Difference between revisions of "Team:IIT Madras"

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the wild type compared to the mutants which have developed antibiotic resistance</li>
 
the wild type compared to the mutants which have developed antibiotic resistance</li>
 
</ol>
 
</ol>
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<p>Most pathogenic microbes secrete quorum sensing (QS) molecules like AHL, AI-2 when present in high
 
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cell density. QS molecules are sensed by receptor proteins on the cell surface of pathogens. These
 
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signaling molecules help them in regulating their communal activities.</p>
 
  
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<p>In the lab, we will be using E.Coli DH5alpha strain as pathogenic model. DH5alpha does not secrete
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<p>Consequentially this will lead to a population of predominantly wild type bacteria. Again, as the
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quorum sensing molecules natively. Therefore, we will genetically modify E. coli DH5alpha strain to
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population of wild type goes up, the same cycle of steps 1-3 is repeated. After few cycles, the pathogens
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release Auto-Inducer-2, a signaling molecule.[Ref. 4] We do this so as to create a recombinant that
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should be eliminated.</p>
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mimics many pathogenic bacterium that secrete quorum sensing molecules.</p>
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<p>We will be using Lactococcus lactis MG1363 strain as a receiver. It has a simple circuit to detect high and
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<p>The system we propose will use an anti microbial peptide Alyteserin, which has bactericidal
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low cell density of pathogens and release the appropriate molecules. L. lactis activates the expression
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effects. Alyteserin has a lethal effect on gram negative bacteria.[Ref. 2] We have designed a
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of Alyteserin (antu-microbial peptide) at high cell density and NAly (neutralizing anit-microbila peptide)
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novel short peptide sequence(which shall be called NAly from herein). Molecular Dynamics
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at low cell density. The precise mechanism with the genes involved is explained below.</p>
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simulations of Alyteserin and NAly have been performed under various conditions and conformations,
 +
and we have found that the two peptides interact favourably.</p>
  
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<p>At low cell density, luxP,luxQ,luxU receptor proteins act as kinases that results in the phosphorylation
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<p>The pathogen of interest, which has to be a gram negative bacterium in this case was chosen to
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of luxO (luxO-P) that activates qrr1-5 sRNAs with the help of sigma54 RNAP subunit factor, qrr RNAs degrade
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be E.Coli. We chose E.Coli for its ease of availability in a laboratory setting, but Alyteserin
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the mRNA of LuxR which has been found to activate and repress a number of gene when expressed in the cell.[Ref. 3] </p>
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is a broad antimicrobial peptide and is effective against a wide range of gram negative bacteria
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including E.Coli and Salmonella Typhi.</p>
  
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<p>While at high cell density, the same receptor proteins (LuxP,Q,U) acts as phosphatases which removes
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<p>We chose the carrier of the AMP to be Lactococcus Lactis, for several reasons. L.lactis is a
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the phosphate from LuxO-P and this results into the higher expression of LuxR gene.[Ref. 3]</p>
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gram positive bacterium. This ensures that the AMP would not have lethal effects on L. lactis
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itself. Also, L.lactis is a non-pathogenic bacteria, and will not elicit an inflammatory response
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<p>Through this project, we aim to investigate a few questions and hypothesis:</p>
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from the human immune system. Developing the genetic circuit for timed release of AMP and its
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<ol>
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inhibitory molecule inside L.lactis will provide more scope in the future for developement of a robust
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<li> Is bacterial resistance a big cause of concern when using antimicrobial peptides?</li>
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drug delivery system. </p>
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<li> Are oscillations in cell population observed when our system is used? Are the oscillations damped?</li>
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<li> How effective is the activity of NAly in neutralising the effects of Alyteserin?</li>
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</ol>
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Revision as of 08:15, 15 July 2015

The Problem

Our iGEM project aims to tackle the emerging problem of antibiotic resistance by leveraging the power of natural selection under selective pressure. It is known that higher exposure to antibiotics leads to the resistance against that antibiotic in bacterial populations.

Anti microbial peptides are small protein molecules that have been shown to have anti-microbial activity. They are also also known to exhibit lower tendency to develop antibiotic resistance. Recently, However, it has also been reported that a prolonged exposure to anti-microbial peptides could also lead to the emergence of resistance in bacteria

Our Solution

Here, we come up with a solution to this problem. We will synthesize a bacterial system that:

  1. Senses the cell density of pathogenic bacteria
  2. Our system releases anti-microbial peptides which kill pathogens, when it has sensed high cell density
  3. As the population goes down we release a peptide that neutralizes the activity of anti-microbial peptides, resulting into a stress free environment.The stress-free environment would be favourable to the wild type compared to the mutants which have developed antibiotic resistance

Consequentially this will lead to a population of predominantly wild type bacteria. Again, as the population of wild type goes up, the same cycle of steps 1-3 is repeated. After few cycles, the pathogens should be eliminated.

The system we propose will use an anti microbial peptide Alyteserin, which has bactericidal effects. Alyteserin has a lethal effect on gram negative bacteria.[Ref. 2] We have designed a novel short peptide sequence(which shall be called NAly from herein). Molecular Dynamics simulations of Alyteserin and NAly have been performed under various conditions and conformations, and we have found that the two peptides interact favourably.

The pathogen of interest, which has to be a gram negative bacterium in this case was chosen to be E.Coli. We chose E.Coli for its ease of availability in a laboratory setting, but Alyteserin is a broad antimicrobial peptide and is effective against a wide range of gram negative bacteria including E.Coli and Salmonella Typhi.

We chose the carrier of the AMP to be Lactococcus Lactis, for several reasons. L.lactis is a gram positive bacterium. This ensures that the AMP would not have lethal effects on L. lactis itself. Also, L.lactis is a non-pathogenic bacteria, and will not elicit an inflammatory response from the human immune system. Developing the genetic circuit for timed release of AMP and its inhibitory molecule inside L.lactis will provide more scope in the future for developement of a robust drug delivery system.