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| | 70.000 of those need hospitalization and 800 die. | | 70.000 of those need hospitalization and 800 die. |
| | </p> | | </p> |
| - | <h1>Project Overview</h1>
| |
| - | <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
| |
| - | is composed of approximately 500 anaerobic and aerobic bacteria species,
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| - | most of them localized in large intestine.
| |
| - | <br/>
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| - | Many studies on animals
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| - | bred under germ-free conditions have shown that microflora has specific
| |
| - | functions:
| |
| - | <ul class="fancy">
| |
| - | <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;
| |
| - | </li>
| |
| - | <li>
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| - | Trophic: control of epithelial cell proliferation and differentiation;
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| - | development and homoeostasis of the immune system;
| |
| - | </li>
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| - | <li>Protective: protection against pathogens. </li>
| |
| - | </ul>
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| - |
| |
| - | 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>
| |
| - | <h3>Project overview</h3>
| |
| - | <p class="fancy">
| |
| - | 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. </br>
| |
| - | The gene guard system is divided in two parts:
| |
| - | <ul class="fancy">
| |
| - | <li>the killing mechanism</li>
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| - | <li>the regulating pattern</li>
| |
| - | </ul>
| |
| - | </p>
| |
| - | <p class="fancy">
| |
| - | The killing mechanism is placed on the same plasmid used for protein
| |
| - | expression. It consists of two different proteins, T4 Holin and
| |
| - | Cathelicidin LL-37, both toxic for prokaryotic cells. These antimicrobial
| |
| - | proteins are both regulated by a cumate responsive promoter (T5 promoter
| |
| - | Cumate Operator) 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.
| |
| - | <br/>
| |
| - | The cumate-responsive regulator CymR,
| |
| - | 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.
| |
| - | </p>
| |
| - | <p class="fancy">
| |
| - | <strong>No Cumate Expression Mode:</strong>
| |
| - | 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!
| |
| - | </p>
| |
| - | <p class="fancy">
| |
| - | <strong>Cumate-Induction Kill Mode:</strong>
| |
| - | 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!
| |
| - | </p>
| |
| - | <p class="fancy">
| |
| - | <strong>Plasmid Transfer:</strong>
| |
| - | <ol class="fancy">
| |
| - | <li>
| |
| - | 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 responsiv promoter (T5 Cumate
| |
| - | Operator) is derepressed and the T4 Holin and Cathelicidin
| |
| - | LL-37 expression is activated.<br/>Receiving bacteria die!
| |
| - | </li>
| |
| - | <li>
| |
| - | E. coli Nissle looses the plasmid. It continues to produce the
| |
| - | CymR repressor, which does not appear to be toxic for prokaryotic
| |
| - | cells.<br/>Bacteria live and is harmless!
| |
| - | </li>
| |
| - | </ol>
| |
| - | </p>
| |
| - | <h3>Application</h3>
| |
| - | <p class="fancy">
| |
| - | The safe probiotic constructed here can be used to produce nutritious,
| |
| - | preventive or therapeutic molecules. For example, this E.coli can
| |
| - | express antitumorals, antibodies against different intestinal pathogens,
| |
| - | imunomodulators, antigens (recombinant proteins which act as mucosal
| |
| - | vaccine) which are important for the correct development of the immune
| |
| - | system etc. It can express also different enzymes as lactase or enzymes
| |
| - | needed to recreate the metabolic pathways to produce nutrients such
| |
| - | as vitamins.
| |
| - | </p>
| |
| - | <p class="fancy">
| |
| - | To give a proof-of-concept, we have used our safe probiotic to deliver
| |
| - | to gut mucosa a neutralizing antibody (Ab 54,6) against an emerging
| |
| - | Norovirus (NoV), one of the most common causes of gastroenteritis in the
| |
| - | world*. To this end, we used a LPP-OmpA based cell display system* to
| |
| - | express the scFv (single chain fragment variable) format of the antibody
| |
| - | attached to the bacterial surface. According to the literature, scFvs
| |
| - | anchored to the bacterial surface can bind multiple viral particles
| |
| - | and protect efficiently against infection. The chosen scFv sequence,
| |
| - | which was already known to inhibit Norovirus (NoV) interaction with
| |
| - | cells*, was inserted in frame downstream the LPP-OmpA sequence and
| |
| - | this construct was cloned under a constitutive promoter. LPP-OmpA is a
| |
| - | chimeric sequence having part of the major outer membrane lipoprotein
| |
| - | and an outer membrane porin OmpA fragment. This chimeric sequence acts
| |
| - | as a leader sequence and an anchor, it transports the scFv fused at its
| |
| - | C-terminus through the cytoplasm membrane into the periplasm, where the
| |
| - | scFv assumes the right conformation. OmpA portion then introduces itself
| |
| - | into the outer membrane displaying extracellularly its C terminus with
| |
| - | the Ab attached on.
| |
| - | <br/>The anti-NoV antibody was also expressed as a SIP
| |
| - | (Small Immuno Protein) format, which contains the scFv fused to the CH3
| |
| - | domain of the heavy chain of human immunoglobulin A (IgA). Since the
| |
| - | CH3 region is able to homodimerize, it should confer bivalent binding
| |
| - | properties to the anti-NoV scFv54.6. Secreted SIPs have the dual advantage
| |
| - | of being bivalent as full-length antibodies and being small as scFvs. They
| |
| - | have also been shown to have the potential to protect against enteric
| |
| - | infections when administered orally (Bestagno et al 2006). Eventually,
| |
| - | we planned to produce also the secreted versions of the anti-NoV SIP
| |
| - | 54.6 and of the scFv 54.6. Both the scFv and the SIP sequences were
| |
| - | cloned downstream the pelB leader sequence. PelB drives proteins into the
| |
| - | periplasmic space, where it is cleaved off. The resulting proteins can
| |
| - | be released into the extracellular space by passing through porins. This
| |
| - | system can also be used for production of small, soluble molecules.
| |
| - | </p>
| |
| | </div> | | </div> |
| | </div> <!-- end box_main --> | | </div> <!-- end box_main --> |
"
