Applied Design

Blockages of pipes that is caused by hairy, fatty and soapy components is a daily problem that commonly occurs in every household. By now these blockages are usually removed using chemical tube cleaners that are harmful to health and environment or by laborious mechanical work that can be expensive. By inventing a biological tube cleaner that is based on a holistic approach using the fictionally created E.pipelight that expresses all the desired enzymes, we want to provide for a safe, economic and sustainable tube cleaner that will work more efficient as the nowadays biological tube cleaner do (verlinkung interview cordes).

During our project we talked with various experts from different fields, amongst others Mr. Meyer from the department of wastewater technology of the University Stuttgart, Dr. Schäfer from the German Federal Environmental Foundation and Dr. Arno Cordes from ASA enzymes (see human practices). In the past Dr. Arno Cordes worked on the invention of a biological tube cleaner - based on purified enzymes though-, being supported by the German Federal Environmental Foundation. Additionally we had a great information exchange with the Rohr-Fuchs Company a drain cleaning company, that is located in Stuttgart (see human practices).

All of them provided us with important information what had to be considered when inventing a genetically modified microbial tube cleaner and how this could have effects on society and the environment. Doing research we tried to improve biological tube cleaner approaches that already exist, gaining important information of previous iGEM teams that had worked on similar projects to degrade the clogging of pipes with the help of enzymes that are expressed by microorganisms (e.g. iGEM Team Canmore 2015/16). As the secretion of different enzymes such as keratinases, esterases and lipases has been already shown to be successful in E.coli in the past, we decided to use this gram negative and practical to handle bacteria as our host organism.

Furthermore we knew that by metabolising phenylalanine (a by-product that results during the keratine degrading process), E.coli could produce a rose-like fragrance. Based on this information we planned our project design (fig. 1) using promoters with different strength and distinct signal sequences to provide for the most efficient extracellular enzyme secretion without being toxic to the host organism. After cultivating the different enzyme expressing E.coli strains, harvesting and cell centrifugation was performed to evaluate different enzyme activity assays intracellular and extracellular (see results Esterases and Keratinases). Enzyme activity was shown to be expressed in both locations, so to take advantage of all the expressed enzymes following project approach could be considered, to invent a safe and efficient biological tube cleaner in the future.

If we assume that our product will be actually marketable in the future we would provide for following invention strategy. Our genetically modified auxotroph E.pipelight strain that encodes for a keratinase, an esterase, a lipase and a fragrance gene and that furthermore has a deletion of the asd gene will be cultivated in complex medium containing DPA (see safety) (Fig 2).

With this we provide for an efficient cell growth, as our E.pipelight strain is not lacking any essential components and can proliferate (fig. 3). As soon as we reach a certain cell amount, calculated defined amount of cells will be lyophilized and stored in form of capsules at 4°C. This is an efficient method to preserve cells in a silent mode without destroying their viability or having a destructive effect on the expressed enzymes and has been already successfully shown in different Lactobacili strains for the preparation of oral capsule probiotics (Jalali et al. 2012). Using this storing method the cells can be stored for several months without growing or developing mutations. This would allow us to prepack the E.pipelight capsules and provide them in a cooled supermarket shelf or fridge.

To use E.pipelight in a common household, the encapsuled and freeze-dried cells should be diluted in a certain amount of a standard minimal media to ,awaken’ them. Therefore we would provide all the required components for a complex medium + DPA in a dried form. The amount of DPA is exactly adapted to the cell amount of each E.pipelight capsule. By adding a certain amount of warm water to the DPA containing complex medium, we get a solution in which the E.pipelight can regenerate (fig. 4). The E.pipelight capsule is dissolved a provided lid and poured into the tube afterwards. The calculated amount of DPA in the mixture will provide that these cells can go through just a few more cell divisions to ensure an appropriate amount of cells that can degrade the clogging.

After a defined time, when all of the available DPA is consumed by the E.pipelight, the cells will undergo lysis as the required molecule for cell wall and lysine synthesis is missing. This is an additional benefit, as all of the produced enzymes that haven’t been extracellularly secreted via the signal peptides by now will be released and can contribute to degrade the clogging in the pipe (fig. 5).

This tube cleaner could work more efficient and faster than recent biological tube cleaner do so far, without harming the consumer nor the location of its usage (fig. 6). We know there are still huge amounts of of results and methods that have to be improved and that the invention of a tube cleaner like this still provides certain unpredictable risks that we might have not considered yet. Still we think it could be a great and sustainable alternative in the future for consumer and environment.