Coding
YFP:tetR

Part:BBa_K606025:Experience

Designed by: Kévin Yauy   Group: iGEM11_Paris_Bettencourt   (2011-07-27)
Revision as of 15:59, 22 September 2011 by Cyrpaut (Talk | contribs)

Characterization: Biobricked TetO Array's running way

Microscopy of double transformated pFX234 / Biobricked TetO Array E. Coli

In order to do this characterization, we took pictures of different plasmids containing only TetO; TetR + YFP; TetO + TetR + YFP. in each case we made a control by non inducing the promoter with arabinose in E. coli (double transformated with pFX234 and TetO Array).

tetO array : 37°C
tetO / TetO array inducted with no arabinose on E. Coli .
tetO / TetO array inducted with 0,2% arabinose on E. Coli .
tetR:YFP : 37°C
tetR:YFP / TetR-YFP inducted with no arabinose on E. Coli .
tetR:YFP / TetR-YFP inducted with 0,2% arabinose on E. Coli .
tetR:YFP / TetO array : 37°C
TetR:YFP-tetO/ full construct inducted with no arabinose on E. Coli .
TetR:YFP-tetO / full construct inducted with 0,2% arabinose on E. Coli .
tetR:YFP and tetR:YFP / TetO array : 37°C - Zoom and comparison
TetR:YFP inducted with 0,2% arabinose on E. Coli .
tetR:YFP-TetO / full construct inducted with 0,2% arabinose on E. Coli .

The pictures of TetO show no YFP activity, which is normal because there is no YFP sequence in these plasmids.
The TetR-YFP construct which constitutes the transmitter part, occasionally shows gross aggregated YFP. This is not what we expected at first, but that does not prevent us to characterize the full construct.
After observing the full construct's pictures, we can obviously distinguish glowing dots in some cells. They reflect the behavior we expected. Indeed, appearance of dots (red arrow) shows that the receiver (TetO array) actually links tightly to TetR-YFP which is the emitted protein. Not all the dots are highlighted with red arrows but all are fluorescence loci !

Microscopy of ibpA mCherry double transformated in E. Coli

We transformated ibpA mCherry cells (expressing a mcherry in a agregation chaperon protein, with courtesy of Anne-Sophie Coquel, Inserm U1001) with pFX234 YFP:TetR and biobricked TetO Array plasmids to differenciate TetO array foci than agregation.

  • Case of YFP:TetR over-expression by arabinose induction
Microscopy yfp ibpa.jpg

Microscopy shows overlap for most foci and mCherry agregation but there are some foci that are alone indicating a TetR-YFP/TetO binding activity.
Hopefully we don't expect to get high concentration of YFP:tetR in receiver cell so it will be ok.

  • Case of YFP:TetR low expression by arabinose induction


Microscopy yfp ibpa2.jpg

Microscopy shows that most of agregation are gone and we have more not-overlaping foci.
We could manage to get less agregation if we deal with the arabinose induction.

Future

Next step is to biobrick the YFP:TetR fusion protein so we can finish the cloning plan and put the system in B. subtilis. Hopefully we can improve our GFP diffusion experiments and have a better characterisation of nanotubes !

References and acknowledgments

  1. Kinetics of plasmid segregation in Escherichia coli, Scott Gordon, JerĂ´me Rech, David Lane and Andrew Wright, Molecular Biology, available here
    2. Thanks to David Lane, Andrew Wright and François-Xavier Barre for information and great help they gave to us