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Revision as of 01:31, 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 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.

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.

Backbone construct

A quick look at the partregistery shows that for Bacillus subtilis there aren’t that many backbones to pick from. This 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. None of the backbones in the partsregistry had these, 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) don’t have to worry about placing a said promoter in front of their constructs any 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.

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.