Team:HKUST-Hong Kong/overview.html

From 2011.igem.org

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<b><h3>OVERVIEW</h3></b>
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<b><h3>Overview</h3></b>
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<h4 align=left><a name=back></a>1. Background </h4>
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<a href=#top>[Top]</a>
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<h4><a name=build></a><b>Building the horse - Engineering a novel strain for antibiotics-free selection</b></h4>
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<h4 align=left><a name=build></a>2. Building the horse - Engineering a novel strain for antibiotics-free selection</h4>
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<h4 align=left>3. The army inside - Toluene-4-Monooxygenase (T4MO)</h4>
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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.</p>
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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 or reduce 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. </p>
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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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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 <i>E. coli</i>[3], and quite possibly for other strains of <i>E. coli</i> 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.<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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<b><a name=army></a>3.1 Soldiers need shields...maybe - Multidrug Efflux Pump (Bcr)</b>
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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.
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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=>[here]</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>
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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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[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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<a href=#back>1. Background</a><br>
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<a href=#build>2. Building the horse - Engineering a novel strain for antibiotics-free selection</a><br>
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<a href=#build>Building the horse - Engineering a novel strain for antibiotics-free selection</a><br>
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<a href=#army>3. The army inside - Toluene-4-Monooxygenase (T4MO)</a><br>
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Revision as of 03:57, 5 October 2011

Overview

1. Background

[Top]

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

3. 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 or reduce 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]

3.1 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] Mutations of Toluene-4-Monooxygenase That Alter Regiospecificity of Indole Oxidation and Lead to Production of Novel Indigoid Pigments
[3] Enterohemorrhagic Escherichia coli Biofilms Are Inhibited by 7-Hydroxyindole and Stimulated by Isatin

Overview



1. Background
2. Building the horse - Engineering a novel strain for antibiotics-free selection
3. The army inside - Toluene-4-Monooxygenase (T4MO)


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