Team:Groningen/Project

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<h1>Introduction</h1>
<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 dollar a year, the undesired effects also cause great discomfort and a 25% increase in mortality[].
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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.
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>
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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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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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)
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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 (Vepari & Kaplan 2007, Mandal et al 2012).  
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In order to achieve this goal, we engineered a micro-organism that can not only produce silk but also secrete it. Additionally we designed a system for coating the implant with the spider silk. This to counter the possible low yield of the spider silk production and ensure the implants being coated evenly.
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<a href="https://2013.igem.org/Team:Groningen/SchematicOverview" class="myButton" color="white">Schematic overview</a>
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For this job it is important to choose a fitting chassis, for this we choose the bacterium <i>Bacillus subtilis</i>. The main reason for using <i>B. subtilis</i> is because it is a gram-positive bacterium, so it only has one cell membrane the spider silk proteins have to traverse. This is very beneficial when attempting secretion. Another advantage is that <i>B. subtilis</i> is highly motile and is able to form a biofilm, two properties that are exploited for the coating mechanism. Lastly <i>B. subtilis</i> is a model organism and is frequently used in industrial processes dealing with harvesting proteins and it is a bacterium that is 'Generally regarded as safe’ (GRASS).
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<h2>Backbone construct</h2>
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<b>Our goal is to develop a silk coating for medical implants and a coating mechanism with the help of bacteria.</b>
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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 href="https://2013.igem.org/Team:Groningen/Navigation/Construct" class="myButton" color="white">Read More</a>  
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Production of spider silk on a grand scale, however, is unfeasible 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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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.
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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.
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In that way the silk will only be produced on site increasing the efficiency and saving energy.
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So our project consists of two subproject, 1 being the production of silk and 2 the coating mechanism
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<h2>Spider silk genes</h2>
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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.
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 <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.
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>
<h2>Heat Motility</h2>
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In order to have a system for targeted secretion, we came up with a system that would move according to the temperature of the environment. This is a nice idea since implants can be easily heated. First we made a system in which the motility of <i>Bacillus subtilis</i> could be controlled via knocking out the motility gene <i>cheY</i>. We could now introduce any particular promoter in front of our cheY gene and thus controlling the motility. This system in combination with our general chemotaxis model allow for good control of the cell. The promoter from the thermosensing des pathway, that is natively present in <i>Bascillus subtilis</i>, was fused to a <i>cheY</i> gene. In the end the thermotaxis model showed that this approach for controllable motility worked.  
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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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<h2>Backbone construct</h2>
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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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A quick look at the partregistery shows that for <i>Bacillus subtilis</i> there aren’t that many backbones to pick from. This i 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. None of the backbones in the partsregistry had these, so we made a backbone that has a inducible IPTG promoter in it. In the long run this saves a tremendous amount of time and effort, since we (and future iGEM teams) don’t have to worry about placing a said promoter in front of their constructs any more.
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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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