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| - | <h1>Engineering Bacillus subtilis to self-assemble into a biofilm that coats medical implants with spider silk.</h1>
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| - | <h1>Abstract</h1>
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| - | Approximately half of all implanted medical devices result in one or more medical complications. Complications lengthen the hospital stay, and increase mortality rates by 25%.
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| - | A possible solution for these complications is to form a protective biocompatible layer between the implant and the body by means of a spider silk coating. In the designed system, that envisages employing the Gram-positive model bacterium Bacillus subtilis, to secrete silk.
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| - | B. subtilis is generally regarded as safe (GRAS) and is often used in industry for the commercial production of extracellular proteins.
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| - | Using mathematical modelling and synthetic biology approaches, a system will be engineered by which B. subtilis cells will sense the implant, move towards the implant and secrete silk to cover the medical implant.
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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 known silk comes from the silk moth pupa but arthropods are also capable of producing silk. One of the arthropods well known 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 success 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 secretes 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 secrete 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 applied 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 BioBrick 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 subtilis''. For variation, strep tags 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 secrete 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>Heat Motility</h1>
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| - | <p>Since the silk only needs to be applied to the implants itself it would be beneficial to have a kind of targeting mechanism. A way to achieve this is using the natural motility of ''Bacillus subtilis''. However the attractants and repellents of bacterial motility are in general chemical compounds, and a chemotaxis would be hard to induce and maintain. If however bacillus would be attracted to heat, or repelled by cold, that would provide an easy way to target the implants. Since implants need to be sterile they can stand high temperatures, so perfect for heating.</p>
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| - | <p>The way to achieve heat motility is to knock out the native genes coding for motility proteins. Mainly cheY, which in phosphorylated state causes strait swimming. The knock out in combination with a cheY gene coupled to the cold sensing promoter of the Des gene, will provided the ability to move only when the cell is in a cold area. Once the cells have reached a warmer area they will not move, since cheY is not present anymore, therefore this is a great system to target heated implants.</p>
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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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| - | <p>
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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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| - | </p><p>
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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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| - | </p>
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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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| - | 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>
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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 since it'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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| - | <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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| - | <h2> References </h2> | + | <h1>Engineering Bacillus subtilis to self-assemble into a biofilm that coats medical implants with spider silk.</h1> |
| - | <p>[1] Frank K. Ko, et al, (2001). "Engineering properties of spider silk". MRS Proceedings, vol. 702 </p>
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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>
| + | Introduction |
| - | <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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| | + | Approximately half of all implanted medical devices results in one or more medical complications, such as blood clots, infections, poor healing, and excessive cell growth. Complications lengthen the hospital stay costing the american society an additional $30 billion dollar every year, and an increase in mortality rates by 25%. (ref) |
| | + | A possible solution is to form a protective biocompatible layer between the implant and the body by means of a coating. Applying a biocompatible, biodegradable coating onto medical implants addresses these problems. Although such coatings are currently being applied for example with collagen, they are still inadequate as complications arise. A potent alternative for the coating is spider silk, besides high tensile strength and extensibility, spider silk has good biocompatibility, cell adhesion, and will not induce immune responses in the human body. |
| | + | Production of spider silk on a grand scale, however, is infeasible due to the territorial behavior of spiders, and due to the fact that each thread silk has to be extracted individually by hand. Recognizing the potential of spider silk, researchers have begun developing silk producing bacteria. The yield, however, is still low, and the bacteria must be lysed in order to obtain it. Furthermore, the formation of a silk coating requires “polymerized proteins” rather than actual silk threads. To realize this, secretion of silk is required, which will presumably increase to production yield. |
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| | + | Our goal is to develop a silk coating for medical implants and a coating mechanism with the help of bacteria. |
| | + | Bacillus subtilis is the bacterium of choice, because it is a gram-positive bacteria. Gram positive bacteria are often used in industry for the commercial production of extracellular proteins. A codon optimised silk sequence is transformed to silk to and with the use of the natural secretion pathway the silk will be secreted. |
| | + | With the recent development of porous 3d printed implants, for example cartilage implants. Cartilage implants are used to regrow cartilage inside the human body. They consist of a biodegradable porous polymer (ref). To coat such an implant with such a porous structure is off course hard. A system system has been designed that exploits the chemotaxis system of Bacillus in order to guide Bacillus towards the implant. The environmental control factor for this system is heat, which is sensed by the DesK system, which, in turn, is coupled to the chemotaxis system of Bacillus. |
| | + | In that way the silk will only be produced on site increasing the efficiency and saving energy. |
| | + | So our project consists of two subproject, 1 being the production of silk and 2 the coating mechanism |
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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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| - | <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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| | </html> | | </html> |
Introduction
Approximately half of all implanted medical devices results in one or more medical complications, such as blood clots, infections, poor healing, and excessive cell growth. Complications lengthen the hospital stay costing the american society an additional $30 billion dollar every year, and an increase in mortality rates by 25%. (ref)
A possible solution is to form a protective biocompatible layer between the implant and the body by means of a coating. Applying a biocompatible, biodegradable coating onto medical implants addresses these problems. Although such coatings are currently being applied for example with collagen, they are still inadequate as complications arise. A potent alternative for the coating is spider silk, besides high tensile strength and extensibility, spider silk has good biocompatibility, cell adhesion, and will not induce immune responses in the human body.
Production of spider silk on a grand scale, however, is infeasible due to the territorial behavior of spiders, and due to the fact that each thread silk has to be extracted individually by hand. Recognizing the potential of spider silk, researchers have begun developing silk producing bacteria. The yield, however, is still low, and the bacteria must be lysed in order to obtain it. Furthermore, the formation of a silk coating requires “polymerized proteins” rather than actual silk threads. To realize this, secretion of silk is required, which will presumably increase to production yield.
Our goal is to develop a silk coating for medical implants and a coating mechanism with the help of bacteria.
Bacillus subtilis is the bacterium of choice, because it is a gram-positive bacteria. Gram positive bacteria are often used in industry for the commercial production of extracellular proteins. A codon optimised silk sequence is transformed to silk to and with the use of the natural secretion pathway the silk will be secreted.
With the recent development of porous 3d printed implants, for example cartilage implants. Cartilage implants are used to regrow cartilage inside the human body. They consist of a biodegradable porous polymer (ref). To coat such an implant with such a porous structure is off course hard. A system system has been designed that exploits the chemotaxis system of Bacillus in order to guide Bacillus towards the implant. The environmental control factor for this system is heat, which is sensed by the DesK system, which, in turn, is coupled to the chemotaxis system of Bacillus.
In that way the silk will only be produced on site increasing the efficiency and saving energy.
So our project consists of two subproject, 1 being the production of silk and 2 the coating mechanism
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