From 2013.igem.org

Revision as of 15:21, 2 October 2013

Preventing complication from implants

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).

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.

For this job it is important to choose a fitting chassis, for this we choose the bacterium Bacillus subtilis. The main reason for using B. subtilis 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 B. subtilis is highly motile and is able to form a biofilm, two properties that are exploited for the coating mechanism. Lastly B. subtilis 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).

Our goal is to develop a silk coating for medical implants and a coating mechanism with the help of bacteria.

Spider silk genes

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 construct

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.