Difference between revisions of "Team:REC-CHENNAI/Description"

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<h1>GENETIC ENGINEERING</h1>
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<h1>QUORUM SENSING</h1>
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<p>Our project involves the production of Lt2a in genetically modified organisms. We chose E.coli DH5a to be our chassis due to its multiple mutations that correspond to its distinct characteristics. These mutations allow for blue-white screening for recombinants and ensure high plasmid transfer rates by lowering endonuclease activity.</p>
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<p>Our project involves genetically engineering <i>E.coli DH5a </i> cells to produce Latarcin and the mutant. We chose <i> E.coli </i> to be our chassis due to its higher transformation efficiency. However, a major problem that we had to overcome is the toxicity of this peptide to the chassis itself. In order to overcome this, a specially designed quorum sensing mechanism is employed. </p>
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<p>A major issue that was to be tackled during the course of our project was Host toxicity. Latarcin being antimicrobial would naturally be lethal to the chassis itself. In order to overcome this, a specially designed quorum sensing mechanism is employed.</p>
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<p> Bacterial quorum sensing is defined as a cell-to-cell communication mechanism wherein gene expression is regulated by fluctuations in cell concentration. Bacteria that use quorum sensing mechanisms release certain signalling molecules known as autoinducers into their surroundings. Beyond a threshold limit, these autoinducers alter gene expression by binding to specific promoters within the genome. </p>
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                      <p> Quorum sensing is an essential element in our project because the Latarcin that is produced by the E. coli cells is deadly to the bacteria itself. This will not allow for large scale production of Latarcin. When the quorum sensing mechanism is incorporated, Latarcin production will begin only after the cells reach a high concentration. Thus, large quantities of Latarcin can be synthesized. </p>
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                      <p> The proposed genetic circuit for quorum sensing consists of three functional devices. The first of these three contains a constitutive promoter (Anderson promoter) coding for the gene for AHL synthesis (LuxI). The second device has another Anderson promoter that regulates the LuxR repressor gene. The last has a Lux promoter that is activated by the binding of AHL and LuxR. This regulates the expression of the mutant Latarcin. </p>
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                      <p> At low cell density, the quantity of AHL produced is too little for it to bring about Latarcin production. As the cell density increases, the cells divide and simultaneously produce both AHL and LuxR. Without the AHL, LuxR is unstable and gets degraded shortly after production. But beyond a certain threshold level, AHL begins forming a complex with LuxR, preventing its degradation. This complex now binds to the Lux promoter that regulates Latarcin synthesis. On binding, the promoter immediately switches ON and begins to produce a large amount of Latarcin. A small portion of the synthesised Latarcin destroys the host, and the rest can now be isolated and purified. </p>
 
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Revision as of 16:21, 1 November 2017