Difference between revisions of "Part:BBa K606027:Experience"

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__NOTOC__
 
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 
how you used this part and how it worked out.
 
  
===Applications of BBa_K606027===
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==Characterization: Biobricked YFP:TetR==
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'''Parts construction''':<br>
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<html>
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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>Cloning plan of YFP:TetR construction</center></p>
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</html>
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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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'''Microscopy of double transformed pDAG470 / Biobricked YFP:TetR in <i>E. Coli</i>:'''
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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 (BBa_K606027).</p>
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<center>
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{| border="1" class="wikitable" style="text-align: center;" align="center"
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|+ Biobricked YFP:TetR only
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|-
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|[[Image:yfptetrbb_trans.jpg|350px|thumb|center|Biobricked expression YFP:tetR system in E. coli.]]
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|[[Image:yfptetrbb_Fluo2.jpg|350px|thumb|center|Biobricked expression YFP:tetR system in E. coli.]]
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|}
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{| border="1" class="wikitable" style="text-align: center;" align="center"
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|+ Biobricked YFP:TetR and TetO Array
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|-
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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: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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|}
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</center>
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The TetR-YFP construct which (emitter part) occasionally shows gross aggregated YFP. <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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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===
 
===User Reviews===
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<h1>The YFP Concentrator design</h1>
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<h2>Introduction</h2>
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<p>One of the main result of Dubey and Ben-Yehuda papers proving the existence of nanotubes is the evidence of GFP diffusion from  B.subtilis cells one to another.
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However the resulting fluorescence from this diffusion is quite weak supposedly due to the limited number of molecules passing through nanotubes.
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One alternative to get a better fluorescence signal with the same amount of fluorescent molecules is to concentrate them in foci. In order to do this we decided to use an already existing system based on a <em>YFP:TetR</em> fusion protein.
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<p> YFP:TetR is composed of Yellow Fluorescent Protein (YFP) and the Tetracycline Repressor Protein (TetR) that binds to the Tet operator sequence (TetO). Using the TetO array composed of a 10kb repeat of TetO sequences, we can concentrate YFP:TetR in several loci and increase the fluorescence sensitivity.
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The two different constructs, YFP:TetR and TetO Array, come from François-Xavier Barre, Andrew Wright and Dave Lane (Kinetics of plasmid segregation, Molecular Microbiology, 2004)</p>
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<p> We use this design as a proof of the nanotube concept between <i>B.Subtilis - B.Subtilis</i> and <i>B.Subtilis - E. Coli</i>.</p>
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<h2>Making the YFP:tetR diffuse through the tube</h2>
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<p><em>In the emitter cell <i>(B. Subtilis)</i></em>, we have inserted an expression system for the YFP:tetR. It contains the constitutive promoter pVeg, the RBS for <i>B.subtilis</i> and the YFP:tetR protein. Constitutively expressed YFP:tetR molecules will diffuse through the nanotube to the receiver cell.</p>
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<p><em>In the receiver cell <i>(B. Subtilis or E. Coli)</i></em>, there is the TetO array where diffused YFP:tetR will concentrate. The YFP is the monitor of the signal.</p>
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<p>The principle of the design is summed up in the image below</p>
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<br/>
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<center><img src="https://static.igem.org/mediawiki/2011/5/56/TetR-YFP4.jpg">
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<p><u>Fig1:</u> Schematics of the YFP concentration design</center></p>
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<br/>
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<h2>Model and experiments</h2>
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<p>To know more about what we have done on this system and in the experiments, we invite you to visit the corresponding <em>diffusion modeling</em> and <em>experiment</em> pages:</p>
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<ul>
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<li><em><a href="http://2011.igem.org/Team:Paris_Bettencourt/Modeling/Diffusion">Diffusion modelling</a></em></li>
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<li><em><a href="http://2011.igem.org/Team:Paris_Bettencourt/Experiments/YFP_TetR_diffusion">Experiments</a></em></li>
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</ul>
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Latest revision as of 16:27, 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

UNIQ7c50b2bb24fea559-partinfo-00000002-QINU UNIQ7c50b2bb24fea559-partinfo-00000003-QINU