Team:Groningen/Project

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<h1>Abstract</h1>
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<p>The unique properties of silk have provided it with a utility that goes far beyond that of any other natural fibers known to man. Its amazing mechanical properties, 'silky smooth' softness, and bio-compatibility, has led to applications ranging from from simple clothing to high tech biomedical devices.</p>
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The industry from which silk is obtained, however, is less than ideal. Scientists have therefore begun to design silk-producing micro-organisms. The 2012 iGEM team from Utah have indeed successfully designed BioBricks for this very purpose. However, these micro-organisms remain inadequate in the secretion of silk, which is a major limiting factor on the range of potential biosynthetic designs and applications.
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Our goal is to solve the secretion issue and to use it for the formation of a silk biofilm. The beauty of such a biofilm can be attributed to the properties of silk, and to the fact that any imaginable shape of seamless silk could be created since the biofilm grows in such a way as to fit its mold. We will be exploiting these properties to coat biomedical prosthetic devices, with the goal to prevent infections, and hence to prevent the required operations in dealing with such infections.
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<h1> Silk</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 Ling Shi, 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 Ling Shi 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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<h1>Introduction</h1>
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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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<p>Princess Xi Ling Shi. [3]</p>
 
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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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<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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<h2>Backbone construct</h2>
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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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<h2> The production of silk</h2>
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<p>The farming of silk is an arduous, time consuming, and costly process. Although a single cocoon may produce up to one mile of filament, 4 to 8 filaments are needed to produce a single thread, and approximately 5500 cocoons are needed for one kilogram of silk. Eight fully grown mulberry trees would have been needed for this single kilogram, and 48 hours of man-labor required to hand-reel it. Finally, the caterpillars required a full month to mature and three to five days to spin their cocoons, after which they were brutally boiled alive.
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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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Harvesting the more desired and rare silk from spiders requires an even more labor-extensive process. Each thread actually has to be pulled individually by hand from the spiders gland - needless to say, not a viable business plan!
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<p>The silk industry itself has undergone very little development over the past few millennia. Indeed, the manner in which it is obtained follows the very same process as that of princes Xi Ling Shi's initial discovery, albeit at a much grander scale with more specialized equipment. Scientists have therefore begun to design their own silk producing organisms [2]. Moreover, the 2012 iGEM team from Utah successfully designed the first spider-silk producing Biobricks for Escherichia coli  (for more information, please visit their [https://2012.igem.org/Team:Utah_State wiki]). Such advancements are needed to provide the industries and manufacturers with sufficient silk proteins for their applications. </p>
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<h2> Applications for silk</h2>
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<h2>Silk Assembly shop</h2>
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<p>Silk's journey as a product began as luxurious clothing reserved exclusively for the emperor subsequent to its initial discovery. As the sericulture developed, however, it was soon adopted by all classes of society. New applications were discovered, and it was spun into many different products; fishing lines, musical instruments, and bowstrings to name a few. It's utility and value were also recognized by other kingdoms, and a world-wide, ever increasing demand for the material began. Indeed, the western demand for silk was so great, that the main set of trade routes between Europe and Asia became known as the Silk Road. </p>
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<p> .... Nowadays, the variety of silk applications is even more extensive; bullet-proof clothing, all sorts of ropes and cables, artificial tendons and ligaments, bandages, sewing thread, seat belts, parachutes, biodegradable bottles, and much more..... </p>
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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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<h2> References </h2>
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<p>[1] Frank K. Ko, et al, (2001). "Engineering properties of spider silk". MRS Proceedings, vol. 702 </p>
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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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<p>[2] Charlotte Vendrely & Thomas Scheibel, (2007). Biotechnological production of spider-silk proteins enables new applications. Macromol. Biosci, vol 7, pp 401-409.</p>
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[3] [http://www.chinancient.com/chinese-ancient-four-beauties-xi-shi/xi-shi-02-2/ Current online source.] Waiting for author for more details.
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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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