Team:Newcastle University/ProofOfConceptBrick

From 2008.igem.org

Revision as of 22:22, 29 October 2008 by Mtaschuk (Talk | contribs)

Bugbuster-logo-red.png
Ncl uni logo.jpg


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.

We wanted to show that it was possible to build a system to sense quorum peptides. Bacillus subtilis ATCC 6633 produces a small peptide antibiotic, subtilin, that is also used in quorum sensing. We decided to develop a system that would sense subtilin and incorporate it into B. subtilis 168 that does not naturally possess this peptide sensing system.

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.


TCSOverview-withBrick.png

Figure 1: Design and implementation of the subtilin receiver BioBrick, 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.