Difference between revisions of "Team:IIT Kharagpur"

 
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ILorem Ipsum is simply dummy text of the printing and typesetting industry. Lorem Ipsum has been the industry's standard dummy text ever since the 1500s, when an unknown printer took a galley of type and scrambled it to make a type specimen book. It has survived not only five centuries, but also the leap into electronic typesetting, remaining essentially unchanged. It was popularised in the 1960s with the release of Letraset sheets containing Lorem Ipsum passages, and more recently with desktop publishing software like Aldus PageMaker including versions of Lorem Ipsum.<br><br>
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Cobweb….sounds familiar? With it’s spiral design to it’s sticky threads. They seem to be the most useless and good for nothing artwork on our walls and ceilings. But what if someone say that they are a marvel of protein engineering by nature. A structural combination of molecules arranged in such a fashion that even the modern day megastructures take inspiration from them. The sticky thread on these webs can be scientifically called as spider silk. As the name suggests, spider silk is woven by spider for the purpose of catching prey. This is very much different from the mulberry silk extracted from silkworm. The spider silk subunits are composed of Masp2 protein crystals. These protein molecules have anti parallel beta sheets in them The extensive beta linkages in them gives enormous tensile strength( even greater than reinforced steel). Feasible extraction of this silk protein can have numerous applications and we our in our way to provide a method for feasible extraction of spider silk
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Lorem Ipsum is simply dummy text of the printing and typesetting industry. Lorem Ipsum has been the industry's standard dummy text ever since the 1500s, when an unknown printer took a galley of type and scrambled it to make a type specimen book. It has survived not only five centuries, but also the leap into electronic typesetting, remaining essentially unchanged. It was popularised in the 1960s with the release of Letraset sheets containing Lorem Ipsum passages, and more recently with desktop publishing software like Aldus PageMaker including versions of Lorem Ipsum.
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<h3 class="step-title color-scheme">Our Idea</h3>
 
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<p class="step-description" style="font-family:Dosis; font-size:20px;"> Spiders, cobwebs .. these are not pleasant things to encounter in your home.These cobwebs or scientifically spider silk which everyone feels is good for nothing has many uses and potential applications.With its’ strength, spider silk vest could be a saviour to soldiers. And that’s not the end of it, it’s biocompatibility and biodegradability make it a very good scaffold material in bone tissue engineering, cartilage repair. So what is stopping us from synthesizing them? It is the spider silk itself. Spider silk that is produced by spiders is very thin and the production of these materials requires bulk quantities of spider silk. Previously there were many attempts made to produce spider silk in bacteria but all of them involved killing of bacteria for it’s extraction.<br>
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<p class="justify" style="font-family:Dosis; font-size:20px;"> Our idea focuses on devising a method  to produce silk proteins along with a monitoring mechanism with minimal cost and maximum output. We plan to produce the spider silk protein Masp2 from recombinant
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E.coli. We are targeting the mass production of spider silk protein Masp2 and a mechanism to extract it without the need of cell lysis. At present, we are restricting our idea up to monomer production, rather than thread production. We have also devised a method to characterise the amount of silk being produced and a way to monitor it.  
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We iGEM IIT Kharagpur took this a step forward and devised a method for production and extraction of spider silk protein without killing the bacteria and to proof the concept of protease cleave extracellularly we develope a detection system using FRET dye pair.  </p>
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Currently industries are using silkworm silk for preparing silk clothes, manufacture of fishing fibres, parachutes, cartridge bags, insulation coils for telephones and wireless receivers, tyres of racing cars etc. The reason of using silk threads is that they are very fine, soft and light in weight. They are very thin but strong having high elastic property. But it has been found that the spider silk is much stronger and elastic than the normal silk and thus have great potential to serve better in these industries but the harvest of spider silk is very impractical due to the small quantities produced by spiders, combined with their tendency to eat one another. Collecting silk from webs is a time-consuming task. It took 8 years to make a golden spider silk cape from 1.2 million golden orb webs. Spider dragline silk is considered to be the toughest biopolymer on Earth due to an extraordinary combination of strength and elasticity. Moreover, silks are biocompatible and biodegradable protein-based materials. Recent advances in genetic engineering make it possible to produce recombinant silks in heterologous hosts, opening up opportunities for large-scale production of recombinant silks for various biomedical and material science applications.
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The Masp2 protein also known as the major ampullate silk protein has acidic residues. In aqueous solution, the pH of the silk protein comes out to be near 4. Due to the presence of large number of acidic amino acids, the protein if produced inside recombinant e-coli,will remain in the cytoplasm.The only way to get them out is to lyse the cell. Our idea is to add a transmembrane protein Omp-A along with the silk protein, in order to anchor it to the membrane outside the cell The silk protein can be extracted by cleaving the linkage between Omp-A and Masp2 with an HIV protease. We have also put a histidine tag on the Masp-2 protein. The chelating effect of Histidine with Nickel atom will help us to extract the silk protein after cleavage. And isolate it on a nickel column. The silk protein construct can be added with multiple silk monomers according to the number of subunits desired. <br>
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The HIV protease required for cleavage action, will also be anchored to the membrane via the Omp-A protein. The protease will first carry out self cleavage in order to free itself from the membrane . Then the protease will cleave the silk protein from the membrane, releasing it into the solution. Nickel columns would be used in order to extract the silk monomers.  
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Spider silks are remarkable natural polymers that consist of three domains: a repetitive middle core domain that dominates the protein chain, and non-repetitive N-terminal and C-terminal domains. A variety of heterologous host systems have been explored to produce different types of recombinant silks.E. coli is a well-established host for industrial scale production of proteins.E. coli is easy to manipulate, has a short generation time, is relatively low cost and can be scaled up for larger amounts protein production.There are four main steps in the process: (i) design and assembly of synthetic silk-like genes into genetic ‘cassettes’, (ii) insertion of this segment into a DNA vector, (iii) transformation of this recombinant DNA molecule into a host cell and (iv) expression and purification of the selected clones.But the major problem is that the silk production is intracellular and thus for collecting and purification of the silk formed ,the cell lysis is necessary which is non economical. <br>
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In order to characterise the amount of HIV protease required for this action, we have created a novel detection system based on FRET(Fluorescence Resonance Energy Transfer) pairs. This characterisation construct will have CFP and YFP attached with the same HIV cleavage site that we used in the silk construct and the protease construct. The construct will also have Omp-A in order to anchor it to the membrane. If the concentration of the protease will be high enough to perform the cleavage action on all the three constructs, then the YFP part will get cleaved and we will see a cyan fluorescence other wise due to the intact YFP part we will see a yellow fluorescence.  
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In our project we are trying to secrete the spider silk extracellularly thus the bacteria can be used again and again for future production, leading to much economical process.
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The silk construct along with the FRET and the protease construct will provide us a mechanism for a continuous production of silk monomers without cell lysis and to monitor the silk production process.
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<h5 style="color:white; text-align:left;"><b>C. </b>Air Bags:</h5>
 
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If the air bags are made of spider silk than due to their higher elasticity and strength the air bags would absorb more energy and thus the sudden impact  would be less injurious.
 
If the air bags are made of spider silk than due to their higher elasticity and strength the air bags would absorb more energy and thus the sudden impact  would be less injurious.
  
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2. As the spider silk is thinner, stronger and more elastic it can be used as surgical thread to stitch the wounds together.<br>
 
2. As the spider silk is thinner, stronger and more elastic it can be used as surgical thread to stitch the wounds together.<br>
 
Diverse and unique biomechanical properties together with biocompatibility and a slow rate of degradation make spider silks excellent candidates as biomaterials for tissue engineering, guided tissue repair and drug delivery, for cosmetic products
 
Diverse and unique biomechanical properties together with biocompatibility and a slow rate of degradation make spider silks excellent candidates as biomaterials for tissue engineering, guided tissue repair and drug delivery, for cosmetic products
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<h5 style="color:white; text-align:left;"><b>E. </b>Manufacturing Practices:</h5>
 
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For production of silk, cultivation of silkworm is done known as sericulture. The first stage of silk production is the laying of silkworm eggs, in a controlled environment such as an aluminum box, which are then examined to ensure they are free from disease. The female deposits 300 to 400 eggs at a time.<br>
 
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In an area the size of your monitor screen, 100 moths would deposit some 40,000 eggs, each about the size of a pinhead. The female dies almost immediately after depositing the eggs and the male lives only a short time after. The adult possesses rudimentary mouthparts and does not eat during the short period of its mature existence.<br>
 
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The tiny eggs of the silkworm moth are incubated (about 10 days) until they hatch into larvae. Once hatched, the larvae are placed under a fine layer of gauze and fed huge amounts of chopped mulberry leaves during which time they shed their skin four times. The larvae may also feed on Osage orange or lettuce. Larvae fed on mulberry leaves produce the very finest silk. The larva will eat 50,000 times its initial weight in plant material.<br>
 
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For about six weeks the silkworm eats almost continually. After growing to its maximum size of about 3 inches at around 6 weeks, it stops eating and changes colour. The silkworm attaches itself to a compartmented frame, twig, tree or shrub in a rearing house to spin a silk cocoon over a 3 to 8 day period. Steadily over the next four days, the silkworm rotates its body in a figure-8 movement some 300,000 times, constructing a cocoon and producing about a kilometer of silk filament. At this stage, the cocoon is treated with hot air, steam, or boiling water. The silk is then unbound from the cocoon by softening the sericin and then delicately and carefully unwinding, or 'reeling' the filaments from 4 - 8 cocoons at once, sometimes with a slight twist, to create a single strand.
 
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Latest revision as of 02:23, 20 October 2016

IGEM-IIT Kharagpur

Our Inspiration

Cobweb….sounds familiar? With it’s spiral design to it’s sticky threads. They seem to be the most useless and good for nothing artwork on our walls and ceilings. But what if someone say that they are a marvel of protein engineering by nature. A structural combination of molecules arranged in such a fashion that even the modern day megastructures take inspiration from them. The sticky thread on these webs can be scientifically called as spider silk. As the name suggests, spider silk is woven by spider for the purpose of catching prey. This is very much different from the mulberry silk extracted from silkworm. The spider silk subunits are composed of Masp2 protein crystals. These protein molecules have anti parallel beta sheets in them The extensive beta linkages in them gives enormous tensile strength( even greater than reinforced steel). Feasible extraction of this silk protein can have numerous applications and we our in our way to provide a method for feasible extraction of spider silk

1

Our Idea

Our idea focuses on devising a method to produce silk proteins along with a monitoring mechanism with minimal cost and maximum output. We plan to produce the spider silk protein Masp2 from recombinant E.coli. We are targeting the mass production of spider silk protein Masp2 and a mechanism to extract it without the need of cell lysis. At present, we are restricting our idea up to monomer production, rather than thread production. We have also devised a method to characterise the amount of silk being produced and a way to monitor it.

Project Overview

The Masp2 protein also known as the major ampullate silk protein has acidic residues. In aqueous solution, the pH of the silk protein comes out to be near 4. Due to the presence of large number of acidic amino acids, the protein if produced inside recombinant e-coli,will remain in the cytoplasm.The only way to get them out is to lyse the cell. Our idea is to add a transmembrane protein Omp-A along with the silk protein, in order to anchor it to the membrane outside the cell The silk protein can be extracted by cleaving the linkage between Omp-A and Masp2 with an HIV protease. We have also put a histidine tag on the Masp-2 protein. The chelating effect of Histidine with Nickel atom will help us to extract the silk protein after cleavage. And isolate it on a nickel column. The silk protein construct can be added with multiple silk monomers according to the number of subunits desired.
The HIV protease required for cleavage action, will also be anchored to the membrane via the Omp-A protein. The protease will first carry out self cleavage in order to free itself from the membrane . Then the protease will cleave the silk protein from the membrane, releasing it into the solution. Nickel columns would be used in order to extract the silk monomers.

In order to characterise the amount of HIV protease required for this action, we have created a novel detection system based on FRET(Fluorescence Resonance Energy Transfer) pairs. This characterisation construct will have CFP and YFP attached with the same HIV cleavage site that we used in the silk construct and the protease construct. The construct will also have Omp-A in order to anchor it to the membrane. If the concentration of the protease will be high enough to perform the cleavage action on all the three constructs, then the YFP part will get cleaved and we will see a cyan fluorescence other wise due to the intact YFP part we will see a yellow fluorescence. The silk construct along with the FRET and the protease construct will provide us a mechanism for a continuous production of silk monomers without cell lysis and to monitor the silk production process.

Describe with a video

Application

Stronger than steel but elastic than a rubber band, spider silk have dozen of potential application from construction, medicine to military. Scientists have suggested using spider silk to construct product as diverse as biodegradable water bottles, flexible bridge suspension cables and unrippable writing paper.
The early Roman and Greek populations used the silk to weave fabrics, create nets , create fishing lines and seal wounds. The material is used to make gloves and stockings, though the process take some time and required many spiders. The cross hairs on many weapons used spider silk, and to this day some military units still keep Black Widow spiders on site in case repairs to old weaponry are needed.

Potential Applications

A. Lighter and Stronger Bulletproof Clothing:

Researcher melded human skin with the spider silk and the hybrid skin was able to repel a slow moving bullet fire by a .22-caliber rifle. If scaled up, spider silk body armor could be three times stronger than Kevlar. This technology can be very useful for military and automobile and flight industries. Artificial Skin: Growing artificial skin for burn victims is a tough task. We need a specific kind of scaffolding upon which to build the healthy skin tissue that can merge with the body. Collagen and synthetic fibres are typically used to provide support for growing skin tissue, but these materials are not biodegradable. The spider silk’s strength, flexibility and biodegradability may make it a nearly ideal matrix for growing skin and healing wounds. Air bags: if the air bags are made of spider silk than due to their higher elasticity and strength the air bags would absorb more energy and thus the sudden impact would be less injurious.

B. Artificial Skin:

Growing artificial skin for burn victims is a tough task. We need a specific kind of scaffolding upon which to build the healthy skin tissue that can merge with the body. Collagen and synthetic fibres are typically used to provide support for growing skin tissue, but these materials are not biodegradable. The spider silk’s strength, flexibility and biodegradability may make it a nearly ideal matrix for growing skin and healing wounds.

C. Air Bags:

If the air bags are made of spider silk than due to their higher elasticity and strength the air bags would absorb more energy and thus the sudden impact would be less injurious.

D. Medical Uses:

1. In injuries like torn interior cruciate ligament, spider silk due to its properties can be used to make a structure so that it can used as a artificial ligament.
2. As the spider silk is thinner, stronger and more elastic it can be used as surgical thread to stitch the wounds together.
Diverse and unique biomechanical properties together with biocompatibility and a slow rate of degradation make spider silks excellent candidates as biomaterials for tissue engineering, guided tissue repair and drug delivery, for cosmetic products