Team:Paris Bettencourt/Experiments/YFP TetR diffusion experiments

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Team IGEM Paris 2011

Testing nanotubes with the YFP concentrator system

Summary

Results for the YFP concentrator:

  • We've done E.coli to B.subtilis diffusion experiments (with negative results)
  • We successfully BioBricked both the YFP:TetR (BBa_K606025) and the TetO Array (BBa_K606026) constructs and sent them to the registry
  • We characterized the YFP:TetR fusion protein both in E.coli and B.subtilis
  • We characterized the TetO array in E.coli


Design overview

YFP:TetR/TetO array system

More information on the design here.

Emitter & receiver constructs in B.subtilis (receiver in plasmid)

Emitter construct in E.coli - Receiver construct in B.subtilis (plasmid)

We success to integrate the TetO Array in B. subtilis with the multihost episomal pHM3 plasmid.
Experiments of E. coli (pFX234 plasmid, D. Lane strains) to B. subtilis (TetO Array, pHM3) to follow an eventual fluorescence diffusion by nanotube has been tried.

At the beginning of the movie : t = 0 min
Mix of E. coli (YFP:tetR) and B. subtilis (TetO Array) - Trans image
Mix of E. coli (YFP:tetR) and B. subtilis (TetO Array) - Fluo image
At the end of the movie : t = 125 min
Mix of E. coli (YFP:tetR) and B. subtilis (TetO Array) - Trans image
Mix of E. coli (YFP:tetR) and B. subtilis (TetO Array) - Fluo image

Movie : Experiment of mixed YFP:tetR (E. coli) & TetO Array (B. subtilis)

We cannot detect any tetR-YFP foci in the receiver cells specific of YFP concentration on the TetO Array. This suggests that the YFP-tetR fusion protein from E. coli cannot diffuse into B. subtilis through the potential nanotubes.

Conclusions