Coding
tetO array

Part:BBa_K606026:Experience

Designed by: Kvin Yauy   Group: iGEM11_Paris_Bettencourt   (2011-07-27)
Revision as of 21:10, 21 September 2011 by Kyauy (Talk | contribs)

Characterisation of BBa_K606026 - TetO Array

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).

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 inducted with no arabinose on E. Coli .
tetO / tetO array inducted with no arabinose on E. Coli .
tetO / tetO array inducted with 0,2% 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 no arabinose on E. Coli .
tetR:YFP / tetr-YFP inducted with 0,2% 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 no arabinose on E. Coli .
tetR:YFP-tetO / full construct inducted with 0,2% 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 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.

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

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