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.
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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]