Team:Trieste/project/overview

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         </html>{{Team:Trieste/menu}}<html>
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<div id="content"> <!-- start content -->
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         <h1 id="h1_lf" class="main_tit"><div>Project overview</div></h1>
+
         <h1 id="h1_lf" class="main_tit"><div>Project Overview</div></h1>
             <h1 id="h1_rt" class="main_tit"><div>More</div></h1>
             <h1 id="h1_rt" class="main_tit"><div>More</div></h1>
             <div id="box_main"> <!-- start box_main -->
             <div id="box_main"> <!-- start box_main -->
                 <div class="box_contenuti">
                 <div class="box_contenuti">
              
              
-
    <h1>Background</h1>
+
    <h1>Background</h1>
 +
<h2></h2>
-
    <p class="fancy">
+
    <p class="fancy">
-
The human intestinal microflora is considered an essential “organ”
+
The human intestinal microflora is considered an essential “organ”
-
which plays an important role in human health. This complex ecosystem
+
which plays an important role in human health. This complex ecosystem
-
is composed of approximately 500 anaerobic and aerobic bacteria species,
+
is composed of approximately 500 anaerobic and aerobic bacteria species,
-
most of them localized in large intestine.
+
most of them localized in large intestine.
-
<br/>
+
</p>
-
Many studies on animals
+
<p class="fancy">
-
bred under germ-free conditions have shown that microflora has specific
+
Many studies on animals
-
functions:
+
bred under germ-free conditions have shown that microflora has specific
-
<ul class="fancy">
+
functions:
-
    <li>Metabolic: fermentation of non-digestible dietary residue and
+
<ul class="fancy">
-
endogenous mucus, salvage of energy as short-chain fatty acids,
+
    <li>
-
production of vitamin K, absorption of ions;
+
Metabolic: fermentation of non-digestible dietary residue and
-
    </li>
+
endogenous mucus, salvage of energy as short-chain fatty acids,
-
    <li>
+
production of vitamin K, absorption of ions;
-
Trophic: control of epithelial cell proliferation and differentiation;
+
    </li>
-
development and homoeostasis of the immune system;
+
    <li>
-
    </li>
+
Trophic: control of epithelial cell proliferation and differentiation;
-
    <li>Protective: protection against pathogens. </li>
+
development and homoeostasis of the immune system;
-
</ul>
+
    </li>
 +
    <li>Protective: protection against pathogens. </li>
 +
</ul>
 +
</p>
 +
<p class="fancy">
 +
A disruption or an alteration of human gut microflora equilibrium leads
 +
to severe auto-immune diseases, types of colon cancer and non-allergic
 +
food hypersensitivities; therefore, it is very important to keep its
 +
integrity.  In fact already a century ago, the first probiotics have
 +
been commercialized. Probiotics are bacteria species (normally present
 +
in human gut) that are administered as food components or supplements.
 +
Nowadays, with the emerging research field of synthetic biology the
 +
potential applications of probiotics could further increase. In addition,
 +
the introduction of novel functions to this “organ” via probiotics
 +
could lead the way to new therapeutics in order to cure or prevent many
 +
pathological conditions.
 +
    </p>
 +
<br/>
 +
    <h1>Project overview</h1>
 +
<h2></h2>
-
A disruption or an alteration of human gut microflora equilibrium leads
+
<img src="https://static.igem.org/mediawiki/2012/8/8a/Diapositiva1.jpg"width="680px"/>
-
to severe auto-immune diseases, types of colon cancer and non-allergic
+
 
-
food hypersensitivities; therefore, it is very important to keep its
+
    <p class="fancy">
-
integrity.  In fact already a century ago, the first probiotics have
+
We engineered an indigenous strain from the gut microflora in order to
-
been commercialized. Probiotics are bacteria species (normally present
+
create a safe, controllable and versatile molecular platform, which can
-
in human gut) that are administered as food components or supplements.
+
be used for production of a wide range of molecules.  For this purpose
-
Nowadays, with the emerging research field of synthetic biology the
+
we used the <i>Escherichia coli</i> strain Nissle 1917 (commercialized as
-
potential applications of probiotics could further increase. In addition,
+
Mutaflor) as the host organism for our platform. This particular strain
-
the introduction of novel functions to this “organ” via probiotics
+
has been used as a probiotic for decades and its beneficial effects
-
could lead the way to new therapeutics in order to cure or prevent many
+
on human health are well documented. We introduced into the strain a
-
pathological conditions.
+
gene guard system based on an inducible cumate gene guard switch and we
-
    </p>
+
trasformed it with a particularly designed plasmid for protein expression.
-
    <h1>Project overview</h1>
+
</p>
-
<center>
+
<p class="fancy">
-
<img src="https://static.igem.org/mediawiki/2012/8/8a/Diapositiva1.jpg"width="730px"/>
+
The gene guard system is divided in two parts:
-
</center>
+
<ul class="fancy">
-
    <p class="fancy">
+
    <li><strong>the killing mechanism</strong></li>
-
We engineered an indigenous strain from the gut microflora in order to
+
    <li><strong>the regulating pattern</strong></li>
-
create a safe, controllable and versatile molecular platform, which can
+
</ul>
-
be used for production of a wide range of molecules.  For this purpose
+
    </p>
-
we used the Escherichia coli strain Nissle 1917 (commercialized as
+
    <p class="fancy">
-
Mutaflor) as the host organism for our platform. This particular strain
+
The killing mechanism is placed on the  same plasmid used for protein
-
has been used as a probiotic for decades and its beneficial effects
+
expression. It consists of two different proteins, T4 Holin<sup><a href="#sup1">[1]</a></sup> and
-
on human health are well documented. We introduced into the strain a
+
Cathelicidin LL-37<sup><a href="#sup2">[2]</a></sup>,
-
gene guard system based on an inducible cumate gene guard switch and we
+
both toxic for prokaryotic cells. These antimicrobial
-
trasformed it with a particularly designed plasmid for protein expression. </br>
+
proteins are both regulated by a cumate responsive promoter (T5 promoter
-
The gene guard system is divided in two parts:
+
Cumate Operator<sup><a href="#sup3">[3]</a><a href="sup4">[4]</a></sup>)
-
<ul class="fancy">
+
placed upstream the structural genes. Holin is the
-
    <li>the killing mechanism</li>
+
biobrick BBa_K112000 designed by group iGEM08 UC Berkeley. This protein
-
    <li>the regulating pattern</li>
+
comes from bacteriophage T4; it is a small membrane protein which
-
</ul>
+
depolarizes and permeabilizes the membrane allowing the secretion of
-
    </p>
+
Cathelicidin LL-37 to the periplasm. Cathelicidin LL-37 is an antimicrobial
-
    <p class="fancy">
+
peptide produced by human macrophages. It has an amphypathic structure
-
The killing mechanism is placed on the  same plasmid used for protein
+
that allows it to associate with bacterial membrane and form pores which
-
expression. It consists of two different proteins, T4 Holin and
+
lead to cell lysis; its presence in the periplasm is pivotal for its
-
Cathelicidin LL-37, both toxic for prokaryotic cells. These antimicrobial
+
killing effect. As an alternative killing mechanism the complex T4 Holin +
-
proteins are both regulated by a cumate responsive promoter (T5 promoter
+
Cathelicidin LL-37 can be replaced with TSE 2 toxin, a standard biobrick
-
Cumate Operator) placed upstream the structural genes. Holin is the
+
BBa_K314200 designed and characterized by Group iGEM10 Washington. TSE
-
biobrick BBa_K112000 designed by group iGEM08 UC Berkeley. This protein
+
2 arrests the bacterial growth.
-
comes from bacteriophage T4; it is a small membrane protein which
+
</p>
-
depolarizes and permeabilizes the membrane allowing the secretion of
+
<p class="fancy">
-
Cathelicidin LL-37 to the periplasm. Cathelicidin LL-37 is an antimicrobial
+
The cumate-responsive regulator CymR<sup><a href="#sup3">[3]</a><a href="sup4">[4]</a></sup>,
-
peptide produced by human macrophages. It has an amphypathic structure
+
which regulates the expression of the T4 Holin and Cathelicidin LL-37,
-
that allows it to associate with bacterial membrane and form pores which
+
will be introduced into the bacterial chromosome by recombination using
-
lead to cell lysis; its presence in the periplasm is pivotal for its
+
the UPO-Sevilla miniTn7 Biobrick together with the helper plasmid pTNS2
-
killing effect. As an alternative killing mechanism the complex T4 Holin +
+
which codes for the Tn7 transposase.  The regulator cassette consists
-
Cathelicidin LL-37 can be replaced with TSE 2 toxin, a standard biobrick
+
of a constitutive promoter (J23100) and the cymR gene, put in tandem
-
BBa_K314200 designed and characterized by Group iGEM10 Washington. TSE
+
in order to increase the quantity of the repressor  to tightly control
-
2 arrests the bacterial growth.
+
toxin expression.
-
<br/>
+
    </p>
-
The cumate-responsive regulator CymR,
+
    <p class="fancy">
-
which regulates the expression of the T4 Holin and Cathelicidin LL-37,
+
<h2>No Cumate Expression Mode:</h2>
-
will be introduced into the bacterial chromosome by recombination using
+
CymR is continuously produced. It binds the cumate responsive promoter (T5 Cumate Operator) and inhibits the expression of T4 Holin and Cathelicidin LL-37.<br/>  
-
the UPO-Sevilla miniTn7 Biobrick together with the helper plasmid pTNS2
+
<strong>Bacteria live!</strong><br/>
-
which codes for the Tn7 transposase.  The regulator cassette consists
+
 
-
of a constitutive promoter (J23100) and the cymR gene, put in tandem
+
<img src="https://static.igem.org/mediawiki/2012/d/d3/Diapositiva2.jpg"width="680px"/>
-
in order to increase the quantity of the repressor  to tightly control
+
 
-
toxin expression.
+
    </p>
-
    </p>
+
    <p class="fancy">
-
    <p class="fancy">
+
<h2>Cumate-Induction Kill Mode:</h2><br/>
-
<strong>No Cumate Expression Mode:</strong>
+
Cumate enters by diffusion into bacteria, binds and inactivates the
-
CymR is continuously produced. It binds the cumate responsive promoter (T5 Cumate Operator) and inhibits the expression of T4 Holin and Cathelicidin LL-37.   
+
repressor. The cumate responsive promoter (T5 Cumate Operator) is
-
Bacteria live!
+
derepressed and the expression of T4 Holin and Cathelicidin LL-37
-
    </p>
+
is turned on. T4 Holin interacts with the cytoplasmatic membrane,
-
    <p class="fancy">
+
permeabilizes it allowing the secretion of Cathelicidin LL-37 into the
-
<strong>Cumate-Induction Kill Mode:</strong>
+
periplasmic space. Cathelicidin LL-37 binds the outermembrane and forms
-
Cumate enters by diffusion into bacteria, binds and inactivates the
+
pores, which leads to cell lysis.<br/>
-
repressor. The cumate responsive promoter (T5 Cumate Operator) is
+
<strong>Bacteria die!</strong><br/>
-
derepressed and the expression of T4 Holin and Cathelicidin LL-37
+
<br/>
-
is turned on. T4 Holin interacts with the cytoplasmatic membrane,
+
-
permeabilizes it allowing the secretion of Cathelicidin LL-37 into the
+
<img src="https://static.igem.org/mediawiki/2012/f/f8/Diapositiva3.jpg"width="680px"/>
-
periplasmic space. Cathelicidin LL-37 binds the outermembrane and forms
+
    </p><br/>
-
pores, which leads to cell lysis.
+
<br/>
-
Bacteria die!
+
    <p class="fancy">
-
    </p>
+
<h2>Plasmid Transfer:</h2></br>
-
    <p class="fancy">
+
<ol class="fancy">
-
<strong>Plasmid Transfer:</strong>
+
    <li>
-
<ol class="fancy">
+
The plasmid transfers from the <i>E. coli</i> Nissle into other bacteria
-
    <li>
+
via horizontal transfer. The receiving bacteria do not produce
-
The plasmid transfers from the E. coli Nissle into other bacteria
+
the CymR repressor, so the cumate responsive promoter (T5 Cumate
-
via horizontal transfer. The receiving bacteria do not produce
+
Operator) is derepressed and the T4 Holin and Cathelicidin
-
the CymR repressor, so the cumate responsiv promoter (T5 Cumate
+
LL-37 expression is activated.<br/><strong>Receiving bacteria die!</strong>
-
Operator) is derepressed and the T4 Holin and Cathelicidin
+
<center>
-
LL-37 expression is activated.<br/>Receiving bacteria die!
+
<img src="https://static.igem.org/mediawiki/2012/8/8a/Diapositiva1.jpg"width="650px"/>
-
    </li>
+
<img src="https://static.igem.org/mediawiki/2012/0/0a/Arrowts.png"width="90px"/>
-
    <li>
+
<img src="https://static.igem.org/mediawiki/2012/a/ac/Diapositiva5ts.jpg"width="650px"/>
-
E. coli Nissle looses the plasmid. It continues to produce the
+
</center>
-
CymR repressor, which does not appear to be toxic for prokaryotic
+
    </li>
-
cells.<br/>Bacteria live and is harmless!
+
    <li>
-
    </li>
+
<i>E. coli</i> Nissle looses the plasmid. It continues to produce the
-
</ol>
+
CymR repressor, which does not appear to be toxic for prokaryotic
-
    </p>
+
cells.<br/><strong>Bacteria live and are harmless!</strong>
 +
    </li>
 +
</ol>
 +
    </p>
 +
<div class="footnotes">
 +
                                                <h3>References</h3>
 +
<p id="sup1">
 +
[1]: <strong>Genetic analysis od the T4 holin: timing and topology</strong><br/>
 +
Erlan Ramanculov, Ry Young<br/>
 +
Gene, March 2001, Volume 256, Issues 1-2, Pages 25-36 doi:10.1016-S0378-1119(01)00365-1
 +
</p>
 +
<p id="sup2">
 +
[2]: <strong>LL-37, the only human member of the cathelicidin family of antimicrobial peptides</strong> <br/>
 +
Ulrich H.N. Dürr, U.S. Suadheendra, Ayyalusamy Ramamoorthy <br/>
 +
Biochimica et Biophysica Acta, September 2006, Volume 1758, Issue 9, Pages 1406-1425 doi:10.1016/j.bbamem.2006.003.030
 +
</p>
 +
<p id="sup3">
 +
[3]: <strong>The cumate gene-switch: a system for regulated expression in mammalian cells</strong><br/>
 +
Alaka Mullick, Yan Xu, René Warren, Maria Koutroumanis, Claire Guilbault, Sophie Broussau, Félix Malenfant, Lucie Bourget, Linda Lamoureux, Rita Lo, Antoine W Caron, Amelie Pilotte and Bernard Massie<br/>
 +
BMC Biotechnology, 2006, 6:43  doi:10.1186/1472-6750-6-43
 +
</p>
 +
<p id="sup4">
 +
[4]: <strong>Novel, Versatile, and Tightly Regulated Expression System for Escherichia coli Strains</strong><br/>
 +
Young J. Choi, Lyne Morel, Teffanie Le François Denis Bourque, Lucie Bourget, Denis Groleau, Bernard Massie and Carlos B. Míguez<br/>
 +
Applied and Environmental Microbiology, Aug. 2010, p. 5058–5066 Vol.76  doi:10.1128/AEM.00413-10
 +
</p>
 +
</div>
                 </div>
                 </div>
             </div> <!-- end box_main -->
             </div> <!-- end box_main -->
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             <ul id="sub_menu">
             <ul id="sub_menu">
<li><a href="https://2012.igem.org/Team:Trieste/project">Abstract</a></li>
<li><a href="https://2012.igem.org/Team:Trieste/project">Abstract</a></li>
-
<li><a href="https://2012.igem.org/Team:Trieste/project/overview">Project Overview</a></li>
+
                <li class="select"  ><a href="https://2012.igem.org/Team:Trieste/project/overview">Project Overview</a></li>
<li><a href="https://2012.igem.org/Team:Trieste/project/applications">Applications</a></li>
<li><a href="https://2012.igem.org/Team:Trieste/project/applications">Applications</a></li>
-
</ul>
+
                <li><a href="https://2012.igem.org/Team:Trieste/project/modeling">Modeling</a></li>
 +
                        <li><a href="https://2012.igem.org/Team:Trieste/project/mainres">Main Results</a></li>
 +
                </ul>
                 <img src="https://static.igem.org/mediawiki/2012/b/b0/Team_trieste.jpg" alt="Team iGEM 2012" id="igem_team" />
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Latest revision as of 01:31, 27 September 2012

Project Overview

More

Background

The human intestinal microflora is considered an essential “organ” which plays an important role in human health. This complex ecosystem is composed of approximately 500 anaerobic and aerobic bacteria species, most of them localized in large intestine.

Many studies on animals bred under germ-free conditions have shown that microflora has specific functions:

  • Metabolic: fermentation of non-digestible dietary residue and endogenous mucus, salvage of energy as short-chain fatty acids, production of vitamin K, absorption of ions;
  • Trophic: control of epithelial cell proliferation and differentiation; development and homoeostasis of the immune system;
  • Protective: protection against pathogens.

A disruption or an alteration of human gut microflora equilibrium leads to severe auto-immune diseases, types of colon cancer and non-allergic food hypersensitivities; therefore, it is very important to keep its integrity. In fact already a century ago, the first probiotics have been commercialized. Probiotics are bacteria species (normally present in human gut) that are administered as food components or supplements. Nowadays, with the emerging research field of synthetic biology the potential applications of probiotics could further increase. In addition, the introduction of novel functions to this “organ” via probiotics could lead the way to new therapeutics in order to cure or prevent many pathological conditions.


Project overview

We engineered an indigenous strain from the gut microflora in order to create a safe, controllable and versatile molecular platform, which can be used for production of a wide range of molecules. For this purpose we used the Escherichia coli strain Nissle 1917 (commercialized as Mutaflor) as the host organism for our platform. This particular strain has been used as a probiotic for decades and its beneficial effects on human health are well documented. We introduced into the strain a gene guard system based on an inducible cumate gene guard switch and we trasformed it with a particularly designed plasmid for protein expression.

The gene guard system is divided in two parts:

  • the killing mechanism
  • the regulating pattern

The killing mechanism is placed on the same plasmid used for protein expression. It consists of two different proteins, T4 Holin[1] and Cathelicidin LL-37[2], both toxic for prokaryotic cells. These antimicrobial proteins are both regulated by a cumate responsive promoter (T5 promoter Cumate Operator[3][4]) placed upstream the structural genes. Holin is the biobrick BBa_K112000 designed by group iGEM08 UC Berkeley. This protein comes from bacteriophage T4; it is a small membrane protein which depolarizes and permeabilizes the membrane allowing the secretion of Cathelicidin LL-37 to the periplasm. Cathelicidin LL-37 is an antimicrobial peptide produced by human macrophages. It has an amphypathic structure that allows it to associate with bacterial membrane and form pores which lead to cell lysis; its presence in the periplasm is pivotal for its killing effect. As an alternative killing mechanism the complex T4 Holin + Cathelicidin LL-37 can be replaced with TSE 2 toxin, a standard biobrick BBa_K314200 designed and characterized by Group iGEM10 Washington. TSE 2 arrests the bacterial growth.

The cumate-responsive regulator CymR[3][4], which regulates the expression of the T4 Holin and Cathelicidin LL-37, will be introduced into the bacterial chromosome by recombination using the UPO-Sevilla miniTn7 Biobrick together with the helper plasmid pTNS2 which codes for the Tn7 transposase. The regulator cassette consists of a constitutive promoter (J23100) and the cymR gene, put in tandem in order to increase the quantity of the repressor to tightly control toxin expression.

No Cumate Expression Mode:

CymR is continuously produced. It binds the cumate responsive promoter (T5 Cumate Operator) and inhibits the expression of T4 Holin and Cathelicidin LL-37.
Bacteria live!

Cumate-Induction Kill Mode:


Cumate enters by diffusion into bacteria, binds and inactivates the repressor. The cumate responsive promoter (T5 Cumate Operator) is derepressed and the expression of T4 Holin and Cathelicidin LL-37 is turned on. T4 Holin interacts with the cytoplasmatic membrane, permeabilizes it allowing the secretion of Cathelicidin LL-37 into the periplasmic space. Cathelicidin LL-37 binds the outermembrane and forms pores, which leads to cell lysis.
Bacteria die!



Plasmid Transfer:


  1. The plasmid transfers from the E. coli Nissle into other bacteria via horizontal transfer. The receiving bacteria do not produce the CymR repressor, so the cumate responsive promoter (T5 Cumate Operator) is derepressed and the T4 Holin and Cathelicidin LL-37 expression is activated.
    Receiving bacteria die!
  2. E. coli Nissle looses the plasmid. It continues to produce the CymR repressor, which does not appear to be toxic for prokaryotic cells.
    Bacteria live and are harmless!

References

[1]: Genetic analysis od the T4 holin: timing and topology
Erlan Ramanculov, Ry Young
Gene, March 2001, Volume 256, Issues 1-2, Pages 25-36 doi:10.1016-S0378-1119(01)00365-1

[2]: LL-37, the only human member of the cathelicidin family of antimicrobial peptides
Ulrich H.N. Dürr, U.S. Suadheendra, Ayyalusamy Ramamoorthy
Biochimica et Biophysica Acta, September 2006, Volume 1758, Issue 9, Pages 1406-1425 doi:10.1016/j.bbamem.2006.003.030

[3]: The cumate gene-switch: a system for regulated expression in mammalian cells
Alaka Mullick, Yan Xu, René Warren, Maria Koutroumanis, Claire Guilbault, Sophie Broussau, Félix Malenfant, Lucie Bourget, Linda Lamoureux, Rita Lo, Antoine W Caron, Amelie Pilotte and Bernard Massie
BMC Biotechnology, 2006, 6:43 doi:10.1186/1472-6750-6-43

[4]: Novel, Versatile, and Tightly Regulated Expression System for Escherichia coli Strains
Young J. Choi, Lyne Morel, Teffanie Le François Denis Bourque, Lucie Bourget, Denis Groleau, Bernard Massie and Carlos B. Míguez
Applied and Environmental Microbiology, Aug. 2010, p. 5058–5066 Vol.76 doi:10.1128/AEM.00413-10

Team iGEM 2012

Contact us

For other information, write to:

igem2012@gmail.com
Università degli studi di Trieste ICGEB Illy Fondazione Cassa di Risparmio
iGEM 2012 iGEM 2012 iGEM 2012 iGEM 2012 iGEM 2012 iGEM 2012
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