Team:Paris Bettencourt/Experiments/YFP TetR diffusion

From 2011.igem.org

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<h1>Experiments of the YFP concentrator design</h1>
<h1>Experiments of the YFP concentrator design</h1>
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<p>The planning of the experiments is the following : first we have tested the strains from D. Lane containing YFP:TetR and TetO array. Then we constructed/biobricked the YFP:TetR and TetO array system. To finish with the microscopy step and results of this proof of concept between <i>B. subtilis and B. subtilis / E. coli</i>.</p>
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<p>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 <i>B.subtilis</i> (pHM3). In fact, this plasmid is also compatible with <i>E.coli</i>, which permitted us to successfully characterize it in this strain. The results are presented here in detail.</p>
<h2>Design overview</h2>
<h2>Design overview</h2>
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<h2>Results</h2>
<h2>Results</h2>
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Start with the good news : <br>We success to biobrick the TetO array and the next step is to characterize it. The plan is to do microscopy in ''E. coli'' double transformated with YFP:tetR WT and tetO array BB, ''E. coli'' with YFP:tetR WT only (already have from D. Lane), ''E. coli'' with tetO array BB only, Subtilis with tetO array (in pHM3 or K090403). <br><br>
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Start with the good news : <br>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, <i>B.subtilis</i> with tetO array (in pHM3 or K090403). <br><br>
The bad news : <br>
The bad news : <br>
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We have a lot of trouble to biobrick the YFP:tetR so it will be done if we successed to go to the World Jamboree.
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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.
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<h3>Testing the YFP:tetR and tetO array strains from D. Lane</h3>
<h3>Testing the YFP:tetR and tetO array strains from D. Lane</h3>
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In the article <a href="https://2011.igem.org/Team:Paris_Bettencourt/Experiments/YFP_TetR_diffusion#references">[1]</a>, <i>E. coli</i> strains are growing at 20°C to avoid protein agregation but the problem is that nanotube between <i>B. subtilis</i> has been only proved to exist at 37°C.
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In the article <a href="https://2011.igem.org/Team:Paris_Bettencourt/Experiments/YFP_TetR_diffusion#references">[1]</a>, <i>E. coli</i> strains are growing at 20°C to avoid protein aggregation, but the problem is that nanotubes between <i>B. subtilis</i> have only been shown to exist at 37°C.
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We test different possibilities : at 37°C or 30°C and concentration of arabinose (0% - 0,1% -0,2%) to deal with protein agregation.
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We tested different possibilities : at 37°C or 30°C and concentration of arabinose (0% - 0,1% -0,2%) to deal with protein aggregation.
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<p>With the combinaison of YFP:TetR and TetO array plasmids, we can see few loci 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 agregation is very effective when there is only YFP:tetR at 30°C and 37°C in <i>E. coli</i>.</p>
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<p>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 <i>E. coli</i>.</p>
<p>More pictures and information on the notebook here : <a href="https://2011.igem.org/Team:Paris_Liliane_Bettencourt/Notebook/2011/08/03/#Kevin">link</a></p>
<p>More pictures and information on the notebook here : <a href="https://2011.igem.org/Team:Paris_Liliane_Bettencourt/Notebook/2011/08/03/#Kevin">link</a></p>
<h3>Characterization: Biobricked TetO Array's running way </h3>
<h3>Characterization: Biobricked TetO Array's running way </h3>
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<h4>Microscopy of double transformated pFX234 / Biobricked TetO Array <i>E. Coli</i></h4>
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<h4>Microscopy of double transformed pFX234 / Biobricked TetO Array <i>E. Coli</i></h4>
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The pictures of TetO show no YFP activity, which is normal because there is no YFP sequence in these plasmids.<br>
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The pictures of TetO alone show no YFP activity, which is normal because there is no YFP sequence in these plasmids.<br>
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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.<br>
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The TetR-YFP construct which constitutes the transmitter part, occasionally shows gross aggregated YFP.<br>
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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 (red arrow) shows 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 !
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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).
<h4>Microscopy of ibpA mCherry double transformated in <i>E. Coli</i></h4>
<h4>Microscopy of ibpA mCherry double transformated in <i>E. Coli</i></h4>
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.
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.
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[[File:microscopy_yfp_ibpa.jpg|center|]]
[[File:microscopy_yfp_ibpa.jpg|center|]]
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Microscopy shows overlap for most foci and mCherry agregation but there are some foci that are alone indicating a  TetR-YFP/TetO binding activity.<br>
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Microscopy shows overlap for most foci and mCherry agregation but there is some foci that is alone.<br>
Hopefully we don't expect to get high concentration of YFP:tetR in receiver cell so it will be ok.
Hopefully we don't expect to get high concentration of YFP:tetR in receiver cell so it will be ok.

Revision as of 02:34, 22 September 2011

Team IGEM Paris 2011

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 yfp ibpa.jpg

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 yfp ibpa2.jpg

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.

Future

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 !

References and acknowledgments

  1. Kinetics of plasmid segregation in Escherichia coli, Scott Gordon, Jerôme Rech, David Lane and Andrew Wright, Molecular Biology, available here
  2. Thanks to David Lane, Andrew Wright and François-Xavier Barre for information and great help they gave to us