Team:Groningen/Project

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<h1>Introduction:</h1>
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<p>Silk is a natural protein fibre that is known for its use in textiles. The best know silk comes from the silk moth pupa but other arthropods are also capable of producing silk. One of the arthropods well know for its silk is the spider.</p>
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<p>Spider Silk is amazing but currently large scale production is impossible. Various techniques to produce spider silk are being considered and one of them is letting bacteria produce the silk. This idea is promising although one of the main challenges is the low production rate of bacteria. </p>
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<p>Silk has some really nice attributes and some of these attributes are feasible for medical devices. Silk is an inalergic biomaterial and it is proven to enhance the healing process when it is used as a cover of implants, allowing for better acceptance by the human body (vepari & kaplan 2007, Mandal et al 2012).</p>
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<p>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.</p>
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<p>Though there has been some succes in producing silk in bacteria (Xia et al 2010), currently the bacteria needs to be killed in order to extract the silk. In this project the plan is to have the bacteria secrete the silk so that it can live and continue to produce more silk. Also the the design needs to be made such that most of the silk production happens at the required location to compensate for the low production volume.</p>
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<h1>Project goal:</h1>
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<p>The goal of this project is creating a bacteria that produces silk proteins and secreets it for the purpose of attaching it on the surface of an implant. For this the plan is to create a bacillus subtilis that can produce and secreet a silk like protein. Additionally, it is attempted to make the bacillus move to the location where the silk is needed. This way there should be fewer problems caused by the low silk yield. There are different methods of directing the movement. The one that will be aplied in this project is the use of temperature as the attractant/repellant. The bacillus should move away from the cold and towards the heat. This way the bacillus can move toward the implant and there it can produce and exceed silk.</p>
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<h1>Silk expression:</h1>
 
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<p>For the silk expression the bio-brick from the Utah team of 2012 is used. They successfully created a gene for the production of a silk like protein based on the spider silk gene. This gene was expressed in E. coli. In this project the gene is placed in bacillus suptilis. For variation, strep tacs are added to the silk to allow for binding to objects. This should allow for a coating of, among others, medical implants. </p>
 
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<h1>Silk secretion:</h1>
 
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<p>For the silk to be secreted the sec pathway is used. A signal sequence in the vector is used to secreet the proteins. This allows the bacillus to recognize the protein as an object that needs to be moved outside of the cell. (Pohl and Harwood)</p>
 
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<p>The first Signal sequences that will be attempted are MotB FliZ EstA and LytB.</p>
 
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<h1>Introduction</h1>
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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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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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</p><p>
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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>
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<a href="https://2013.igem.org/Team:Groningen/SchematicOverview" class="myButton" color="white">Schematic overview</a> 
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<h1> Silk</h1>
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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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</html>[[File:Stressstrain.JPG]] <html>
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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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<p>Charu Vepari and David L. Kaplan, Silk as biomaterial, <i>Progress in polymer science</i> (2007), Vol. 32 No. 8-9, pp. 991-1007. </p>
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<p>Biman B. Mandal, Ariela Grinberg, Eun Seok Gil, Bruce Palinaitis and David L. Kaplan, High-strength silk protein scaffolds for bone repair, <i>Proceedings of the National Acadamie of Science of the United States of America</i> (2012),Vol. 109 No. 20, pp. 7699-7704.</p>
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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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<p>Xiao-Xia Xia, Zhi-Gang Qian, Chang Seok Ki, Young Hwan Park, David L. Kaplan, and Sang Yup Lee, Native-sized recombinant spider silk protein produced in metabolically engineered Escherichia coli results in a strong fiber, <i>Proceedings of the National Acadamie of Science of the United States of America</i> (2010), Vol. 107 No. 32, pp. 14059-14063.</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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<p>Susanne Pohl and Colin R. Harwood, Heterologous Protein Secretion by Bacillus Species: From the Cradle to the Grave, <i>Advances in Applied Microbiology</i> (2010),Vol. 73, pp. 1-25.</p>
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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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