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Team:Paris Bettencourt/Experiment/T7 diff subt subt microfluidic
From 2011.igem.org
Nanotube-assisted diffusion of T7 polymerase in Microfluidics
Experimental Scheme
In order to test whether our Bacillus subtilis T7 emitters & receivers can form nanotubes when mixed, we mix them in microfluidic system modified from Jeff Hasty's recent paper [1], shown below. For more detailed information regarding the microfluidic device and experimental procedure, check our methodology page.
We imaged two channels: one experimental channel injected with an emitter strain (RFP constitutive) and a receiver strain (pT7-T7polyermase-GFP); the other is a control channel injected with only the receiver strain. The receiver strain contains the T7 autoloop, which will gain a strong fluorescence when activated.
Results
The receiver strain by itself in our control channel have never produced any GFP positive cells, in contrast to the leaky situation where the T7 autoloop is encoded by a plasmid in E.coli. This is due to the low copy number of T7 polymerase gene on the chromosome. Yet the channel with the mixed population start to produce GFP positive cells immediately after injection. Yet due to the large population in the main channel compared to our single layer chambers, we never actually saw a turning-on event. Below is the summary of the total GFP fluorescent signals in 3 imaging positions in each channel.
Conclusion
The fact that when mixed with emitters, the receiver cells can turn on GFP signals in such large numbers and in such short time, indicates:
1. Our pT7-T7polyermase-GFP construct worked when integrated into bacillus subtilis chromosome.
2. We can't find another explanation other than nanotube-assisted diffusion of T7 polymerase in the biofilm in the main channel for the turning on events we saw in the channels.
References
- Entrainment of a population of synthetic genetic oscillators. Mondragón-Palomino, O., Danino, T., Selimkhanov, J., Tsimring, L. & Hasty, J. Science 333, 1315-1319 (2011).
