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From 2013.igem.org

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 (Vepari & Kaplan 2007, Mandal et al 2012).

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.

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

Silk construct

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.

Heat Motility

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 Bacillus subtilis could be controlled via knocking out the motility gene cheY. 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 Bascillus subtilis, was fused to a cheY gene. In the end the thermotaxis model showed that this approach for controllable motility worked.

backbone + IPTG induced promoter

A quick look at the partregistery shows that for Bacillus subtilis there aren’t that many backbones to pick from. This i 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. 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.

Alternative 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 dollar 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.

Unlike the silk from silkworms, the spider silk is difficult to acquire, as spiders have a cannibalistic nature which complicates breeding on a large scale. In the recent years biotechnological approaches have been used to produce recombinant spider silk, and properties of the proteins were examined. Still, production is very limited and optimization is required to realize each of the many possible applications of spider silk.

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 seeking 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. Bacillus subtilis is inherently able to sense temperature[], and by coupling this ability to its movement system, the cells will move towards a heated object. This trick allows efficient and localised 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 improved implant is ready for use.