Difference between revisions of "Team:IIT Madras/Description"

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<h2>How does the circuit work?</h2>
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<h1>Project Overview</h1>
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Our team aims to construct an in vitro insulin expression system that responds to different glucose concentrations. Insulin plays a crucial role in regulating blood glucose concentration and a lack of insulin secretion can cause diabetes. Currently diabetes patients have to inject insulin to keep their blood sugar level stable at approriate time (e.g., before meal). We hope to design an insulin expression system with the ability to detect surrounding glucose concentration, and insulin is produced only when glucose concentration reaches a threshold. To achive this, we combine the CRP activator, a regulatory protein activated by cAMP, with the gene sequence coding for insulin. Since glucose concentration is reversely related with cAMP level, a change of glucose level can influence the binding of CRP with target operon, thus altering the expression of downstream sequence. By expressing our desinged gene circuit with <i>in vitro</i> protein expression system, we will then test the expression level of the gene and find out how it relates with glucose concentration.
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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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quorum sensing molecules natively. Therefore, we will genetically modify E. coli DH5alpha strain to
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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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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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low cell density of pathogens and release the appropriate molecules. L. lactis activates the expression
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of Alyteserin (antu-microbial peptide) at high cell density and NAly (neutralizing anit-microbila peptide)
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at low cell density. The precise mechanism with the genes involved is explained below.</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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of luxO (luxO-P) that activates qrr1-5 sRNAs with the help of sigma54 RNAP subunit factor, qrr RNAs degrade
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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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<p>While at high cell density, the same receptor proteins (LuxP,Q,U) acts as phosphatases which removes
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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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<p>Through this project, we aim to investigate a few questions and hypothesis:</p>
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<ol>
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<li> Is bacterial resistance a big cause of concern when using antimicrobial peptides?</li>
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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 15:42, 22 August 2015

How does the circuit work?

Most pathogenic microbes secrete quorum sensing (QS) molecules like AHL, AI-2 when present in high cell density. QS molecules are sensed by receptor proteins on the cell surface of pathogens. These signaling molecules help them in regulating their communal activities.

In the lab, we will be using E.Coli DH5alpha strain as pathogenic model. DH5alpha does not secrete quorum sensing molecules natively. Therefore, we will genetically modify E. coli DH5alpha strain to release Auto-Inducer-2, a signaling molecule.[Ref. 4] We do this so as to create a recombinant that mimics many pathogenic bacterium that secrete quorum sensing molecules.

We will be using Lactococcus lactis MG1363 strain as a receiver. It has a simple circuit to detect high and low cell density of pathogens and release the appropriate molecules. L. lactis activates the expression of Alyteserin (antu-microbial peptide) at high cell density and NAly (neutralizing anit-microbila peptide) at low cell density. The precise mechanism with the genes involved is explained below.

At low cell density, luxP,luxQ,luxU receptor proteins act as kinases that results in the phosphorylation of luxO (luxO-P) that activates qrr1-5 sRNAs with the help of sigma54 RNAP subunit factor, qrr RNAs degrade the mRNA of LuxR which has been found to activate and repress a number of gene when expressed in the cell.[Ref. 3]

While at high cell density, the same receptor proteins (LuxP,Q,U) acts as phosphatases which removes the phosphate from LuxO-P and this results into the higher expression of LuxR gene.[Ref. 3]

Through this project, we aim to investigate a few questions and hypothesis:

  1. Is bacterial resistance a big cause of concern when using antimicrobial peptides?
  2. Are oscillations in cell population observed when our system is used? Are the oscillations damped?
  3. How effective is the activity of NAly in neutralising the effects of Alyteserin?