Team:HKUST-Hong Kong/overview.html

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<b><h3>Overview</h3></b>
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<a href=#back><img src="https://static.igem.org/mediawiki/2011/4/4a/Ust_back.gif" width=100 height=100 alt="Background"></a>
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<a href=#plan><img src="https://static.igem.org/mediawiki/2011/6/68/Ust_battle.gif" width=100 alt="The Battle Plan" height=100></a>
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<img src="https://static.igem.org/mediawiki/2011/e/e4/Ust_build.gif" width=100 height=100 alt=" Building the horse - Engineering a novel strain for antibiotics-free selection"></a>
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<img src="https://static.igem.org/mediawiki/2011/2/23/Ust_army.gif" width=100 height=100 alt="The army inside - Toluene-4-Monooxygenase (T4MO)"></a>
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<b><font size=14> &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;Overview</font></b>
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&nbsp;  &nbsp;
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<a href=#back>Background</a> · &nbsp;
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<a href=#plan>The Battle Plan</a> · &nbsp;
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<a href=#build>Building the horse</a> · &nbsp;
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<a href=#army>The army inside</a>
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<h4 align="left"><a name=back></a>1. Background </h4>
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<b><a name=back></a>1. Background </b>
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In the past decade, the number of medical cases involving multi-antibiotic resistant microbes has seen a steady increase, and misuse of antibiotics has repeatedly been mentioned as one of the major reasons for the arise of such organisms. This situtation provided the inspiration for our project, which is to attempt to improve the effectiveness of antibiotics, so that we can apply less of it to achieve the same effect as we do now.
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In the past decade, the number of medical cases involving multi-antibiotic resistant microbes has seen a steady increase, and misuse of antibiotics has repeatedly been mentioned as one of the major reasons for the arise of such organisms. This situation provided the inspiration for our project, which is to attempt to improve the effectiveness of antibiotics, so that we can apply less of it to achieve the same effect as we do now.
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<b><a name=plan></a>2. The Battle Plan</b>
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Indole has been proposed as a key signalling molecule for <i>E. coli</i> in mediating resistance to environmental stress. In particular, indole is found to stimulate passive resistance against antibiotics by triggering stress-response mechanisms inside cells, including increased expression of drug efflux pumps and oxidative-damage repairing systems.  
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Indole has been proposed as a key signalling molecule for <i>E. coli</i> in mediating resistance to environmental stress.[1] In particular, indole is found to stimulate passive resistance against antibiotics by triggering stress-response mechanisms inside cells, including increased expression of drug efflux pumps and oxidative-damage repair systems.  
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In a normal <i>E. coli</i>  popoulation containing wild type and naturally resistant mutants, the mutants will supply indole to nearby cells that are under antibiotic stress. This is considered a 'charity' action as the mutant does so at a fitness cost to itsef. Our aim is to introduce E. trojan into the population and proceed to degrade indole via an enzyme (T4MO) and remove the charity effect. If the charity effect is significant, removal of its presence will cause a notable increase in the effectiveness of antibiotics.
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In a normal <i>E. coli</i>  population containing wild type and naturally resistant mutants, the mutants will supply indole to nearby cells that are under antibiotic stress. This is considered to be a 'charity' action as the mutant does so at a fitness cost to itself. Our aim is to introduce E. trojan into the population and proceed to degrade indole via an enzyme (T4MO) and remove the charity effect. If the charity effect is significant, removal of its presence will cause a notable increase in the effectiveness of antibiotics.
<a href=#top>[Top]</a>
<a href=#top>[Top]</a>
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<h4 align=left><a name=build></a>3. Building the horse - Engineering a novel strain for antibiotics-free selection</h4>
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<b><a name=build></a>3. Building the horse - Engineering a novel strain for antibiotics-free selection</b>
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Our <i>E. Trojan</i> that carries the T4MO gene must remain sensitive to antibiotics while it degrades intracellular indole, otherwise it would mean violation of our original intention since it would introduce another high resistant strain into the population, and decrease the effectiveness of antibiotics.
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To study the interaction and population dynamics of the naturally-coexisting HR (high resistance) and LR (low resistance) <i>E. coli</i> strains under antibiotic selection pressure, the E. trojan - which is to be introduced to HR/ LR mixed culture - should harbor the T4MO gene to allow it to perform quorum sensing disruption between the two strains. In addition, the E. trojan should not possess a wide spectrum of antibiotic resistance as this would give it an inherent selective advantage, thus interfering with the relationship between MICs and indole degradation in our study.
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Nonetheless, molecular cloning needed to be performed for bringing the T4MO gene and the bcr gene into antibiotics-sensitive bacteria. Here we encounter a problem: how to perform cloning without using antibiotics as the selection marker?
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To fulfill the above criteria, it becomes necessary for us to first construct a new, pre-E. trojan bacterial strain (E. CRAFT) that is capable of performing plasmid selection without antibiotics and consequently contains as few antibiotic resistance genes as possible. Due to some limitations of the various alternative (non-antibiotic) selection methods[2] available today, we have designed E. CRAFT in a way that causes it to perform selection by imposing internal pressure of holding an essential gene for survival. Upon the completion of this precursor strain, the E. trojan can be easily constructed by transformation of the T4MO gene into it. In the long run, however, we hold a hope that this novel strain will prove to be a versatile, effective alternative to the widely- used antibiotic selection method.<a href=#top>[Top]</a>
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Other common and readily available selection methods include auxotrophic and those that utilize toxin-antitoxins interactions, yet they have their own drawbacks in our study of bacterial population dynamics.
 
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Thus we propose the construction of EX, which would be a new bacterial strain that in theory can be selected for plasmid while remains possesses no antibiotic resistance. Selection can be performed under a simple change in physical environment – increasing the incubation temperature. The trick behind the mechanism originates from yeast artificial chromosome. It is the removal of an essential gene <i>nadE</i> and its relocation to a piece of extra-chromosomal DNA that we wish to maintain. <a href=#top>[Top]</a>
 
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<h4 align=left><a name=army></a>4. The army inside - Toluene-4-Monooxygenase (T4MO)</h4>
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<b><a name=army></a>4. The army inside - Toluene-4-Monooxygenase (T4MO)</b>
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As our novel strain will have one of its essential genes maintained in a plasmid, a lot of uncertainties are present regarding its fitness. It is quite possible that it will be more vulnerable to antibiotics compared with wild type and thus will be selected against when cultured together with wild type <i>E. coli</i>. This would reduce the effectiveness of our construct and is not desirable. Hence we planed an additional component to be maintained in the plasmid, known as Bcr, which we hope will boost the resistance of <i>E. Trojan</i>. You will find more details on its construction <a href="asm.html"  target=_top><b>here</b></a>.<a href=#top>[Top]</a></p>
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As our novel strain will have one of its essential genes maintained in a plasmid, a lot of uncertainties are present regarding its fitness. It is quite possible that it will be more vulnerable to antibiotics compared with wild type and thus will be selected against when cultured together with wild type <i>E. coli</i>. This would reduce the effectiveness of our construct and is not desirable. Hence we planed an additional component to be maintained in the plasmid, known as Bcr, which we hope will boost the resistance of E. trojan. You will find more details on its construction <a href="asm.html"  target=_top><b>here</b></a>.<a href=#top>[Top]</a></p>
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[1] <a href=http://www.nature.com/nature/journal/v467/n7311/pdf/nature09354.pdf>Bacterial charity work leads to population-wide resistance</a>
[1] <a href=http://www.nature.com/nature/journal/v467/n7311/pdf/nature09354.pdf>Bacterial charity work leads to population-wide resistance</a>
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[2] <a href=aem.asm.org/cgi/reprint/71/9/5476.pdf>Mutations of Toluene-4-Monooxygenase That Alter Regiospecificity of Indole Oxidation and Lead to Production of Novel Indigoid Pigments</a>
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[2] <a href=http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2941680/pdf/1475-2859-9-65.pdf>Antibiotic-free selection in <i>E. coli</i>: new considerations for optimal design and improved production</a>
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[3] <a href=aem.asm.org/cgi/reprint/73/13/4100.pdf>Enterohemorrhagic Escherichia coli Biofilms Are Inhibited by 7-Hydroxyindole and Stimulated by Isatin</a><br><br>
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[3] <a href=aem.asm.org/cgi/reprint/71/9/5476.pdf>Mutations of Toluene-4-Monooxygenase That Alter Regiospecificity of Indole Oxidation and Lead to Production of Novel Indigoid Pigments</a>
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[4] <a href=aem.asm.org/cgi/reprint/73/13/4100.pdf>Enterohemorrhagic Escherichia coli Biofilms Are Inhibited by 7-Hydroxyindole and Stimulated by Isatin</a><br><br>
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<b><font color="green">Our Project</font></b></p>
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<a href="https://2011.igem.org/Team:HKUST-Hong_Kong/overview.html" target=_top>Overview</a><font color="green"> | </font>
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<b><font color="green">Experiments and Results</font></b></p>
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Latest revision as of 17:51, 28 October 2011


Background The Battle Plan  Building the horse - Engineering a novel strain for antibiotics-free selection The army inside - Toluene-4-Monooxygenase (T4MO)              Overview
    Background ·   The Battle Plan ·   Building the horse ·   The army inside



1. Background

In the past decade, the number of medical cases involving multi-antibiotic resistant microbes has seen a steady increase, and misuse of antibiotics has repeatedly been mentioned as one of the major reasons for the arise of such organisms. This situation provided the inspiration for our project, which is to attempt to improve the effectiveness of antibiotics, so that we can apply less of it to achieve the same effect as we do now.

Our project name - E. trojan - has its origins rooted in the Trojan Horse found in the tale of the Trojan War. Similar to how the Greeks destroyed the city of Troy from within using a wooden horse filled with soldiers, our E. trojan also works to destroy an E. coli population that is under antibiotic stress from within through indole quorum-sensing disruption. [Top]

2. The Battle Plan

Indole has been proposed as a key signalling molecule for E. coli in mediating resistance to environmental stress.[1] In particular, indole is found to stimulate passive resistance against antibiotics by triggering stress-response mechanisms inside cells, including increased expression of drug efflux pumps and oxidative-damage repair systems.

In a normal E. coli population containing wild type and naturally resistant mutants, the mutants will supply indole to nearby cells that are under antibiotic stress. This is considered to be a 'charity' action as the mutant does so at a fitness cost to itself. Our aim is to introduce E. trojan into the population and proceed to degrade indole via an enzyme (T4MO) and remove the charity effect. If the charity effect is significant, removal of its presence will cause a notable increase in the effectiveness of antibiotics. [Top]

3. Building the horse - Engineering a novel strain for antibiotics-free selection

To study the interaction and population dynamics of the naturally-coexisting HR (high resistance) and LR (low resistance) E. coli strains under antibiotic selection pressure, the E. trojan - which is to be introduced to HR/ LR mixed culture - should harbor the T4MO gene to allow it to perform quorum sensing disruption between the two strains. In addition, the E. trojan should not possess a wide spectrum of antibiotic resistance as this would give it an inherent selective advantage, thus interfering with the relationship between MICs and indole degradation in our study.

To fulfill the above criteria, it becomes necessary for us to first construct a new, pre-E. trojan bacterial strain (E. CRAFT) that is capable of performing plasmid selection without antibiotics and consequently contains as few antibiotic resistance genes as possible. Due to some limitations of the various alternative (non-antibiotic) selection methods[2] available today, we have designed E. CRAFT in a way that causes it to perform selection by imposing internal pressure of holding an essential gene for survival. Upon the completion of this precursor strain, the E. trojan can be easily constructed by transformation of the T4MO gene into it. In the long run, however, we hold a hope that this novel strain will prove to be a versatile, effective alternative to the widely- used antibiotic selection method.[Top]

4. The army inside - Toluene-4-Monooxygenase (T4MO)

Toluene-4-Monooxygenase (T4MO) is a three-component enzyme system that is naturally able to degrade a variety of hydrocarbons. In particular, its ability to oxidize small aromatic compounds has caught the attention of scientists looking for biological means of producing certain useful organic chemicals for industrial use, resulting in extensive research on modifying its activity and affinity.

For our project, a mutated form of T4MO (double mutations at G103L & A107G) was chosen as the candidate for indole degradation. While there were several possible candidates, this particular one was known to convert almost 50% of the indole into 7-hydroxyindole (7-HI)[2]. This is of great interest to us as 7-HI has been documented to inhibit biofilm formation in Enterohemorrhagic E. coli[3], and quite possibly for other strains of E. coli as well. Biofilm formation is a major element that reduces the efficiency of antibiotics due to reduced area of action, and hence we were keen to take advantage of this synergistic effect to improve our construct.[Top]

4.1 Soldiers need shields...maybe - Multidrug Efflux Pump (Bcr)

As our novel strain will have one of its essential genes maintained in a plasmid, a lot of uncertainties are present regarding its fitness. It is quite possible that it will be more vulnerable to antibiotics compared with wild type and thus will be selected against when cultured together with wild type E. coli. This would reduce the effectiveness of our construct and is not desirable. Hence we planed an additional component to be maintained in the plasmid, known as Bcr, which we hope will boost the resistance of E. trojan. You will find more details on its construction here.[Top]


[1] Bacterial charity work leads to population-wide resistance
[2] Antibiotic-free selection in E. coli: new considerations for optimal design and improved production
[3] Mutations of Toluene-4-Monooxygenase That Alter Regiospecificity of Indole Oxidation and Lead to Production of Novel Indigoid Pigments
[4] Enterohemorrhagic Escherichia coli Biofilms Are Inhibited by 7-Hydroxyindole and Stimulated by Isatin

Home

Our Project

Overview | Data Page

Experiments and Results

Strain Construction | Culture Tests | Modeling

Miscellaneous

Notebook

iGEM Resources

Acknowledgements

The Team

iGEM Member List | Contributions

Achievements

Medal Requirements | BioSafety

BioBricks

Master List & Characterization Data

Human Practice

Workshop | Survey