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

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<div class="site_title">PROJECT DESCRIPTION</div>
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<h1>LATARCOLI: A KNIGHT ON GUARD AGAINST FOOD PATHOGENS</h1>
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<h3>INTRODUCTION</h3>
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In today’s world, despite all the available, sophisticated food processing techniques, right from the farm to the plate, food contamination and spoilage is a major issue. There are thousands of people in the world who die because of hunger every day, which is a shame because around 1.3 billion tonnes of food is wasted every year out of which 50-60% is spoilage due to microbial contamination.
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<h3>SCOPE IN INDIA</h3>
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In a country where 20 crore people go to sleep hungry any given night, the actual worth of the food wasted in India annually is estimated at a whopping Rs. 58,000 crore. Of the total food production that not only involves resources like lot of water, electricity, agricultural tools but also manpower and land, 40% of the total food produced is wasted. Even though India comes second in the total agricultural output worldwide, it still ranks 63<sup>rd</sup> among 88 countries in Global Hunger Index. With the amount of food wasted and considering the lack of priority that is given towards proper food storage and preservation, food is more often than not subjected to microbial contamination, thus unsurprisingly leading to eventual spoilage.
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<h3>SOLUTION</h3>
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<p>This year, we thus plan to tackle this problem to an extent. Though we cannot completely solve the wastage of food, we can surely provide a solution to bring a halt to wastage pf food by microbes. How? Well, the basic idea is to make<i> Escherichia coli</i> produce an antimicrobial peptide (AMP) called Latarcin, otherwise known as M-Zodatoxin. Latarcin is a short, basic, linear peptide isolated from a spider called<i> Lachesana tarabaevi</i>. It acts on bacteria via the carpet mechanism and effectively kills them. Thus we plan to create a wrapper for food, which will have Latarcin producing<i> Escherichia coli </i>incorporated within that will help in acting against microbial infection. It is an efficient and simple solution that we are sure will help in bringing down the total amount of food wasted due to spoilage considerably.
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<h1>PROBLEM STATEMENT</h1>
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<p>The emergence of multidrug resistance in food-borne bacteria is an increasing problem globally. Described by WHO,CDC and other International agencies as one of the World’s most pressing public health threats, antibiotic resistant infections from food-borne bacteria cause an estimated 430,000 illnesses each year, in the USA alone. This issue, thus, has attracted significant attention towards Antimicrobial peptides (AMPs).</p>
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<h1>ANTIMICROBIAL PEPTIDES- A SOLUTION</h1>
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<p>AMPs are gaining popularity due to their effectiveness against a wide range of pathogenic organisms that are resistant to conventional drugs. Since their mode of action, for the most part, exploits general but fundamental structural characteristics such as the bacterial cell membrane and in many cases they may have multiple targets within cells, the likelihood of the emergence of resistance is thought to be considerably reduced compared with that for many current antibiotics, which have more specific molecular targets (Håvard Jenssen et al).</p>
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<p>For easy biosynthetic production, we decided to work with a small peptide, devoid of cysteine knots. In addition, we preferred that it not be easily degraded by proteases. Keeping these in mind, we looked for peptides that possess antimicrobial activity .Of the many that we came across, we were interested in working Latarcin2a.</p>
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<h1>ABOUT OUR PEPTIDE</h1>
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<p>Latarcin2a (commonly known as M-Zodatoxin) is a 26 amino acid peptide isolated from the venom of the Asian spider <i>Lachesana tarabaevi</i>. Latarcins (Lt-2a) adopt an amphipathic alpha helical structure in membrane-mimicking environments.</p>
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<p>NMR structure of Latarcin2a</p>
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<p>Amphipathicity corresponds to the segregation of hydrophobic and polar residues between the two opposite faces of the a-helix, a distribution well suited for membrane binding. The polar part is shown in magenta color, and hydrophobic part is shown in wheat color.</p>
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<p>They produce lytic effects on both gram positive and gram negative bacteria via a carpet mechanism. As explained by Tamba and Yamazaki, this mechanism involves the external binding of the peptide to the membrane, followed by its critical destabilization.</p>
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<p>Carpet model of antimicrobial-induced killing</p>
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<p> In this model, the peptides disrupt the membrane by orienting parallel to the surface of the lipid bilayer and forming an extensive layer or carpet. Hydrophilic regions of the peptide are shown coloured red, hydrophobic regions of the peptide are shown coloured blue.</p>
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<p>However, Lt-2a has a disadvantage; it is found to possess 20% haemolytic and cytotoxic activity. A study done by A.A. Polyansky et al./ FEBS 583(2009) showed that a mutation in the sequence of native Lt-2a resulted in multifold reduction of its cytotoxic and haemolytic effects. Hence, we chose to work with this mutant, Lt-2a F10K (Phe replaced by Lys) as well.</p>
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<p>position of the F10 in the peptide is shown in green color.</p>
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<h1>GENETIC ENGINEERING</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>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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Revision as of 07:50, 27 October 2017