Design and implementation of the subtilin receiver BioBrick, [http://partsregistry.org/Part:BBa_K104001 BBa_K104001]. This contains a promoter, driving a reduced spa operon, and the PspaS promoter. The reduced spa operon contains the CDS for spaR and spaK only, with spaR being the subtilin sensor which activates spaK, the transcription factor. The PspaS promoter is activated by the spaK transcription factor.Cartoon of the BioBrick in action. On sensing the subtilin signal peptide, the spaR subtilin sensor activates the spaK regulatory protein by phosphorylation. This activate form now activates the transcription of other genes. In the full system, these would be other sensors or transciption factors. In the proof-of-principle positive controls, these would be reporters, such as GFP.
