Team:NTU-Taida/PEPDEX

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{{:Team:NTU-Taida/Templates/BSHero|Title=Overall Project|Content=}}
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{{:Team:NTU-Taida/Templates/BSHero|Title=Circuit|Content=<p>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.
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{{:Team:NTU-Taida/Templates/BSHero|Title=Stability and Safety|Content=<p>Every GM system that will function outside lab will face two major problems:system stability and safety! Without selective pressure ,we have to deal with plasmid instability ; to make our coli colonized bowel we have to use recA+ strains which may cause plasmid multimer. Our GM coli will also contact with many kinds of bacteria at the risk of horizontal gene transfer.</p>
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==Introduction==
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<p>The following segment is our struggle against these obstacles.</p>}}
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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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==Stability of Delivery System==
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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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===Briefing===
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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 &amp; mobile gene element, we cope up with vector instability by incorporating '''partition system''', '''Multimer resolution system''' and '''toxin antitoxin system''' these modules into our design.
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===Obstacles===
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===Circuit===
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Sources of plasmid instability:
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# Segregational instability <br> Plasmids are unevenly distributed inside bacterium, after cell division, some progeny might loss plasmid.
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====Fat Extinguisher====
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# Burden Effect <br> The plasmid free cells sure will grow better than those bearing plasmids because they don't have to spend energy/resources on our pepdEX system, so this growth rate difference will finally eliminate plasmid-bearing bacteria in population
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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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#
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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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====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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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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{{:Team:NTU-Taida/Templates/ContentEnd}}{{:Team:NTU-Taida/Templates/Footer|ActiveNavbar=Project, #nav-Project-Overview}}

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.