Team:IIT Kharagpur/Project
PROJECT
Introduction
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.
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.
Our Idea
Spider silk is weaved commonly from a silk protein Masp. We aim to mimic the production of this spider silk protein in E.coli and extract it without cell lysing. Firstly a construct with HIV cleavage site, spider silk protein with histidine tag and Omp-A is used for production of spider silk. spider silk protein secretion is guided to the cell membrane and is anchored to it by Omp-A protein. The HIV cleavage site to which the spider silk protein is anchored or attached is a retroviral protease. We made the bacteria to produce protease by using another construct with HIV cleavage site , protease and Omp-A . This protease cleaves its own site first and enter into the solution and then cleaves silk protein site releasing it into solution.
Spider silk released into the solution is purified by using nickel columns which has high affinity for histidine. The spider silk protein released into the solution has multiple histidine residues which we introduced in the construct. This purified spider silk can be further treated in downstream process and can be transformed into silk threads.
To validate the activity of the protease and to determine the concentration of the protease required to effectively cleave the HIV cleavage site of the Masp protein, we used FRET based detection system. For this detection system we used two constructs, one for protease production and other for fluorescence activity. The construct for fluorescence emission is designed as shown in figure. The emission wavelength of CFP coincides with excitation wavelength of YFP. When the solution is excited with excitation wavelength of CFP, it produces yellow fluorescence. When the HIV cleavage site is cleaved, CFP is left anchored to the membrane. Now, on exciting with excitation wavelength of CFP, cells will fluoresce cyan validating the activity of the protease.
Project Description
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.
