Team:Groningen/Project

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!align="center"|[[Team:Groningen|Home]]
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!align="center"|[[Team:Groningen/Team|Team]]
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!align="center"|[https://igem.org/Team.cgi?year=2013&team_name=Groningen Official Team Profile]
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!align="center"|[[Team:Groningen/Project|Project]]
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!align="center"|[[Team:Groningen/Parts|Parts Submitted to the Registry]]
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== Background ==
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<h1>Introduction</h1>
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<h2> The origin of silk </h2>
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<p>Ancient Chinese legend has it that a princess named Xi Linshi, who was having a relaxing afternoon under a Mulberry tree, first discovered silk 4500 years ago when a cocoon suddenly fell in her tee. After some time, Xi Linshi extracted a silk thread from her still steaming cup of thee, and unraveled the secret of silk along with its cocoon.</p>
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The knowledge that silk could be extracted from insect cocoons was closely guarded, and harsh conditions were set (the penalty of death) to anyone who was caught smuggling the eggs or cocoons. By such means, China realized a two thousand year monopoly of the silk industry, which was, according to yet another legend, put to an end when a Chinese princess smuggled moth eggs and mulberry seeds as a gift for her future husband. Subsequent to the princesses betrayal, the secret of silk was still kept secret from the west for another good thousand years or so, as it was only in the 12th century ACE that sericulture (the production of silk) began to develop.
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Bone fractures and other physical problems are often solved with implants. Unfortunately about half of the implants give rise to complications, such as inflammations, infections and rejection by the host. Beside the delays in recovery, which cost the American society alone $30 billion a year, the undesired effects also cause great discomfort and a 25% increase in mortality.
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Silk has thus inspired many legends and myths. Whether the stories are true or not, it is a fact that the discovery of silk has had world-wide impacts on culture, economy, development, and trade due to its much desired properties.
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To reduce negative effects a protective and biocompatible coating can be applied to the implant, prior to insertion into the body. A very potent material to use for this coating is spider silk. Not only does it exert great biomedical properties, it also has high tensile strength, elasticity and is biodegradable.
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The focus of this project is to coat an implant with recombinant spider silk. <i>Bacillus subtilis</i> cells were transformed to enable spider silk production, and to introduce a novel heat triggered system.</u>
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By addition of a signal sequence to the silk protein gene the bacterium is able to export the protein out of its cell. Also a Strep-tag® is added to the silk protein sequence. <i>B. subtilis</i> is inherently able to sense temperature, and by coupling this sensor to its movement system the cells will become immobilized near the implant. This trick allows efficient and localized production of spider silk near the heated implant, to which the Strep-tagged silk proteins can attach. After processing and thorough sterilization, which the spider silk coating can withstand, the coated implant is ready for use.
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<a href="https://2013.igem.org/Team:Groningen/SchematicOverview" class="myButton" color="white">Schematic overview</a> 
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<h2> The properties of silk </h2>
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<h2>Backbone construct</h2>
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<p>The unique properties of silk are a result of its highly constant and repetitive amino-acid structure. The sequence of amino-acids determines what secondary structures will arise, and thus the final preferred protein conformation. The secondary structures may be beta sheets, beta-spirals, and beta-helices, of which the sheets realize the silk's amazing tensile strength, and the spirals and helices its elongation.</p>
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<p>In the figure below a stress-strain diagram can be found (Frank K. Ko, et at. 2001) where Clavipus spider silk is compared to, Kevlar 29, normal silkworm silk, PET (polyethylene terephthalate), Nylon 6, and Merino wool. The stress-strain diagram relates the degree of deformation to the amount of energy absorbed. </p>
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[[File:Stressstrain.JPG]]
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When used as clothing, silk has many beneficial properties. Its smooth, compact surface feels and looks nice, and it enables easy removal of dirt. It is a bad conductor of heat, making it cool in the summer and warm in the winter. Furthermore, it has a water absorption efficiency similar to that of wool, and is resistant to insects and mildew.
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A quick look at the partregistery shows that for <i>Bacillus subtilis</i> there aren’t that many backbones to pick from. This is in contrast to the legion of backbones available when working with <i>E. coli</i>. It was necessary for the coordinated expression of spider silk to have a inducible promoter. So we made a backbone that has a IPTG inducible promoter in it. In the long run this saves a tremendous amount of time and effort, since we (and future iGEM teams) do not have to worry about placing a said promoter in front of their constructs any more.
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A final general property of silk it that it can be integrated with the human body - it will not induce an immune response - potentially making it an ideal choice for many biomedical applications. Its compatibility extends to the gastrointestinal tract, that is, it is even safe to eat!
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<h3> References  </h3>
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<a href="https://2013.igem.org/Team:Groningen/Navigation/Construct" class="myButton" color="white">Read More</a> 
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[1] Frank K. Ko, et al. (2001). "Engineering properties of spider silk". MRS Proceedings, vol. 702
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<h1>   </h1>
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<h2>Silk Assembly shop</h2>
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The spider silk construct needs to have 3 abilities: it needs to be produced, it needs to be secreted and it needs to be attached to an implant.
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Working with the spider silk gene posed a couple of difficulties, due to its high repetitiveness. Codon optimisation was used to overcome most of these problems. For the secretion of the spider silk we utilized the already present sec pathway in <i>Bacillus subtilis</i>. This is accomplished by adding a signal sequence in front of the protein.  For the attachment of the silk protein to the implant we used a strep-tag which was attached to the end of the protein. Strep binds to streptavidin with which we coat the implant.
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<a href="https://2013.igem.org/Team:Groningen/Navigation/SilkAssemblyShop" class="myButton" color="white">Read More</a> 
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<h2>Heat Motility</h2>
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In order to realize some form of targeted secretion, we came up with a system that would move according to the temperature of the environment. First we made a system in which the motility of <i>Bacillus subtilis</i> could be controlled by knocking out the motility gene <i>cheY</i>, and placing it under the control of a different promoter. For this we use the promoter from the thermosensing des pathway, which is natively present in <i>Bascillus subtilis</i>. </p>
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<a href="https://2013.igem.org/Team:Groningen/Navigation/Motility" class="myButton" color="white">Read More</a> 
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Latest revision as of 03:07, 5 October 2013

Introduction

Bone fractures and other physical problems are often solved with implants. Unfortunately about half of the implants give rise to complications, such as inflammations, infections and rejection by the host. Beside the delays in recovery, which cost the American society alone $30 billion a year, the undesired effects also cause great discomfort and a 25% increase in mortality.

To reduce negative effects a protective and biocompatible coating can be applied to the implant, prior to insertion into the body. A very potent material to use for this coating is spider silk. Not only does it exert great biomedical properties, it also has high tensile strength, elasticity and is biodegradable.

The focus of this project is to coat an implant with recombinant spider silk. Bacillus subtilis cells were transformed to enable spider silk production, and to introduce a novel heat triggered system.

By addition of a signal sequence to the silk protein gene the bacterium is able to export the protein out of its cell. Also a Strep-tag® is added to the silk protein sequence. B. subtilis is inherently able to sense temperature, and by coupling this sensor to its movement system the cells will become immobilized near the implant. This trick allows efficient and localized production of spider silk near the heated implant, to which the Strep-tagged silk proteins can attach. After processing and thorough sterilization, which the spider silk coating can withstand, the coated implant is ready for use.

Schematic overview



Backbone construct

A quick look at the partregistery shows that for Bacillus subtilis there aren’t that many backbones to pick from. This is in contrast to the legion of backbones available when working with E. coli. It was necessary for the coordinated expression of spider silk to have a inducible promoter. So we made a backbone that has a IPTG inducible promoter in it. In the long run this saves a tremendous amount of time and effort, since we (and future iGEM teams) do not have to worry about placing a said promoter in front of their constructs any more.

Read More


Silk Assembly shop

The spider silk construct needs to have 3 abilities: it needs to be produced, it needs to be secreted and it needs to be attached to an implant. Working with the spider silk gene posed a couple of difficulties, due to its high repetitiveness. Codon optimisation was used to overcome most of these problems. For the secretion of the spider silk we utilized the already present sec pathway in Bacillus subtilis. This is accomplished by adding a signal sequence in front of the protein. For the attachment of the silk protein to the implant we used a strep-tag which was attached to the end of the protein. Strep binds to streptavidin with which we coat the implant.

Read More


Heat Motility

In order to realize some form of targeted secretion, we came up with a system that would move according to the temperature of the environment. First we made a system in which the motility of Bacillus subtilis could be controlled by knocking out the motility gene cheY, and placing it under the control of a different promoter. For this we use the promoter from the thermosensing des pathway, which is natively present in Bascillus subtilis.

Read More



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