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The Anti Germ Coating
We’re going to work on an antimicrobial surface-coating. It is our goal to produce the substances Nisin (antimicrobial peptide), Rhamnolipid (glycolipid) and Chitosan (biopolymer) in E. coli and P. putida, combine them and transform them into a form suitable for application. The resulting coating should be usable in places, where contact with germs is a commonplace thing, like door handles or various surfaces in hospitals. The application of our coating could reduce or inhibit the growth of multiresistant germs.
Team Stuttgart has been approved and we are ready to create new ideas, biobricks... in the 2018 iGEM season!
Frequently used surfaces in hospitals or other public places are prone to hosting a multitude of germs. Some of them are not dangerous for humans, but others might be pathogenic or have several resistances against antibiotics and are therefore a risk for our health. In this project, we aim to manufacture an antimicrobial and stain-resistant surface coating to prevent the spreading of multiresistant germs in public places. To achieve that, we want to introduce the production of Rhamnolipids and Nisin, both substances with antimicrobial properties, in suitable host organisms, produce and link them to a Chitosan scaffold. The resulting coating should exhibit the same or even enhanced anti-microbial properties as the used substances and prevent the growth and attachment of germs to often used surfaces.
Contaminated surfaces allow germs including pathogens to spread easily. Affected Surfaces can be found not only in hospitals where the proliferation of multi-resistant germs is a major problem but also in public spaces (handholds, railings), schools, workplaces and more. Already existing methods to prevent the spreading of these germs are often based on metals such as silver, copper or titanium. Those are in doubt because of their environmental compatibility and cost-efficiency wherefore a biological and effective alternative will be developed in this project. Antimicrobial molecules such as Rhamnolipid or Nisin will be linked to the carbohydrate chitosan which has strong antimicrobial properties itself (Rafaat et al., 2008) to create a surface with the desired antimicrobial characteristics. 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 was described (Magalhaes and Nitschke, 2012) so that the approach of using these two molecules together to generate a surface coating looks promising. Different ratios between these molecules are tested to get the best result.
Rhamnolipids are a class of glycolipids normally produced by pathogen 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 ''De Rienzo and Martin'', Curr Microbiol (73), pp. 183-189. Rhamnolipid production is to be established in non-pathogenic P. putida KT2440 starting from existent biobricks. Improvement of previous bricks will be accomplished by introducing Rhamnosyltransferase C for synthesis of Di-Rhamnolipids. Analysis is performed using CTAB-Agar plates and thin-layer chromatography (TLC) and a mathematical model of production kinetics is developed. Nisin is an antibiotic normally produced in Lactococcus lactis. It has antimicrobial properties towards gram-positive bacteria. It inhibits the cell wall formation and generates transient pores to disrupt the cell membrane. We aim to insert Tyrosin into the sequence of Nisin as a starting point for chemical and enzymatical linkage to the chitosan matrix. A special challenge is the creation of new biobricks which contain the whole processing machinery for the biological active Nisin. 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 polymers length, which also results in different chemical and physical properties. Short to midrange chitosan with low degrees of acetylation have strong antimicrobial properties. [Raafat et al. (2008): Insights into the Mode of Action of Chitosan as an Antibacterial Compound. In : Applied and Environmental Microbiology (74) , pp. 3764-3773] Therefore, it could be an important part of the anti-germ coating. Production of chitosan in E. coli is to be established based on existing biobricks(iGEM Team Darmstadt 2017) . The expression and detection of chitosan will be shown by using SDS-Page. The focus is on improving biobricks (including productivity). The combination of N-acetylglucosaminyl transferase (NodC) and chitooligosaccharide deacetylase (NodB) will be established to create one cell, which can produce Chitosan.
To create an antimicrobial surface, the mentioned compounds are linked by chemical or enzymatic reactions. Hereby, the chitosan polymer (black) works as a scaffold structure to which Nisin (green) and the Rhamnolipids (red) are coupled. There are three different reaction approaches for the linkage: One of them is the transesterification with divinyl adipate. Here, rhamnolipids and chitosan are connected using divinyl adipate (grey) as linker in ionic liquids. As analyzing method an infrared (IR) spectroscopy will be used. As biological alternative is the enzyme driven catalytic formation of an ester bond between rhamnolipids and chitosan. In this case, hydrolases such as pig liver esterase and lipase B from Candida antarctica will be used. To show the feasibility of this reaction, molecular dynamics (MD) simulations will be performed for investigation if rhamnolipids are accepted as substrate.
Nisin will also be coupled to chitosan enzymatically. Therefore, modified Nisin with Tyrosine at the N-terminus will be used and oxidated in a first step by tyrosinase. The resulting DOPaquinoyl residue is able to perform a nucleophilic attack on the amine group of chitosan. The detection of formation of this … bond will be measured by UV/Vis spectroscopy due to the fact that the aromatic systems absorbs light in a different matter compared to the double bonds between the ring system and the oxygens in the intermediate.