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Experiments of the YFP concentrator design

This system is an improvement of the original experiment with the GFP. In fact, to see the fluorescence better, we decided to concentrate the YFP fluorescent molecules fused to TetR on the TetO array to make them more visible in the cell. We have been kindly given the plasmids containing YFP:TetR construct and TetO array by D. Lane. In order to use this system according to our designs, we biobricked the TetO array and incorporated it in a plasmid compatible with B.subtilis (pHM3). In fact, this plasmid is also compatible with E.coli, which permitted us to successfully characterize it in this strain. The results are presented here in detail.

Design overview

Cloning of TetO array construction

More information on the design here : link

Parts and biobrick system construction

YFP:TetR construction

Cloning plan of YFP:TetR construction

TetO array construction

Cloning plan of TetO array construction

Results

Start with the good news :
We succeeded to biobrick the TetO array and the next step is to characterize it. The plan is to do microscopy in E. coli double transformed with YFP:tetR WT and tetO array (Biobricked), E. coli with YFP:tetR WT only, E. coli with tetO array BB only, B.subtilis with tetO array (in pHM3 or K090403).

The bad news :
We have a lot of trouble to biobrick the YFP:tetR so it will be done if we succeed to go to the World Jamboree.

Testing the YFP:tetR and tetO array strains from D. Lane

In the article [1], E. coli strains are growing at 20°C to avoid protein aggregation, but the problem is that nanotubes between B. subtilis have only been shown to exist at 37°C. We tested different possibilities : at 37°C or 30°C and concentration of arabinose (0% - 0,1% -0,2%) to deal with protein aggregation.

tetR:YFP / TetO array : 37°C

tetR:YFP / TetO array inducted with no arabinose on E. Coli from Dave Lane plasmids.

tetR:YFP / TetO array inducted with 0,2% arabinose on E. Coli from Dave Lane plasmids.

tetR:YFP / TetO array : 30°C

tetR:YFP / TetO array inducted with no arabinose on E. Coli from Dave Lane plasmids.

tetR:YFP / TetO array inducted with 0,2% arabinose on E. Coli from Dave Lane plasmids.

With the combination of YFP:TetR and TetO array plasmids, we can see few spots of fluorescence with 0,2% arabinose and because there are in the cell extremity, we can suppose that it is concentrated fluorescence in TetO array. Nevertheless the protein aggregation is very effective when there is only YFP:tetR at 30°C and 37°C in E. coli.

More pictures and information on the notebook here : link

Characterization: Biobricked TetO Array's running way

Microscopy of double transformed pFX234 / Biobricked TetO Array E. Coli

In order to do this characterization, we took pictures of different plasmids containing only TetO; TetR + YFP; TetO + TetR + YFP. in each case we made a control by non inducing the promoter with arabinose in E. coli (double transformated with pFX234 and TetO Array).

tetO array : 37°C

tetO / TetO array inducted with no 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 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 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 alone 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.
After observing the cells carrying both the TetO and YFP:TetR constructs, we can obviously distinguish glowing dots in some cells. They reflect the behavior we expected. Indeed, appearance of dots (red arrow) shows that the receiver (TetO array) actually links tightly to TetR-YFP which is the emitted protein! In fact, you can see a lot of glowing dots, each of them being a concentration of fluorescent molecules (we have highlighted some of them with red arrows).

Microscopy of ibpA mCherry double transformated in E. Coli

We transformated ibpA mCherry cells (expressing a mcherry 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 is some foci that is alone.
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.

To be continued...

Next step is to biobrick the YFP:TetR fusion protein so we can finish the cloning plan and put the system in B. subtilis. Hopefully we can improve our GFP diffusion experiments and have a better characterisation of nanotubes !