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>
-
<center>
+
<h2></h2>
-
<img src="https://static.igem.org/mediawiki/2012/8/8a/Diapositiva1.jpg"width="7300px"/>
+
-
</center>
+
-
    <p class="fancy">
+
-
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.
+
-
<br/>
+
-
Many studies on animals
+
-
bred under germ-free conditions have shown that microflora has specific
+
-
functions:
+
-
<ul class="fancy">
+
-
    <li>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;
+
-
    </li>
+
-
    <li>
+
-
Trophic: control of epithelial cell proliferation and differentiation;
+
-
development and homoeostasis of the immune system;
+
-
    </li>
+
-
    <li>Protective: protection against pathogens. </li>
+
-
</ul>
+
-
A disruption or an alteration of human gut microflora equilibrium leads
+
    <p class="fancy">
-
to severe auto-immune diseases, types of colon cancer and non-allergic
+
The human intestinal microflora is considered an essential “organ”
-
food hypersensitivities; therefore, it is very important to keep its
+
which plays an important role in human health. This complex ecosystem
-
integrity.  In fact already a century ago, the first probiotics have
+
is composed of approximately 500 anaerobic and aerobic bacteria species,
-
been commercialized. Probiotics are bacteria species (normally present
+
most of them localized in large intestine.
-
in human gut) that are administered as food components or supplements.
+
</p>
-
Nowadays, with the emerging research field of synthetic biology the
+
<p class="fancy">
-
potential applications of probiotics could further increase. In addition,
+
Many studies on animals
-
the introduction of novel functions to this “organ” via probiotics
+
bred under germ-free conditions have shown that microflora has specific
-
could lead the way to new therapeutics in order to cure or prevent many
+
functions:
-
pathological conditions.
+
<ul class="fancy">
-
    </p>
+
    <li>
-
    <h3>Project overview</h3>
+
Metabolic: fermentation of non-digestible dietary residue and
-
    <p class="fancy">
+
endogenous mucus, salvage of energy as short-chain fatty acids,
-
We engineered an indigenous strain from the gut microflora in order to
+
production of vitamin K, absorption of ions;
-
create a safe, controllable and versatile molecular platform, which can
+
    </li>
-
be used for production of a wide range of molecules.  For this purpose
+
    <li>
-
we used the Escherichia coli strain Nissle 1917 (commercialized as
+
Trophic: control of epithelial cell proliferation and differentiation;
-
Mutaflor) as the host organism for our platform. This particular strain
+
development and homoeostasis of the immune system;
-
has been used as a probiotic for decades and its beneficial effects
+
    </li>
-
on human health are well documented. We introduced into the strain a
+
    <li>Protective: protection against pathogens. </li>
-
gene guard system based on an inducible cumate gene guard switch and we
+
</ul>
-
trasformed it with a particularly designed plasmid for protein expression. </br>
+
</p>
-
The gene guard system is divided in two parts:
+
<p class="fancy">
-
<ul class="fancy">
+
A disruption or an alteration of human gut microflora equilibrium leads
-
    <li>the killing mechanism</li>
+
to severe auto-immune diseases, types of colon cancer and non-allergic
-
    <li>the regulating pattern</li>
+
food hypersensitivities; therefore, it is very important to keep its
-
</ul>
+
integrity.  In fact already a century ago, the first probiotics have
-
    </p>
+
been commercialized. Probiotics are bacteria species (normally present
-
    <p class="fancy">
+
in human gut) that are administered as food components or supplements.
-
The killing mechanism is placed on the  same plasmid used for protein
+
Nowadays, with the emerging research field of synthetic biology the
-
expression. It consists of two different proteins, T4 Holin and
+
potential applications of probiotics could further increase. In addition,
-
Cathelicidin LL-37, both toxic for prokaryotic cells. These antimicrobial
+
the introduction of novel functions to this “organ” via probiotics
-
proteins are both regulated by a cumate responsive promoter (T5 promoter
+
could lead the way to new therapeutics in order to cure or prevent many
-
Cumate Operator) placed upstream the structural genes. Holin is the
+
pathological conditions.
-
biobrick BBa_K112000 designed by group iGEM08 UC Berkeley. This protein
+
    </p>
-
comes from bacteriophage T4; it is a small membrane protein which
+
<br/>
-
depolarizes and permeabilizes the membrane allowing the secretion of
+
    <h1>Project overview</h1>
-
Cathelicidin LL-37 to the periplasm. Cathelicidin LL-37 is an antimicrobial
+
<h2></h2>
-
peptide produced by human macrophages. It has an amphypathic structure
+
 
-
that allows it to associate with bacterial membrane and form pores which
+
<img src="https://static.igem.org/mediawiki/2012/8/8a/Diapositiva1.jpg"width="680px"/>
-
lead to cell lysis; its presence in the periplasm is pivotal for its
+
 
-
killing effect. As an alternative killing mechanism the complex T4 Holin +
+
    <p class="fancy">
-
Cathelicidin LL-37 can be replaced with TSE 2 toxin, a standard biobrick
+
We engineered an indigenous strain from the gut microflora in order to
-
BBa_K314200 designed and characterized by Group iGEM10 Washington. TSE
+
create a safe, controllable and versatile molecular platform, which can
-
2 arrests the bacterial growth.
+
be used for production of a wide range of molecules.  For this purpose
-
<br/>
+
we used the <i>Escherichia coli</i> strain Nissle 1917 (commercialized as
-
The cumate-responsive regulator CymR,
+
Mutaflor) as the host organism for our platform. This particular strain
-
which regulates the expression of the T4 Holin and Cathelicidin LL-37,
+
has been used as a probiotic for decades and its beneficial effects
-
will be introduced into the bacterial chromosome by recombination using
+
on human health are well documented. We introduced into the strain a
-
the UPO-Sevilla miniTn7 Biobrick together with the helper plasmid pTNS2
+
gene guard system based on an inducible cumate gene guard switch and we
-
which codes for the Tn7 transposase.  The regulator cassette consists
+
trasformed it with a particularly designed plasmid for protein expression.
-
of a constitutive promoter (J23100) and the cymR gene, put in tandem
+
</p>
-
in order to increase the quantity of the repressor  to tightly control
+
<p class="fancy">
-
toxin expression.
+
The gene guard system is divided in two parts:
-
    </p>
+
<ul class="fancy">
-
    <p class="fancy">
+
    <li><strong>the killing mechanism</strong></li>
-
<strong>No Cumate Expression Mode:</strong>
+
    <li><strong>the regulating pattern</strong></li>
-
CymR is continuously produced. It binds the cumate responsive promoter (T5 Cumate Operator) and inhibits the expression of T4 Holin and Cathelicidin LL-37.   
+
</ul>
-
Bacteria live!
+
    </p>
-
    </p>
+
    <p class="fancy">
-
    <p class="fancy">
+
The killing mechanism is placed on the  same plasmid used for protein
-
<strong>Cumate-Induction Kill Mode:</strong>
+
expression. It consists of two different proteins, T4 Holin<sup><a href="#sup1">[1]</a></sup> and
-
Cumate enters by diffusion into bacteria, binds and inactivates the
+
Cathelicidin LL-37<sup><a href="#sup2">[2]</a></sup>,
-
repressor. The cumate responsive promoter (T5 Cumate Operator) is
+
both toxic for prokaryotic cells. These antimicrobial
-
derepressed and the expression of T4 Holin and Cathelicidin LL-37
+
proteins are both regulated by a cumate responsive promoter (T5 promoter
-
is turned on. T4 Holin interacts with the cytoplasmatic membrane,
+
Cumate Operator<sup><a href="#sup3">[3]</a><a href="sup4">[4]</a></sup>)
-
permeabilizes it allowing the secretion of Cathelicidin LL-37 into the
+
placed upstream the structural genes. Holin is the
-
periplasmic space. Cathelicidin LL-37 binds the outermembrane and forms
+
biobrick BBa_K112000 designed by group iGEM08 UC Berkeley. This protein
-
pores, which leads to cell lysis.
+
comes from bacteriophage T4; it is a small membrane protein which
-
Bacteria die!
+
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
-
<strong>Plasmid Transfer:</strong>
+
that allows it to associate with bacterial membrane and form pores which
-
<ol class="fancy">
+
lead to cell lysis; its presence in the periplasm is pivotal for its
-
    <li>
+
killing effect. As an alternative killing mechanism the complex T4 Holin +
-
The plasmid transfers from the E. coli Nissle into other bacteria
+
Cathelicidin LL-37 can be replaced with TSE 2 toxin, a standard biobrick
-
via horizontal transfer. The receiving bacteria do not produce
+
BBa_K314200 designed and characterized by Group iGEM10 Washington. TSE
-
the CymR repressor, so the cumate responsiv promoter (T5 Cumate
+
2 arrests the bacterial growth.
-
Operator) is derepressed and the T4 Holin and Cathelicidin
+
</p>
-
LL-37 expression is activated.<br/>Receiving bacteria die!
+
<p class="fancy">
-
    </li>
+
The cumate-responsive regulator CymR<sup><a href="#sup3">[3]</a><a href="sup4">[4]</a></sup>,
-
    <li>
+
which regulates the expression of the T4 Holin and Cathelicidin LL-37,
-
E. coli Nissle looses the plasmid. It continues to produce the
+
will be introduced into the bacterial chromosome by recombination using
-
CymR repressor, which does not appear to be toxic for prokaryotic
+
the UPO-Sevilla miniTn7 Biobrick together with the helper plasmid pTNS2
-
cells.<br/>Bacteria live and is harmless!
+
which codes for the Tn7 transposase.  The regulator cassette consists
-
    </li>
+
of a constitutive promoter (J23100) and the cymR gene, put in tandem
-
</ol>
+
in order to increase the quantity of the repressor  to tightly control
-
    </p>
+
toxin expression.
 +
    </p>
 +
    <p class="fancy">
 +
<h2>No Cumate Expression Mode:</h2>
 +
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/>  
 +
<strong>Bacteria live!</strong><br/>
 +
 
 +
<img src="https://static.igem.org/mediawiki/2012/d/d3/Diapositiva2.jpg"width="680px"/>
 +
 
 +
    </p>
 +
    <p class="fancy">
 +
<h2>Cumate-Induction Kill Mode:</h2><br/>
 +
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.<br/>
 +
<strong>Bacteria die!</strong><br/>
 +
<br/>
 +
 +
<img src="https://static.igem.org/mediawiki/2012/f/f8/Diapositiva3.jpg"width="680px"/>
 +
    </p><br/>
 +
<br/>
 +
    <p class="fancy">
 +
<h2>Plasmid Transfer:</h2></br>
 +
<ol class="fancy">
 +
    <li>
 +
The plasmid transfers from the <i>E. coli</i> 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.<br/><strong>Receiving bacteria die!</strong>
 +
<center>
 +
<img src="https://static.igem.org/mediawiki/2012/8/8a/Diapositiva1.jpg"width="650px"/>
 +
<img src="https://static.igem.org/mediawiki/2012/0/0a/Arrowts.png"width="90px"/>
 +
<img src="https://static.igem.org/mediawiki/2012/a/ac/Diapositiva5ts.jpg"width="650px"/>
 +
</center>
 +
    </li>
 +
    <li>
 +
<i>E. coli</i> Nissle looses the plasmid. It continues to produce the
 +
CymR repressor, which does not appear to be toxic for prokaryotic
 +
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" />
                 <img src="https://static.igem.org/mediawiki/2012/b/b0/Team_trieste.jpg" alt="Team iGEM 2012" id="igem_team" />
                 <div class="box_contacts">
                 <div class="box_contacts">
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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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