Team:NTU-Taida/PEPDEX

From 2012.igem.org

(Difference between revisions)
(3. Post-Segregation Killing:srnBC toxin-antitoxin system)
 
(70 intermediate revisions not shown)
Line 1: Line 1:
-
{{:Team:NTU-Taida/Templates/Header}}{{:Team:NTU-Taida/Templates/Navbar}}{{:Team:NTU-Taida/Templates/ProjectSidebar}}{{:Team:NTU-Taida/Templates/ContentStart}}
+
__FORCETOC__{{:Team:NTU-Taida/Templates/Header}}{{:Team:NTU-Taida/Templates/Navbar}}{{:Team:NTU-Taida/Templates/Sidebar|Title=Overal Project}}{{:Team:NTU-Taida/Templates/ContentStart}}
-
{{: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.
+
-
</p>}}
+
-
{{: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>
+
{{:Team:NTU-Taida/Templates/BSHero|Title=Overall Project|Content=}}
-
<p>The following segment is our struggle against these obstacles.</p>}}
+
-
==Stability of Delivery System==
+
==Introduction==
-
===Briefing===
+
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.  
-
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.
+
-
===Obstacles===
+
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.
-
Sources of plasmid instability:
+
-
# Segregational instability <p> Plasmids are unevenly distributed inside bacterium, after cell division, some progeny might loss plasmid.</p>[[File:NTU-Taida-Par1.png|500px|center]]
+
-
# Burden Effect <p>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</p>[[File:NTU-Taida-Burden3.png|500px|center]]
+
-
# Dimer Catastrophe<p>Homologues recombination may cause plasmid multimers which will increases segregational instability and burden, this is the reason why most lab coli are recA1 mutants. But so that our coli must able to colonize gut, it should be recA+ wild type strain. That's the problem!</p>[[File:NTU-Taida-Mrs-1.png|500px|center]]
+
-
===Stabilization Modules===
+
===Circuit===
-
====1. Multimer Resolution System:Tn1000(gamma delta) resolution system====
+
====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.
-
[[FIle:NTU-Taida-Mrs-2.png|350px|thumb|right|structure of MRS]]
+
====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 (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.
-
:To deal with multimerization, we clone an cassette which encodes an autoregulated resolvase(serine type recombinase)from E.coli F plasmid transposon tn1000(tn3 family). Its promoter region consists of 3 sub-sites(res site) and can process recombination. This cassette can resolve multimer that formed during replicative transposition, so it can also resolve plasmid multimer providing plasmid stability by avoiding dimer catastrophe. For this reason, it is multimer resolution system(MRS) that provide analogous function as E.coli chromosome XerCD/dif but acts independent of cell cycle, DNA localization and may have higher efficiency on plasmids compared with slow XerCD system.
+
===Stability and Safety===
-
[[FIle:NTU-Taida-Mrs-4.png|200px|thumb|center]]
+
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 />
-
[[FIle:NTU-Taida-Mrs-3.png|600px|thumb|center]]
 
-
====2. Partition System:from '''Pseudomonas putida'''KT2440====
+
<!-- EOF -->
-
 
+
{{:Team:NTU-Taida/Templates/ContentEnd}}{{:Team:NTU-Taida/Templates/Footer|ActiveNavbar=Project, #nav-Project-Overview}}
-
:Just like many low copy plasmid, bacterial chromosome distribute chromosome evenly to their progeny by certain dynamic system. These systems include SMC like proteins, type Ia partition system and so on. Type Ia partition system segregate chromosome/plasmid in a process akin to Eukaryotic mitosis,it can be found on most eubacteria but E.coli is not the case.
+
-
 
+
-
[[FIle:NTU-Taida-Par2.png|700px|thumb|center]]
+
-
 
+
-
:Previous study have shown that when provide parAB of P.putida in trans, the low copy plasmid(mini F)carrying conserved parS site can be stabilized in E.coli.(Anne-Marie etl. 2002)pSB2K3 is mini F plasmid thus can be stabilized by this way too, we make a new version of pSB2K3 with parS site. This plasmid that have lower gene dosage(copy number)and can be partitioned is an ideal vector to harbor our pepdEX system.
+
-
 
+
-
[[FIle:NTU-Taida-Par3.png|500px|thumb|center]]
+
-
 
+
-
[[FIle:NTU-Taida-Par4.png|500px|thumb|center]]
+
-
 
+
-
====3. Post-Segregation Killing:srnBC toxin-antitoxin system====
+
-
 
+
-
:No matter how well partition system and multimer resolution system work, inevitably there still will have some bacteria losing plasmid. We sentence them to death to solve the problem.
+
-
 
+
-
[[FIle:NTU-Taida-TAgrenade.png|200px]][[FIle:NTU-Taida-bacteria-work-hard.png|200px|Work hard or Die hard!!!]]
+
-
 
+
-
:Type I toxin-antitoxin srnBC is an ideal executor which belongs to hok/sok homologues, it expresses stable toxin encoded RNA and short lived antitoxin RNA that can neutralize toxin RNA by RNA interaction and RNase III cleavage. It acts as post segregation killing system, which kills bacteria when it loss the DNA(genomic islands, plasmids, mobile gene elements) that contains it. Therefore we use it to reduce plasmid loss rate and make applications without antibiotic selection more feasible.
+
-
 
+
-
[[FIle:NTU-Taida-SrnBC-Mechanism1.png|600px|thumb|center]]
+
-
 
+
-
[[FIle:NTU-Taida-SrnBC-Mechanism2.png|600px|thumb|center]]
+
-
 
+
-
 
+
-
{{:Team:NTU-Taida/Templates/ContentEnd}}{{:Team:NTU-Taida/Templates/Footer|ActiveNavbar=Project}}
+

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.