Difference between revisions of "Part:BBa K606025:Experience"
 
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==Characterization: Biobricked YFP:TetR==
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'''Parts construction''':<br>
 
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<h1>Experiments of the YFP concentrator design</h1>
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Origins of YFP:TetR are from pFX234 of D. Lane (Toulouse 2 University).
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<center><img src="https://static.igem.org/mediawiki/2011/c/cb/YFPtetR10.jpg">
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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>Cloning plan of YFP:TetR construction</center></p>  
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<h2>Design overview</h2>
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<center><img src="https://static.igem.org/mediawiki/2011/9/93/YFP_concentration_principle.jpg">
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<p>Cloning of TetO array construction</center></p>
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<p> More information on the design here : <a href="http://2011.igem.org/Team:Paris_Bettencourt/GFPLac_diffusion ">link</a></p>
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<h2>Parts and biobrick system construction</h2>
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<h3>TetO array construction</h3>
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<center><img src="https://static.igem.org/mediawiki/2011/a/a0/TetOarray2.jpg">
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<p>Cloning plan of TetO array construction</center></p>
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<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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<br><br>We characterize it with TetO Array from pDAG479 plasmid of F-X Barre and D. Lane (Kinetics of plasmid segregation, Molecular Microbiology, 2004).<br><br>
  
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The bad news : <br>
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'''Microscopy of double transformed pDAG470 / Biobricked YFP:TetR in <i>E. Coli</i>:'''
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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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<h3>Testing the YFP:tetR and tetO array strains from D. Lane</h3>
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<p>To characterize this part properly, we took pictures of different strains containing YFP:TetR alone and both TetO array (from pDAG479, D.Lane) and expression YFP:TetR system (<partinfo>BBa_K606027</partinfo>).</p>
  
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In the article <a href="http://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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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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{| border="1" class="wikitable" style="text-align: center;" align="center"
 
{| border="1" class="wikitable" style="text-align: center;" align="center"
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|+tetR:YFP / TetO array : 37°C
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|+ Biobricked YFP:TetR only
 
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|[[Image:TetR0_YFP02.jpg|350px|thumb|center|tetR:YFP / TetO array inducted with no arabinose on E. Coli from Dave Lane plasmids.]]
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|[[Image:yfptetrbb_trans.jpg|350px|thumb|center|Biobricked expression YFP:tetR system in E. coli.]]
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|[[Image:TetR02_YFP03.jpg|350px|thumb|center|tetR:YFP / TetO array inducted with 0,2% arabinose on E. Coli from Dave Lane plasmids.]]
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|[[Image:yfptetrbb_Fluo2.jpg|350px|thumb|center|Biobricked expression YFP:tetR system in E. coli.]]
 
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{| border="1" class="wikitable" style="text-align: center;"
 
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|+tetR:YFP / TetO array : 30°C
 
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|[[Image:TetRTetO0_YFP01.jpg|350px|thumb|center|tetR:YFP / TetO array inducted with no arabinose on E. Coli from Dave Lane plasmids.]]
 
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|[[Image:TetRTetO02_YFP01.jpg|350px|thumb|center|tetR:YFP / TetO array inducted with 0,2% arabinose on E. Coli from Dave Lane plasmids.]]
 
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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>More pictures and information on the notebook here : <a href="http://2011.igem.org/Team:Paris_Liliane_Bettencourt/Notebook/2011/08/03/#Kevin">link</a></p>
 
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<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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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).
 
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{| border="1" class="wikitable" style="text-align: center;" align="center"
 
{| border="1" class="wikitable" style="text-align: center;" align="center"
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|+ tetO array : 37°C
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|+ Biobricked YFP:TetR and TetO Array
 
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|[[Image:teto_minus_Fluo20.jpg|350px|thumb|center|tetO / TetO array inducted with no arabinose on E. Coli .]]
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|[[Image:yfptetrbb_teto_trans.jpg|350px|thumb|center|Biobricked expression YFP:tetR system and TetO Array from D. Lane in E. coli.]]
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|[[Image:teto_arab_Fluo20.jpg|350px|thumb|center|tetO / TetO array inducted with 0,2% arabinose on E. Coli .]]
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|[[Image:yfptetrbb_teto__Fluo2.jpg|350px|thumb|center|Biobricked expression YFP:tetR system and TetO Array from D. Lane in E. coli.]]
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{| border="1" class="wikitable" style="text-align: center;"
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|+tetR:YFP : 37°C
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|[[Image:yfp_tetr_minus_fluo20.jpg|350px|thumb|center|tetR:YFP / TetR-YFP inducted with no arabinose on E. Coli .]]
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|[[Image:yfp_tetr_Arab_fluo20.jpg|350px|thumb|center|tetR:YFP / TetR-YFP inducted with 0,2% arabinose on E. Coli .]]
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{| border="1" class="wikitable" style="text-align: center;"
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|+tetR:YFP / TetO array : 37°C
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|[[Image:yfp_tetr_teto_minus_fluo20_2s.jpg|350px|thumb|center|TetR:YFP-tetO/ full construct inducted with no arabinose on E. Coli .]]
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|[[Image:yfp_tetr_teto_Arab_fluo20_2-1.jpg|350px|thumb|center|TetR:YFP-tetO / full construct inducted with 0,2% arabinose on E. Coli .]]
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{| border="1" class="wikitable" style="text-align: center;"
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|+tetR:YFP and tetR:YFP / TetO array : 37°C - Zoom and comparison
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|[[Image:yfp_tetr_zoom.jpg|350px|thumb|center|TetR:YFP inducted with 0,2% arabinose on E. Coli .]]
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|[[Image:yfp_tetr_teto_zoom.jpg|350px|thumb|center|tetR:YFP-TetO / full construct inducted with 0,2% arabinose on E. Coli .]]
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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 TetR-YFP construct which (emitter part) occasionally shows gross aggregated YFP. <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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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.
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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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<h4>Microscopy of ibpA mCherry double transformated in <i>E. Coli</i></h4>
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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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*Case of YFP:TetR over-expression by arabinose induction
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[[Image: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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Hopefully we don't expect to get high concentration of YFP:tetR in receiver cell so it will be ok.
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*Case of YFP:TetR low expression by arabinose induction
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[[Image:microscopy_yfp_ibpa2.jpg|center|]]
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Microscopy shows that most of agregation are gone and we have more not-overlaping foci.<br> We could manage to get less agregation if we deal with the arabinose induction.<br>
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<h2>Future</h2>
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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 !
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<p id="references">References and acknowledgments</p>
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<ol>
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<li><i>Kinetics of plasmid segregation in Escherichia coli</i>, Scott Gordon, Jerôme Rech, David Lane and Andrew Wright, Molecular Biology, available <a href="http://onlinelibrary.wiley.com/doi/10.1046/j.1365-2958.2003.03837.x/pdf">here</a></li>
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Thanks to David Lane, Andrew Wright and François-Xavier Barre for information and great help they gave to us
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===Applications of BBa_K606025===
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More information on : TetO Array characterization (<partinfo>BBa_K606026</partinfo>) and iGEM Paris Bettencourt 2011 wiki [http://2011.igem.org/Team:Paris_Bettencourt/Experiments/YFP_TetR_diffusion]
  
 
===User Reviews===
 
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Latest revision as of 16:28, 24 October 2011

Characterization: Biobricked YFP:TetR

Parts construction:
Origins of YFP:TetR are from pFX234 of D. Lane (Toulouse 2 University).

Cloning plan of YFP:TetR construction



We characterize it with TetO Array from pDAG479 plasmid of F-X Barre and D. Lane (Kinetics of plasmid segregation, Molecular Microbiology, 2004).

Microscopy of double transformed pDAG470 / Biobricked YFP:TetR in E. Coli:

To characterize this part properly, we took pictures of different strains containing YFP:TetR alone and both TetO array (from pDAG479, D.Lane) and expression YFP:TetR system (BBa_K606027).

Biobricked YFP:TetR only
Biobricked expression YFP:tetR system in E. coli.
Biobricked expression YFP:tetR system in E. coli.
Biobricked YFP:TetR and TetO Array
Biobricked expression YFP:tetR system and TetO Array from D. Lane in E. coli.
Biobricked expression YFP:tetR system and TetO Array from D. Lane in E. coli.

The TetR-YFP construct which (emitter 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.


More information on : TetO Array characterization (BBa_K606026) and iGEM Paris Bettencourt 2011 wiki [http://2011.igem.org/Team:Paris_Bettencourt/Experiments/YFP_TetR_diffusion]

User Reviews

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