Part:BBa_K606027:Experience

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Parts construction:
Origins of TetO Array are from pDAG479 of D. Lane (Toulouse 2 University).

Cloning plan of TetO array construction

We characterize it with arabinose-induced YFP:TetR Wild Type from pFX234 plasmid of F-X Barre and D. Lane (Kinetics of plasmid segregation, Molecular Microbiology, 2004).

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 array (K606026); TetR + YFP (pFX234); TetO + TetR + YFP (K606026 and pFX234). In each case we made a control by non inducing the promoter with arabinose. Cells were grown at 37°C and induced at least 45min.

tetO array : 37°C

tetO / TetO array induced without arabinose on E. Coli .	tetO / TetO array induced without arabinose on E. Coli .
tetO / TetO array induced with 0,2% arabinose on E. Coli .	tetO / TetO array induced with 0,2% arabinose on E. Coli .

TetR:YFP : 37°C

tetR:YFP / TetR-YFP induced without arabinose on E. Coli .	tetR:YFP / TetR-YFP induced without arabinose on E. Coli .
tetR:YFP / TetR-YFP induced with 0,2% arabinose on E. Coli .	tetR:YFP / TetR-YFP induced with 0,2% arabinose on E. Coli .

tetR:YFP / TetO array : 37°C

tetR:YFP-tetO/ full construct induced without arabinose on E. Coli .	tetR:YFP-tetO/ full construct induced without arabinose on E. Coli .
tetR:YFP-tetO / full construct induced with 0,2% arabinose on E. Coli .	tetR:YFP-tetO / full construct induced with 0,2% arabinose on E. Coli .

tetR:YFP and tetR:YFP / TetO array : 37°C - Zoom and comparison

tetR:YFP induced with 0,2% arabinose on E. Coli .

tetR:YFP-tetO / full construct induced 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 shows (red arrow) 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 RFP 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 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 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.

More information on : iGEM Paris Bettencourt 2011 wiki [http://2011.igem.org/Team:Paris_Bettencourt/Experiments/YFP_TetR_diffusion]