Team:IIT Kharagpur

IIT Kharagpur

SILKOTRON

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

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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