Testing nanotubes with T7 RNA polymerase diffusion

Design overview

Schematic summary of the T7 diffusion device

All of the parts of the above design have been BioBricked, characterized both in E.coli and B.subtilis, and sent to the registry.

More details on the design are available here.

Summary

Emitter construct in E.coli - Receiver construct in B.subtilis (plasmid)

Emitter & receiver constructs in B.subtilis (receiver in plasmid)

We used our microscopy protocol to do this experiment. The emitter strain is a B.subtilis PY9 strain where our emitter construct is integrated in the genome. The receiver strain is a B.subtilis PY9 strain where our T7 autoloop (receiver) construct is integrated in the genome.

We followed our cultures on three microscopic slides: one for the emitter, one for the receiver and one for the mix.

Emitter & receiver constructs in B.subtilis (receiver in genome)

Using the microfluidic chip (B.subtilis/B.subtilis)

Concentrating the cells more for microscopy (B.subtilis/B.subtilis)

Conclusions

Diffusion experiments

Results negative at first

This design was successfully implemented both in E.coli and B.subtilis. We were therefore able to test the diffusion of the T7 RNA polymerase through nanotubes. We ran several experiments:

Diffusion from E.coli to B.subtilis with our receptor construct on a plasmid. Find more about it here
Diffusion from B.subtilis to B.subtilis with our receptor construct on a plasmid. Find more about it here.
Diffusion from B.subtilis to B.subtilis with our receptor construct integrated in the genome. Find more about it here.

The first three experiments gave us negative results. We saw no obvious increase of the GFP expression in receiver cells when our construct was on a plasmid and absolutely no GFP fluorescence when it was integrated in the genome.

Unexepected breakthrough

However, our microfluidic experiment gave unexepected and encouraging results. We used for this experiment two B.subtilis strains (one emitter, one receiver, both integrated in the genome). Our chromosomic T7 autoloop was brightly activated during this experiment, but only in densely packed mix of emitter and receiver cells. Find more about this experiment here.

Seeing that cell concentration seemed to be the key factor and taking advantage of our perfectly not leaky chromosomic autoloop we conducted a final set of experiments where we concentrated our cells even more. We invite you to see our final results here.