Team:Trieste/project

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                    <h1 id="abstract">Abstract</h1>
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    <h2>Background</h2>
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</br>
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    <h3>Background:</h3>
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    <p class="fancy">  
    <p class="fancy">  
Recent studies have evidenced that the intestinal microflora can
Recent studies have evidenced that the intestinal microflora can
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is therefore believed that having a beneficial and healthy intestinal
is therefore believed that having a beneficial and healthy intestinal
microflora is very important for human health.
microflora is very important for human health.
 +
</br>
 +
</br>
    </p>
    </p>
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    <h3>Project:</h3>
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    <h2>Project</h2>
    <p class="fancy">
    <p class="fancy">
Our aim is to modify a bacteria normally found in human gut and create
Our aim is to modify a bacteria normally found in human gut and create
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    </p>
    </p>
    <p class="fancy">
    <p class="fancy">
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For this purpose we have chosen the E. coli strain Nissle 1917 which has been
+
For this purpose we have chosen the <i>E. coli </i> strain Nissle 1917 which has been
used for many years as a probiotic. We designed a robust gene guard system
used for many years as a probiotic. We designed a robust gene guard system
regulated by a novel and easy to control inducible switch that activates
regulated by a novel and easy to control inducible switch that activates
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because the receiving bacteria do not produce the CymR repressor needed to
because the receiving bacteria do not produce the CymR repressor needed to
repress the expression of the antimicrobial peptide.
repress the expression of the antimicrobial peptide.
 +
</br>
 +
</br>
    </p>
    </p>
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    <h3>Application:</h3>
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    <h2>Application</h2>
    <p class="fancy">
    <p class="fancy">
The safe probiotic constructed here can be used to produce nutritious,
The safe probiotic constructed here can be used to produce nutritious,
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70.000 of those need hospitalization and 800 die.
70.000 of those need hospitalization and 800 die.
    </p>
    </p>
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                    <h1 id="overview">Project overview</h1>
 
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                    <h3>Background</h3>
 
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    <p class="fancy">
 
-
The human intestinal microflora is considered an essential “organ”
 
-
which plays an important role in human health. This complex ecosystem
 
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is composed of approximately 500 anaerobic and aerobic bacteria species,
 
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most of them localized in large intestine.
 
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<br/>
 
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Many studies on animals
 
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bred under germ-free conditions have shown that microflora has specific
 
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functions:
 
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<ul class="fancy">
 
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    <li>Metabolic: fermentation of non-digestible dietary residue and
 
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endogenous mucus, salvage of energy as short-chain fatty acids,
 
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production of vitamin K, absorption of ions;
 
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    </li>
 
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    <li>
 
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Trophic: control of epithelial cell proliferation and differentiation;
 
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development and homoeostasis of the immune system;
 
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    </li>
 
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    <li>Protective: protection against pathogens. </li>
 
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</ul>
 
-
 
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A disruption or an alteration of human gut microflora equilibrium leads
 
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to severe auto-immune diseases, types of colon cancer and non-allergic
 
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food hypersensitivities; therefore, it is very important to keep its
 
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integrity.  In fact already a century ago, the first probiotics have
 
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been commercialized. Probiotics are bacteria species (normally present
 
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in human gut) that are administered as food components or supplements.
 
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Nowadays, with the emerging research field of synthetic biology the
 
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potential applications of probiotics could further increase. In addition,
 
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the introduction of novel functions to this “organ” via probiotics
 
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could lead the way to new therapeutics in order to cure or prevent many
 
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pathological conditions.
 
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    </p>
 
-
    <h3>Project overview</h3>
 
-
    <p class="fancy">
 
-
We engineered an indigenous strain from the gut microflora in order to
 
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create a safe, controllable and versatile molecular platform, which can
 
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be used for production of a wide range of molecules.  For this purpose
 
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we used the Escherichia coli strain Nissle 1917 (commercialized as
 
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Mutaflor) as the host organism for our platform. This particular strain
 
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has been used as a probiotic for decades and its beneficial effects
 
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on human health are well documented. We introduced into the strain a
 
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gene guard system based on an inducible cumate gene guard switch and we
 
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trasformed it with a particularly designed plasmid for protein expression. </br>
 
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The gene guard system is divided in two parts:
 
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<ul class="fancy">
 
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    <li>the killing mechanism</li>
 
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    <li>the regulating pattern</li>
 
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</ul>
 
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    </p>
 
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    <p class="fancy">
 
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The killing mechanism is placed on the  same plasmid used for protein
 
-
expression. It consists of two different proteins, T4 Holin and
 
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Cathelicidin LL-37, both toxic for prokaryotic cells. These antimicrobial
 
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proteins are both regulated by a cumate responsive promoter (T5 promoter
 
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Cumate Operator) placed upstream the structural genes. Holin is the
 
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biobrick BBa_K112000 designed by group iGEM08 UC Berkeley. This protein
 
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comes from bacteriophage T4; it is a small membrane protein which
 
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depolarizes and permeabilizes the membrane allowing the secretion of
 
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Cathelicidin LL-37 to the periplasm. Cathelicidin LL-37 is an antimicrobial
 
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peptide produced by human macrophages. It has an amphypathic structure
 
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that allows it to associate with bacterial membrane and form pores which
 
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lead to cell lysis; its presence in the periplasm is pivotal for its
 
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killing effect. As an alternative killing mechanism the complex T4 Holin +
 
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Cathelicidin LL-37 can be replaced with TSE 2 toxin, a standard biobrick
 
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BBa_K314200 designed and characterized by Group iGEM10 Washington. TSE
 
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2 arrests the bacterial growth.
 
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<br/>
 
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The cumate-responsive regulator CymR,
 
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which regulates the expression of the T4 Holin and Cathelicidin LL-37,
 
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will be introduced into the bacterial chromosome by recombination using
 
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the UPO-Sevilla miniTn7 Biobrick together with the helper plasmid pTNS2
 
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which codes for the Tn7 transposase.  The regulator cassette consists
 
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of a constitutive promoter (J23100) and the cymR gene, put in tandem
 
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in order to increase the quantity of the repressor  to tightly control
 
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toxin expression.
 
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    </p>
 
-
    <p class="fancy">
 
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<strong>No Cumate Expression Mode:</strong>
 
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CymR is continuously produced. It binds the cumate responsive promoter (T5 Cumate Operator) and inhibits the expression of T4 Holin and Cathelicidin LL-37. 
 
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Bacteria live!
 
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    </p>
 
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    <p class="fancy">
 
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<strong>Cumate-Induction Kill Mode:</strong>
 
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Cumate enters by diffusion into bacteria, binds and inactivates the
 
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repressor. The cumate responsive promoter (T5 Cumate Operator) is
 
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derepressed and the expression of T4 Holin and Cathelicidin LL-37
 
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is turned on. T4 Holin interacts with the cytoplasmatic membrane,
 
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permeabilizes it allowing the secretion of Cathelicidin LL-37 into the
 
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periplasmic space. Cathelicidin LL-37 binds the outermembrane and forms
 
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pores, which leads to cell lysis.
 
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Bacteria die!
 
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    </p>
 
-
    <p class="fancy">
 
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<strong>Plasmid Transfer:</strong>
 
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<ol class="fancy">
 
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    <li>
 
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The plasmid transfers from the E. coli Nissle into other bacteria
 
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via horizontal transfer. The receiving bacteria do not produce
 
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the CymR repressor, so the cumate responsiv promoter (T5 Cumate
 
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Operator) is derepressed and the T4 Holin and Cathelicidin
 
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LL-37 expression is activated.<br/>Receiving bacteria die!
 
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    </li>
 
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    <li>
 
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E. coli Nissle looses the plasmid. It continues to produce the
 
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CymR repressor, which does not appear to be toxic for prokaryotic
 
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cells.<br/>Bacteria live and is harmless!
 
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    </li>
 
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</ol>
 
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    </p>
 
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<h3 id="applications">Application</h3>
 
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<p class="fancy">
 
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    The safe probiotic constructed here can be used to produce nutritious,
 
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    preventive or therapeutic molecules. For example, this E.coli can
 
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    express antitumorals, antibodies against different intestinal pathogens,
 
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    imunomodulators, antigens (recombinant proteins which act as mucosal
 
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    vaccine) which are important for the correct development of the immune
 
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    system etc. It can express also different enzymes as lactase or enzymes
 
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    needed to recreate the metabolic pathways to produce nutrients such
 
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    as vitamins.
 
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</p>
 
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<p class="fancy">
 
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    To give a proof-of-concept, we have used our safe probiotic to deliver
 
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    to gut mucosa a neutralizing antibody (Ab 54,6) against an emerging
 
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    Norovirus (NoV), one of the most common causes of gastroenteritis in the
 
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    world*. To this end, we used a LPP-OmpA based cell display system* to
 
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    express the scFv (single chain fragment variable) format of the antibody
 
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    attached to the bacterial surface. According to the literature, scFvs
 
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    anchored to the bacterial surface can bind multiple viral particles
 
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    and protect efficiently against infection.  The chosen scFv sequence,
 
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    which was already known to inhibit Norovirus (NoV) interaction with
 
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    cells*, was inserted in frame downstream the LPP-OmpA sequence and
 
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    this construct was cloned under a constitutive promoter. LPP-OmpA is a
 
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    chimeric sequence having part of the major outer membrane lipoprotein
 
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    and an outer membrane porin OmpA fragment. This chimeric sequence acts
 
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    as a leader sequence and an anchor, it transports the scFv fused at its
 
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    C-terminus through the cytoplasm membrane into the periplasm, where the
 
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    scFv assumes the right conformation. OmpA portion then introduces itself
 
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    into the outer membrane displaying extracellularly its C terminus with
 
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    the Ab attached on.
 
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    <br/>The anti-NoV antibody was also expressed as a SIP
 
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    (Small Immuno Protein) format, which contains the scFv fused to the CH3
 
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    domain of the heavy chain of human immunoglobulin A (IgA). Since the
 
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    CH3 region is able to homodimerize, it should confer bivalent binding
 
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    properties to the anti-NoV scFv54.6. Secreted SIPs have the dual advantage
 
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    of being bivalent as full-length antibodies and being small as scFvs. They
 
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    have also been shown to have the potential to protect against enteric
 
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    infections when administered orally (Bestagno et al 2006).  Eventually,
 
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    we planned to produce also the secreted versions of the anti-NoV SIP
 
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    54.6 and of the scFv 54.6. Both the scFv and the SIP sequences were
 
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    cloned downstream the pelB leader sequence. PelB drives proteins into the
 
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    periplasmic space, where it is cleaved off. The resulting proteins can
 
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    be released into the extracellular space by passing through porins. This
 
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    system can also be used for production of small, soluble molecules.
 
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</p>
 
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             <ul>
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<li><a href="https://2012.igem.org/Team:Trieste/project">Abstract</a></li>
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<li class="select"  ><a href="https://2012.igem.org/Team:Trieste/project">Abstract</a></li>
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<li><a href="https://2012.igem.org/Team:Trieste/project#overview">Project Overview</a></li>
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<li><a href="https://2012.igem.org/Team:Trieste/project/overview">Project Overview</a></li>
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<li><a href="https://2012.igem.org/Team:Trieste/project#applications">Applications</a></li>
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<li><a href="https://2012.igem.org/Team:Trieste/project/applications">Applications</a></li>
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</ul>
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        <li><a href="https://2012.igem.org/Team:Trieste/project/modeling">Modeling</a></li>
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                        <li><a href="https://2012.igem.org/Team:Trieste/project/mainres">Main Results</a></li>
 +
</ul>
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Latest revision as of 01:30, 27 September 2012

Abstract

More

Background

Recent studies have evidenced that the intestinal microflora can actually be considered an organ of the body. It has several functions in the human gut, mostly metabolic and immunologic, and it constantly interacts with the intestinal mucosa in a delicate equilibrium. It is therefore believed that having a beneficial and healthy intestinal microflora is very important for human health.

Project

Our aim is to modify a bacteria normally found in human gut and create a safe, controllable and versatile molecular platform which can be used to produce a wide range of molecules (as for example other antibodies or enzymes) leading to a beneficial probiotic.

For this purpose we have chosen the E. coli strain Nissle 1917 which has been used for many years as a probiotic. We designed a robust gene guard system regulated by a novel and easy to control inducible switch that activates the production of a human antimicrobial peptide that can kill the bacteria and also avoid horizontal transfer.

Our gene guard system is based on the cumate molecule present in the common spice: cumin. In absence of this molecule our system produces a repressor (CymR) which inhibits the expression of an antimicrobial peptide. When administered and therefore present in human intestine, cumin recognizes and disables the CymR repressor resulting in the production of the antimicrobial peptide cathelicidin LL-37 which causes bacterial death. In case of plasmid transfer into other bacteria of the plasmid carrying the expression system, the expression of the antimicrobial peptide will be activated immediately because the receiving bacteria do not produce the CymR repressor needed to repress the expression of the antimicrobial peptide.

Application

The safe probiotic constructed here can be used to produce nutritious, preventive or therapeutic molecules. For example, we have used it to produce an antibody against the emerging virus called Norovirus; this virus which has a dramatic spreading speed: rates of reported Norovirus outbreaks reach 21 million cases of acute gastroenteritis per year, 70.000 of those need hospitalization and 800 die.

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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