Team:NTU-Taida/PEPDEX

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__FORCETOC__{{:Team:NTU-Taida/Templates/Header}}{{:Team:NTU-Taida/Templates/Navbar}}{{:Team:NTU-Taida/Templates/Sidebar|Title=Overal Project}}{{:Team:NTU-Taida/Templates/ContentStart}}
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== '''Overall project''' ==
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In our project, we aim to utilize a microbe that responds to conditions in human body as an approach to administer smart peptide-based therapies. GLP-1, a human innate neuro-peptide for energy balance, is chosen to combat for obesity and metabolic syndrome. We engineer the non-pathogenic E. coli which senses fatty acids in intestines and secretes synthetic GLP-1. Appropriate signal peptides and penetratin are used to facilitate peptide secretion and intestinal uptake. Furthermore, we design a circuit with quorum sensing and double repressors, which aims to generate quick but sustainable responses and serves as an anti-noise filter. Plasmid stabilization modules including partition system and multimer resolution system are also incorporated to circumvent the undesirable loss or segregational instability of our artificial device. With this general concept of delivery of short peptide into human body, we can also target other human diseases with alternative circuit designs.
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== Project Details==
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==Introduction==
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There has been a wide array of peptides, either innate or synthetic, used as drugs or vaccine in combat of different diseases. For instance, insulin was synthesized and mass produced in bacteria; long, synthetic peptide vaccine has also brought to bedside to help treat patient with invasive cancers. (Nat Rev Cancer. 2008 May;8(5):351-60.) However, the delivery of the peptide into human body is a big issue, since it cannot be administered orally, and has a poor distribution and absorption as compared to small molecules drugs in human body. With the advent of biotechnology and the growing forum of synthetic biology, we try to engineer and finely design different circuits in bacteria, which can deliver peptides through epidermal, buccal, rectal, or enteral. As for a simple exemplification of our ultimate goal of microbial peptide delivery system, we choose GLP-1, the endogenous hormone as our delivery model. Our design features efficiency, quick response to the environment changes, and sustainable release of GLP-1. This is the first model of our peptide delivery system, and opens a new room for the synthetic biology in medicine applications.
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=== '''hormone therapy-body weight control''' ===
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GLP-1, a human innate neuro-peptide for energy balance, is chosen to combat for obesity and metabolic syndrome. We engineer the non-pathogenic ''E. coli'' which senses fatty acids in intestines and secretes synthetic GLP-1. Appropriate signal peptides and penetratin are used to facilitate peptide secretion and intestinal uptake. Furthermore, we design a circuit with quorum sensing and double repressors, which aims to generate quick but sustainable responses and serves as an anti-noise filter. Plasmid stabilization modules including partition system and multimer resolution system are also incorporated to circumvent the undesirable loss or segregational instability of our artificial device. With this general concept of delivery of short peptide into human body, we can also target other human diseases with alternative circuit designs.
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====Background====
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====Design====
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===Circuit===
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CPP
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[[File:https://www.dropbox.com/s/1lnt1d2uo401rz6/123.mov]]
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fadR
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====Fat Extinguisher====
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The main circuit is designed to detect the presence of fatty acid in the intestinal environment and produce the peptide drug (GLP-1 in this case) and cell penetrating peptide (CPP) as response. There are two core systems: double repressors and quorum sensing, which aims to corroborate a quick responsive to fat ingestion and stable functioning circuit.
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We start the circuit with fatty acid sensor (fadR), which is followed by double repressor (tetR and LacI). The fadR is a novel promoter which can detect the presence of fatty acid; the double repressor tetR and LacI combine to make the circuit more stable and form a prominent threshold.
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[[FIle:FadR-DNA.png|400px]]
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====Thermal Avenger====
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We designed a thermal sensitive promoter in our circuit to sense the change of temperature of ambient. The temperature sensitive repressor CI would dimerize in room temperature and suppress the expression of downward gene sequence; however, when the temperature rises, the dimer would break and lose the function of repression. As our experiment proves, the circuit works differently under room temperature from inside human body (37<sup>0</sup>C). As for application of the thermal sensitive device, first, we incorporate toxin and anti-toxin in our thermal promoter, which works as a self destructor. Second, we can utilize the thermal sensitive characteristic of the circuit to make a baseline secretion level of the GLP-1.
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===Stability and Safety===
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{{:Team:NTU-Taida/VideoTest}}
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Every genetically modified (GM) system that functions outside of the laboratories will face two major problems: system stability and safety! Without selective pressure, we have to deal with plasmid segregation instability, which may lead to a incomplete system due to loss of circuit pieces; to make our lab ''E. coli'' colonized bowel we have to use recA+ strains which may cause plasmid multimer. Our GM lab E. coli will also be in contact with many kinds of wild bacteria and is under the risk of horizontal gene transfer.<br />
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====Modeling====
 
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====Results====
 
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====Extension====
 
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=== '''peptide2''' ===
 
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====Background====
 
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====Design====
 
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====Modeling====
 
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====Results====
 
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====Extension====
 
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=== '''delivery circuit'''  ===
 
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====Background====
 
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====Design====
 
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====Modeling====
 
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====Results====
 
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====Extension====
 
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=== '''Stability of delivery system''' ===
 
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====Briefing====
 
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<p>As our system will function outside the labrotory and human gut lack for antibiotic selection pressure, the vector stability is the critical point that determine whether our system is applicable or not. Inspired by natural plasmid & transposable gene element, we cope up with vector instability by incorporating <strong>partition system</strong>, <strong>Multimer resolution system</strong> and <strong>toxin antitoxin system</strong> these modules into our design.</p>
 
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====Background====
 
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plasmid instability
 
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<html>
 
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<dl>
 
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<dt>multimer resolution system</dt>
 
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<dd>A description list is perfect for defining terms.</dd>
 
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<dt>partition system</dt>
 
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<dd>A description list is perfect for defining terms.</dd>
 
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<dt>Post-segregation killing</dt>
 
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<dd>A description list is perfect for defining terms.</dd>
 
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</dl>
 
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</html>
 
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====Design====
 
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<p class="lead">multimer resolution system:Tn1000(gamma delta) resolution system</p>
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https://static.igem.org/mediawiki/2012/b/b6/NTU-Taida-Mrs-1.png
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https://static.igem.org/mediawiki/2012/c/c6/NTU-Taida-Mrs-2.png
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https://static.igem.org/mediawiki/2012/9/9f/NTU-Taida-Mrs-3.png
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https://static.igem.org/mediawiki/2012/1/13/NTU-Taida-Mrs-4.png
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<p class="lead">partition system:from <em>pseudomonas putida </em>KT2440</p>
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<p class="lead">Post-segregation killing:srnBC toxin-antitoxin system</p>
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<html>
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<table class='table table-bordered'>
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<thead>
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<tr>
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<th>HEAD</th>
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<th>head</th>
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<tbody>
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  <tr>
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      <td>1</td>
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      <td>2</td>
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  <tr>
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      <td>3</td>
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      <td>4</td>
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      <td>5</td>
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      <td>6</td>
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<p class="lead">reference</p>
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<ul class="unstyled">
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<li>1.reference one</li>
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<li>2.reference two</li>
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</ul>
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=== '''safety of delivery system''' ===
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suicidal system:
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    <div class="row-fluid">
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    <div class="span12">
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      Level 1 of column
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      <div class="row-fluid">
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      <div class="span6">Level 2</div>
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      <div class="span6">Level 2</div>
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    </div>
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Many turn off strategies have been developed, most of these are the inducible suicide system that can be activated at certain condition. For instance, in our project, we plan to use temperature and small molecule as activating signals( following picture). When the course of treatment ends administration of small amount of tetracycline agonist will induce bacterial death, leaving human body also cause suicide gene activation thereby avoid recombinant strain/gene pollutiing. And splitting suicide system to provide repression in trans can prevent plasmid transfering to wild type strains.
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However, these design cannot totally eliminate the risk of horizontal gene transfer(HGT), which recombinant genes can move to other organisms independent of suicide system. So besides suicide system, we come up with a new idea to deal with these kinds of HGT risks by RNA interaction!!!
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Expression well designed antisense RNAs have been shown to have inhibitory effect on paired RNAs, putting appropriate antisense RNAs on untranslated region of transcripts may interfere target RNA function or translation. We can use this tool to prevent HGT. For instance, laboratory E.coli have inactivated all its hok/sok toxin-antitoxin system by mutation, but wild bacteria especially pathogenic bacteria have many active TA locus on its chromosome like E.coli O157 have many active hok/sok homologs. HGT is more likely to occur between related species, we plan to put a stem loop from hok mRNA which can interact with sok RNA 5’sequence on UTR of antibiotic resistance genes we used. IF wild E.coli steal our antibiotic resistance genes and express it, its antitoxin will be competitive inhibited and its toxin will express and kill the thief thus preventing HGT between lab & wild coli. This idea is can have wide extension. Besides targeting antitoxin (functional RNA) of type I TA. We can also design antisense sequences that target RBS then we can down regulate target protein. Type II TA and essential genes for metabolism can also be targets Even if the
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In the past the repression efficiencies of antisense RNA in bacteria are low, but by using the paired termini antisense RNA method and incorporate U turn/YUNR motif,
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==Modeling==
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==Results==
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<html>
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<table class='table table-bordered'>
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<thead>
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<tr>
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<th>HEAD</th>
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<th>head</th>
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</tr>
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</thead>
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<tbody>
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  <tr>
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      <td>1</td>
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      <td>2</td>
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  </tr>
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  <tr>
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      <td>3</td>
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      <td>4</td>
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  </tr>
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  <tr>
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      <td>5</td>
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      <td>6</td>
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  </tr>
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</tbody>
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</table>
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</html>
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==Extension==
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{{:Team:NTU-Taida/Templates/ContentEnd}}{{:Team:NTU-Taida/Templates/Footer|ActiveNavbar=Project}}
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Latest revision as of 17:15, 25 October 2012

Overal Project

Overall Project

Contents

Introduction

There has been a wide array of peptides, either innate or synthetic, used as drugs or vaccine in combat of different diseases. For instance, insulin was synthesized and mass produced in bacteria; long, synthetic peptide vaccine has also brought to bedside to help treat patient with invasive cancers. (Nat Rev Cancer. 2008 May;8(5):351-60.) However, the delivery of the peptide into human body is a big issue, since it cannot be administered orally, and has a poor distribution and absorption as compared to small molecules drugs in human body. With the advent of biotechnology and the growing forum of synthetic biology, we try to engineer and finely design different circuits in bacteria, which can deliver peptides through epidermal, buccal, rectal, or enteral. As for a simple exemplification of our ultimate goal of microbial peptide delivery system, we choose GLP-1, the endogenous hormone as our delivery model. Our design features efficiency, quick response to the environment changes, and sustainable release of GLP-1. This is the first model of our peptide delivery system, and opens a new room for the synthetic biology in medicine applications.

GLP-1, a human innate neuro-peptide for energy balance, is chosen to combat for obesity and metabolic syndrome. We engineer the non-pathogenic E. coli which senses fatty acids in intestines and secretes synthetic GLP-1. Appropriate signal peptides and penetratin are used to facilitate peptide secretion and intestinal uptake. Furthermore, we design a circuit with quorum sensing and double repressors, which aims to generate quick but sustainable responses and serves as an anti-noise filter. Plasmid stabilization modules including partition system and multimer resolution system are also incorporated to circumvent the undesirable loss or segregational instability of our artificial device. With this general concept of delivery of short peptide into human body, we can also target other human diseases with alternative circuit designs.

Circuit

Fat Extinguisher

The main circuit is designed to detect the presence of fatty acid in the intestinal environment and produce the peptide drug (GLP-1 in this case) and cell penetrating peptide (CPP) as response. There are two core systems: double repressors and quorum sensing, which aims to corroborate a quick responsive to fat ingestion and stable functioning circuit. We start the circuit with fatty acid sensor (fadR), which is followed by double repressor (tetR and LacI). The fadR is a novel promoter which can detect the presence of fatty acid; the double repressor tetR and LacI combine to make the circuit more stable and form a prominent threshold.

Thermal Avenger

We designed a thermal sensitive promoter in our circuit to sense the change of temperature of ambient. The temperature sensitive repressor CI would dimerize in room temperature and suppress the expression of downward gene sequence; however, when the temperature rises, the dimer would break and lose the function of repression. As our experiment proves, the circuit works differently under room temperature from inside human body (370C). As for application of the thermal sensitive device, first, we incorporate toxin and anti-toxin in our thermal promoter, which works as a self destructor. Second, we can utilize the thermal sensitive characteristic of the circuit to make a baseline secretion level of the GLP-1.

Stability and Safety

Every genetically modified (GM) system that functions outside of the laboratories will face two major problems: system stability and safety! Without selective pressure, we have to deal with plasmid segregation instability, which may lead to a incomplete system due to loss of circuit pieces; to make our lab E. coli colonized bowel we have to use recA+ strains which may cause plasmid multimer. Our GM lab E. coli will also be in contact with many kinds of wild bacteria and is under the risk of horizontal gene transfer.