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Team:Trieste/parts/2
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<div class="box_contenuti"> | <div class="box_contenuti"> | ||
<h2>Description </h2> | <h2>Description </h2> | ||
| - | This promoter is composed by phage T5 promoter and Lac operator. It is repressed in the presence of the lac repressor protein. | + | This promoter is composed by phage T5 promoter and Lac operator. It is repressed in the presence of the lac repressor protein and activated in the presence of IPTG which binds the repressor inactivating it. |
<br/> | <br/> | ||
<br/> | <br/> | ||
<h2>Assembly</h2> | <h2>Assembly</h2> | ||
Obtained by synthesis. | Obtained by synthesis. | ||
| + | <br/> | ||
| + | <center><img src="https://static.igem.org/mediawiki/2012/1/1b/T5lacOP.png"width="150px"/></center> | ||
<br/> | <br/> | ||
<br/> | <br/> | ||
<h2>Results</h2> | <h2>Results</h2> | ||
| - | Trieste Team 2012 used this inducible | + | Trieste Team 2012 used this inducible promoter in the first phase of their project to test every construct that could have been toxic for the host strain, if expressed in a constitutive way. |
<br/> | <br/> | ||
<br/> | <br/> | ||
| - | <img src="https://static.igem.org/mediawiki/2012/a/ad/Lac_da_carricare.png" alt="Western blo T5LacO-Omp-scFv" width="450px"/> | + | <center><img src="https://static.igem.org/mediawiki/2012/a/ad/Lac_da_carricare.png" alt="Western blo T5LacO-Omp-scFv" width="450px"/></center> |
| - | <p class="didascalia"><strong>FIG. 1. Expression of LPP-OmpA-scFv 54.6 cloned under | + | <p class="didascalia"><strong>FIG. 1. Expression of LPP-OmpA-scFv 54.6 cloned under T5 Lac Operator.</strong> Western blots of lysates of <i>E.coli</i> W3110 bacterial strain expressing the recombinant protein scFv 54.6, induced or non-induced with IPTG.The blot was reacted with the monoclonal F24-796 anti-6XHIS antibody. |
</p> | </p> | ||
How it works: | How it works: | ||
<br/> | <br/> | ||
| - | The host strain contains the low copy plasmid pREP4 which constitutively expresses the Lac repressor protein encoded by the | + | The host strain contains the low copy plasmid pREP4 which constitutively expresses the Lac repressor protein encoded by the LacI gene. The Lac repressor binds the operator sequence preventing the transcription. The Lac repressor protein is rapidly inactivated by the binding of isopropyl-β-thiogalactoside (IPTG). |
<br/> | <br/> | ||
<br/> | <br/> | ||
| - | < | + | We also tested the T5 Lac Operator Promoter cloned upstream the Tse2 toxin construct. And this is the growth curve of IPTG induced and non-induced cells:</br> |
| - | </br> | + | <br/> |
| + | <center><img src="https://static.igem.org/mediawiki/2012/c/c0/TsTse2021.jpg" width="600px" /></center> | ||
| + | <br/> | ||
<h3><a href="http://partsregistry.org/Part:BBa_K875002"target="_blank">Link to the Registry</a></h3> | <h3><a href="http://partsregistry.org/Part:BBa_K875002"target="_blank">Link to the Registry</a></h3> | ||
| - | < | + | <br/> |
<!-- ******************** CONTENUTO QUI ****************** --> | <!-- ******************** CONTENUTO QUI ****************** --> | ||
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<li><a href="https://2012.igem.org/Team:Trieste/parts">Overwiev</a></li> | <li><a href="https://2012.igem.org/Team:Trieste/parts">Overwiev</a></li> | ||
<li><a href="https://2012.igem.org/Team:Trieste/parts/1">BBa_K875001 - Cumate Op</a></li> | <li><a href="https://2012.igem.org/Team:Trieste/parts/1">BBa_K875001 - Cumate Op</a></li> | ||
| - | <li><a href="https://2012.igem.org/Team:Trieste/parts/2">BBa_K875002 - Lac OP</a></li> | + | <li class="select" ><a href="https://2012.igem.org/Team:Trieste/parts/2">BBa_K875002 - Lac OP</a></li> |
<li><a href="https://2012.igem.org/Team:Trieste/parts/3">BBa_K875003 - CymR</a></li> | <li><a href="https://2012.igem.org/Team:Trieste/parts/3">BBa_K875003 - CymR</a></li> | ||
<li><a href="https://2012.igem.org/Team:Trieste/parts/4">BBa_K875004 - OmpA scFv</a></li> | <li><a href="https://2012.igem.org/Team:Trieste/parts/4">BBa_K875004 - OmpA scFv</a></li> | ||
Latest revision as of 15:35, 26 October 2012
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a safe probiotic platform for protein expression
BBa_K875002
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Description
This promoter is composed by phage T5 promoter and Lac operator. It is repressed in the presence of the lac repressor protein and activated in the presence of IPTG which binds the repressor inactivating it.Assembly
Obtained by synthesis.
Results
Trieste Team 2012 used this inducible promoter in the first phase of their project to test every construct that could have been toxic for the host strain, if expressed in a constitutive way.
FIG. 1. Expression of LPP-OmpA-scFv 54.6 cloned under T5 Lac Operator. Western blots of lysates of E.coli W3110 bacterial strain expressing the recombinant protein scFv 54.6, induced or non-induced with IPTG.The blot was reacted with the monoclonal F24-796 anti-6XHIS antibody.
How it works:The host strain contains the low copy plasmid pREP4 which constitutively expresses the Lac repressor protein encoded by the LacI gene. The Lac repressor binds the operator sequence preventing the transcription. The Lac repressor protein is rapidly inactivated by the binding of isopropyl-β-thiogalactoside (IPTG).
We also tested the T5 Lac Operator Promoter cloned upstream the Tse2 toxin construct. And this is the growth curve of IPTG induced and non-induced cells:
Link to the Registry
