Detailed Description

Bio-glues

Bio adhesives are polymeric materials which are naturally produced from a variety of organisms and have adhesive properties. The monomers of the polymeric bio adhesives consist of a variety of substances, but proteins and carbohydrates are the most common.
Biological adhesion is a phenomenon present in many biological systems. Examples of organisms that produce adhesives can include bacteria, spiders, marine tubeworms, sea cucumbers, barnacles and mussels. These adhesives are well known for being very strong, durable and ecologically safe compared with human-made substances. On top of that, some of them can be applied in an aqueous environment being impervious to water and turbulent forces, they give rise for many qualitative applications.

How do mussels attach themselves to a surface?

At the basis of our project are proteins which are normally produced from blue mussels (Mytilus edulis). Blue mussels produce adhesives comparable in strength to human-made glues . However these adhesives have extra advantages like the absence of carcinogens (formaldehyde) and their ability to sustain adhesive under water. Byssus threads of the blue mussel attach to a (underwater) solid surface due to catechols on adhesive proteins.

The mussel’s byssus is an exogenous attachment structure which was first described in 1711 (Brown 1952 ). Byssus’ main role is the attachment of the organism to surfaces. The byssus is a bundle of extracorporeal threads. This bundle is at the proximal end attached to the mussel’s byssal retractor muscle and at the distal end to a surface by adhesive plaques.
Mytilus edulis has the ability to bind to a very broad range of materials, ranging from glass, plastics, metals, wood and Teflon to biological materials, such as biological tissues, organisms, and other chemical compounds or molecules. As a result, the adhesive abilities of mussels have been an inspiration for the production of the synthetic version of this biological glue.

The mussel attached by its byssus threads (Powel, 2009)

How do mussels attach?

Mussels use a rather flexible method to attach to a surface; they spin a set of threads from an internal muscle to the surface, also known as stem. (Waite, 1983) (Waite,1992) The treads are 2 to 4 cm in length and attach to the surface by the use of plaques (diameter 2-3 mm). Thus, this plaque connects the substrate and the byssal thread. The specific adhesion is determined by the number of threads, which can go up to 30 to 50 threads per mussel. This will lead to a total attachment strength up to 400N. The plaque is divided into three sections. The first part is a pre-coating adhesive layer, which can attach to the surface. On top of this, the byssal threads are anchored in an open, cellular structure. (Benedict & Waite, 1986) The overall protection layer is formed by a “varnish”, which protects against enzymatic and chemical degradation. (Rzepecki & Waite, 1995)

The foot of the mussel (Haemers, 2003 )

Formation of the plaque and bysall threads

As well as the plaque as the threads are built of proteins, which are produced in the mussel’s foot. In the mussel’s foot there is a very complex organ with different types of glands of muscles. In a special part (the ventral groove), the thread and plaque are formed. (Pujol, 1967) This ventral groove runs over the whole foot and can be sealed off from the environment if required. (Waite, 1992) The foot can be bend in such a direction, that the ventral groove contacts the substrate.

In the sixties it was shown that as well as the thread as the plaque is formed by the interaction of secretions from three different kind of cells located in the foot. (Pujol, 1967) There are three specific regions in the foot where the different cell types are clustered together; namely the phenol gland, the collagen gland and the enzyme gland. (Tamarin) The phenol gland produces proteins for the protective varnish and for the plaque. The produced proteins are stored in membrane-bound granules, which are homogeneous, dense, spherical bodies. After the formation of the granules, they are transported to the secretion area.

The attachment to a surface in steps

If the mussel identified a suitable surface, this surface will be inspected by the tip of the mussel foot. After this inspection, the foot will sweep the surface to clean it. This cleaning is probably done to remove all the unbound matter. After the cleaning the foot become motionless for 2-5 minutes, in this time the formation of byssus thread takes place. This means that first the ventral groove contracts to squeeze out all the seawater. The groove is then filled again by the already produced granules, containing the proteins and all the components for the formation of the byssal threads and the plaques.

If this procedure it disturbed within 1 minute, the mussel will detach, leaving behind a plaque which is attached to unfinished thread. (Maheo, 1970) This suggests that the characteristic time for plaque formation is less than a minute. This immediately raises the following question: how is it possible that the content of the granules from the enzyme gland mix fast enough with the proteins released by the phenolic gland? This implies that the cross-linking must be very effective, within a minute, since this is the time the mussel needs for formation of the plaques. (Haemers, 2003)

One should also keep in mind the environmental and other biological influences; both environmental factors (salinity, temperature, pH, season, and substratum choice) and biological factors (age and metabolic state of the organism) also affect the efficiency and strength of byssal attachment to surfaces (Van Winkle 1970 ; Crisp et al. 1985; Carrington 2002).

The mussle foot proteins (mfps)

There are at least five different mussel foot proteins identified in the foot of the mussel, the proteins are present in varying amount. The amount present depends on the “specialisation” of the protein (coating or attachment for example), which is directly linked to the DOPA content and the presence of their additional characteristic residues. (Haemers, 2003)

The similarity between all five mussel foot proteins is that they contain a certain amount of tyrosine. The exact amount of tyrosine relates to the final function of the protein. Tyrosine is namely the compound gives adhesive properties to the protein. After hydroxylation of tyrosine, by tyrosinase and additional required compounds, an extra hydroxyl-group is added to the benzene ring, which will lead to an active catechol-group. (Haemers, 2003) The catechol-groups in the DOPA can form strong bonds with catechol-groups on adjacent DOPA molecules. The internal strength of the mussel glue (so called cohesion) is based on the amount of cross-links between polymer chains of the individual adhesive proteins. The “free” catechol groups, thus those which do not cross-link, determine the adhesive properties of the glue. (Waite, 1985) (Pizzi, Mittal and Dekker, 1994)

The five mussel foot proteins seem to differ on one hand by the content of DOPA present in the protein and on the other hand the presence of the other residues. The other residues determine par example if the protein is able to form disulphide bonds etc. (Haemers. 2003) During our project we focus on mfp5, since this mussel foot protein has the highest content of DOPA, which is beneficial for our StickE. Coli.

Biofilms

Using the term biofilm, matrix-enclosed bacterial populations can be described. This extracellular matrix, which is also referred to as slime, is a complex formation, containing a wide variety of compounds such as extracellular DNA, proteins and polysaccharides . There are numerous bacterial species which form biofilms. Usually, the structure of a biofilm consist of more than one bacterial species. As one can understand, this all together makes formation of and the structure of a biofilm extremely complex and difficult to control and regulate.

On the other hand, the “idea” of biofilms could be very useful for many applications in industry for example water-treatment facilities. As a result, in our project we aim to completely control the attachment and detachment of cells on substrate. Therefore, we will give Escherichia coli a simple, effective and controllable mechanism for binding on surfaces, namely binding by mussel glue. E. coli, expressing mfp-5 on the outer cell surface, can than robustly attach to a wide variety of surfaces including glass, plastic and to itself.

Combination...Our Goal

The word combination is essential in our project. First of all, the basis for our project is combining the beneficial adhesive property from the mussel with our working horse E. coli. To get a most realistic result as possible, we combine our results from the lab together with our mathematical model. This combination allows us to predict the attachment speed and stability as well as cell clustering and settling. The controllable, strong attachment opens up new possibilities for the use of bacterial machines in environmental applications, medicine and industry, since it is easily combined with other properties/features.

To reach our goal, we have four main projects on the lab. The first step is to integrate the mussel foot protein 5 into the outer-membrane of E. coli and to characterize and visualize the mfp5. Therefore we will use the already characterized Biobrick of the Berkeley Wetlab 2009 iGEM team (construct BBa_K197034 in vector BBa_K197039). Since a GFP linker is also present in this construct, this will give us the ability to visualize the protein in the membrane by the use of Total Internal Reflection Fluorescence (TIRF) microscopy.

The TIRF has also a prominent role in our second project: the characterisation of the transport of mfp5. Since it is rather difficult to visualize and characterize a protein in the outer-membrane, several techniques are combined. This includes as mentioned above TIRF microscopy, however this time different combination of dyes are used to visualise the transport. Beside the TIRF microscopy, also electron microscopy is used to get the optimal, visual prove. Further experiments include fractionation of the cells and protein gels.

The third project is the so called activation of mfp5, which means the hydroxylation of the tyrosines present in mfp5. For the hydroxylation the enzyme tyrosinase is required. There are several tyrosinases, all need additional compounds in order to fulfil the hydroxylation. For our project, the melA Biobricks from the Tokyo Tech 2009 iGEM team (BBa_K193601 and 602) are used. Once mfp5 is hydroxylated, it will be adhesive. The previous mentioned L-tyrosines now have an additional hydroxylgroup and are called L-DOPA. To determine the adhesive properties, several experiments are done, including atomic force microscopy and MALDI-TOF. These results are also very useful for our mathematical model, so we’ll get the most realistic estimation/result.

Last but certainly not least, there is the project for localisation of mfp5 in one specific part of the outer-membrane. Therefore, we will combine our construct together with a Biobrick of the SDU Denmark 2010 iGEM team (BBa_K343003). The localisation is achieved by the use of the signal sequence which originates from the complex flagellar system. When the localisation is achieved, we can make a controllable production-line when combined with different properties of E. coli. This is the first step to have a chip with a product-line, which will have many beneficial scale-up possibilities in the future.

References

Benedict , C. V., & Waite, J. H. (1986, Aug.). Location and analysis of byssal structural proteins of Mytilus edulis. Journal of morphology, pp. 171-181.

Haemers, S. (2003). The Adhesive of the Blue Mussel : Controlling Cross-Linking. Delft, The Netherlands: DUP Science (Delft University Press).

Mahéo, R. (1970). Étude de la pose et de l'activité de sécrétion du byssus de Mytilus edulis L. Cahiers de Biologie Marine, 475-483.

Pizzi, A., Mittal, K. L., & Dekker, M. (1994). Handbook of Adhesive Technology. New york, The United States of America: Eds.

Powell, M. (2009, 04 01). Retrieved 09 07, 2011, from Blogfish: Fish, Oceans and Conservations: http://blogfishx.blogspot.com/2009_04_01_archive.html

Pujol, P. J. (n.d.). Formation of the byssus in the common mossel (Mytilus edulis). Nature - 214, pp. 204-205. Rzepecki, L. M., & Waite, J. H. (1995, December 4). Wresting the muscle from mussel beards: research and applications. Molecular Marine Biology and Biotechnology, pp. 313-322.

Tamarin , A., & Keller, P. J. (1972 ). An ultrastructural study of the byssal thread forming system. J Ultrastruct, 401-416.

Waite, J. H. (1983). Biological Review (No. 58), 209.

Waite, J. H. (1985). Catechol Oxidase in the Byssus of the Common Mussel, Mytilus Edulis L. Journal of the Marine Biological Association of the United Kingdom, 359-371.

Waite, J. H. (1992). Results and Problems in Cell Differentiation. In C. S.T., Biopolymers (pp. 27-54). Berlin, Germany: Springer-Verlag.