Team:Stuttgart/Description
Project Description
Why TAGC?
The Anti Germ Coating (TAGC) is a surface coating with antimicrobial properties. It consists of a chitosan scaffold linked to nisin (antimicrobial peptide) and rhamnolipid (glycolipid) molecules.
Frequently used surfaces in hospitals or other public places are prone to host a multitude of germs. Some of them are not dangerous for humans but others might be pathogenic or have several resistances against antibiotics. Therefore, they bear some potential risks for our health and infection with these germs should be avoided.
Current solutions are based on metals like copper, silver or titanium but all of them involve the danger of releasing metallic nanoparticles. Studies show that silver nanoparticles can impair mitochondrial functions and be therefore cytotoxic to human cells [1]. Furthermore, the antimicrobial effects were not as impressive as expected. What's more, the properties of metallic coatings occur after a couple of hours, so germs could spread on the coating in this time (see Human practices).
Our fully bio-based antimicrobial coating is the solution without a risk of nanotoxicity. Applying our coating reduces or even inhibits the growth of germs. That decreases the risk of infection caused by touching contaminated surfaces.
We expect the resulting coating to exhibit the same or even enhanced antimicrobial properties as the individual building blocks we used and thus prevent the growth and attachment of germs to surfaces of any kind.
What is TAGC?
The antimicrobial molecules rhamnolipid and nisin are linked to the antimicrobial biopolymer chitosan [2] to receive a surface with the desired antimicrobial effects. Rhamnolipids are glycolipids from Pseudomonas aeroguinosa with antimicrobial characteristics towards a broad spectrum of microorganisms. Nisin is an antimicrobial peptide from Lactococcus lactis, targeting mainly gram-positive microorganisms. A synergistic effect for the combination of rhamnolipid and nisin is described [3] so that the approach of using these two molecules together to generate a surface coating looks promising. Different ratios between these molecules need to be tested to get the desired results.
Our biobased coating (figure 1) contains a chitosan scaffold (black) linked to the antimicrobial peptide nisin (green) and the glycolipid rhamnolipid (red). Our aim is the connection of the scaffold to the rhamnolipid by using chemical linkage (with divinyladipate as a linker, grey) or an enzymatically driven linkage. We also aim to link nisin enzymatically.
The scaffold polymer - chitosan
Chitosan is a polymeric product of deacetylated chitin. Different types of chitosan can be distinguished by degree and pattern of acetylation as well as the polymer length. It also results in different chemical and physical properties. Short to midrange chitosan with low degrees of acetylation has strong antimicrobial properties [2]. Therefore, it has properties forming a functional base for TAGC.
Antimicrobial substances - rhamnolipid and nisin
Rhamnolipids are a class of glycolipids normally produced by the pathogenic bacterium Pseudomonas aeruginosa. They consist of a glycosyl head group with one or two rhamnose groups and a fatty acid tail (e.g. hydroxydecanoic acid). They act as antimicrobial substances with possible synergistic effects as a combination of mono- and di-rhamnolipids [4].
Nisin is an antibiotic normally produced by Lactococcus lactis and shows antimicrobial properties towards gram-positive bacteria. It inhibits cell wall formation and generates transient pores to disrupt the cell membrane.
Where can TAGC be imagined?
TAGC has many applications in public places, healthcare and industry. Thus, it is able to make a significant improvement in daily life.
Public:
Handles and railings in public places are constantly in contact with many different germs present on human skin.
Public toilettes are one of the places with the largest number of germs. A coating which could reduce the number of the germs would result in an improvement of hygienic conditions.
Daily Life:
Most surfaces in workplaces or institutions (e.g. kindergartens, schools) host a surprisingly wide variety of germs. To prevent the attachment of disease provoking germs to surfaces (for example computer mouse or keyboards) the implementation of an antimicrobial coating seems reasonable.
Healthcare:
The persistence and spreading of multi resistant bacteria in hospitals is a threat to all patients, especially to patients with immunodeficiency. Nowadays, prevention of the spreading of these germs is one of the major challenges. Our coating could provide a significant contribution to face this problem.
Industry:
Aerospace industries and other companies involved in space expeditions need to consider the importance of germ free conditions. Space missions aiming to reach outer space must not contain any germs from earth to prevent disturbances of any kind.
How to produce TAGC?
Our bio-based surface coating consists out of three major compounds. Chitosan (scaffold) is synthesized in Escherichia coli. The production cascade is based on existing BioBricks from the iGEM Team Darmstadt 2017. The expression and detection of chitosan will be visualized by SDS-PAGE. We will focus on improving BioBricks (including productivity). The combination of chitin synthase (NodC) and chitin deacetylase (COD) will be established to create cells capable of producing chitosan.
Our antimicrobial substances are nisin and rhamnolipid. Rhamnolipid production is carried out in the well established non-pathogenic Pseudomonas putida KT2440 based on already existing BioBricks (iGEM Team Nankai 2014, BBa_K1331001, BBa_K1331004). Further approaches to produce a mixture of mono- and di-rhamnolipids are investigated. Improvement of previous BioBricks will be accomplished by introducing rhamnosyltransferase II for the synthesis of di-rhamnolipids. Analysis is performed using CTAB-Agar plates, thin-layer chromatography (TLC) and a mathematical model of production kinetics is developed.
The nisin operon is extracted from Lactococcus lactis (obtained from Prof. Dr. Oscar Kuipers from the University of Groningen) and transferred to an E. coli strain. The nisin is further modified to incorporate a tyrosine for linkage. We aim to insert a tyrosine residue into the sequence of nisin as a starting point for enzymatic linkage to the chitosan matrix. A special challenge is the creation of new BioBricks because multiple different genes and features need to be introduced. First an inducible promoter with strong RBS is placed in front of nisA. Next, a tyrosine tag for linkage and a His6-tag for purification are introduced between the leader peptide (cleaved at export) and the functional nisA gene.
To create an antimicrobial surface, the mentioned compounds are linked by chemical or enzymatic reactions. Hereby, the chitosan polymer works as a scaffold structure to which nisin and the rhamnolipids are coupled. There are two different approaches for linkage.
The first option is the transesterification with divinyladipate [5]. Hereby, rhamnolipids and chitosan are connected using divinyladipate as linker. The chemical reaction takes place in ionic liquid as solute because chitosan is slighty soluble in other liquids.
Nisin is coupled to chitosan enzymatically by tyrosinase [6]. The method works for the connection of tyrosine containing proteins to chitosan. For this method a nisin variant bearing a tyrosine residue was created. The tyrosinase oxidizes tyrosine which can react with the amine group of chitosan after oxidization and form a covalent bond. As a proof of concept the green fluourescence protein (GFP) was linked to chitosan. The reaction can be analyzed by SDS-PAGE.
References
[1] Silver nanoparticles: mechanism of antimicrobial action, synthesis, medical applications, and toxicity effects. Sukumaran Prabhu and Eldho K Poulose. International Nano Letters. 2012. 2:32.
[2] Insights into the Mode of Action of Chitosan as an Antibacterial Compound. D. Raafat, K. von Bargen, A. Haas, H.-G. Sahl. 2008. Applied and Environmental Microbiology. Volume 74, Issue 12, pp 3764–3773. http://doi.org/10.1128/AEM.00453-08.
[3] Synergic effect of rhamnolipids and nisin to control Listeria monocytogenes, Magalhaes and Nitschke. 2012. New Biotechnology. DOI 10.1016/j.nbt.2012.08.344.
[4] Effect of Mono and Di-rhamnolipids on Biofilms Pre-formed by Bacillus subtilis BBK006. Díaz De Rienzo, M. A. and Martin, P. J. 2016. Curr Microbiol 73:183-189. DOI 10.1007/s00284-016-1046-4.
[5] Homogeneous vinyl ester-based synthesis of different cellulose derivatives in 1-ethyl-3-methyl-imidazolium acetate. Hinner et al. 2016. Green Chemistry. DOI: 10.1039/C6GC02005D.
[6] Tyrosinase-catalyzed modification of Bombyx mori silk fibroin: grafting of chitosan under heterogeneous reaction conditions. Freddi et al. 2006. Journal of Biotechnology, DOI: 10.1016/j.jbiotec.2006.03.003.