Integrated Human Practices.

One very simple way to ensure that your scientific work is applied in a responsible way, is to first find out how the public responds to your specific topic and analyze why they do so. With that knowledge in mind, one can then start designing his or her work in a way that, if it may change the world, it will be in a truly beneficial way. We went ahead and gathered information on the attitude of the European population towards genetically modified organisms (GMOs) and also on how it may be changed to a more positive one.

For example, Prof. Dr. Wolfgang Stroebe from the University of Groningen in Netherlands, held a speech on the public’s attitude towards GMOs. He referred us to the work of Dr. Joachim Scholderer.

The view of the European population on the use of GMOs is very ambivalent. There is distrust of information supplied by big companies and a distinct desire to preserve the natural state of the environment among them. This approach is very sensible but in some cases can stand in the way of a reasonable discourse on applications of GMOs. We believe that since most companies use GMOs to slim their processes and increase their market value, the European population just does not see any utility for themselves. Therefore, to make our project useful not only in the lab, but as well to the environment, it has been a major goal to design our work to have a positive effect for every person.

D.I.V.E.R.T. is only a method to handily incorporate random mutations into a target gene. This way, very big libraries of all possible random mutations can be assembled in very little time. These libraries can then be screened for improved properties to pick out the best mutant for specific applications. If selection pressure is applied to the cells, all the while D.I.V.E.R.T. is active, evolution is sped up significantly, and mutations are targeted toward a certain beneficial property of a gene.

To showcase the power of this application, we tried to come up with a way to improve the activity of PETase, in order to make it efficient enough for operative biodegradation of its substrate-polymer. The gene encoding the enzyme PETase is mutated randomly via the D.I.V.E.R.T. method and then screened for enzyme activity on PET-film as substrate. Then, the best mutants are chosen for another round of mutation. This cycle is repeated until no significant improvements can be achieved. Since D.I.V.E.R.T. is intended to work rather fast, the screening method for finding the best PETase mutant should be very time-efficient as well. This imposes several additional problems, but we managed to gather enough information to come up with solutions for all the foreseeable problems -though that has yet to be established in the lab.

Step 1 - D.I.V.E.R.T.

For the purpose of using D.I.V.E.R.T. it may be beneficial to employ Escherichia coli as chassis for the improvement of PETase. In order to apply selection pressure to the E. coli growing on the PET-film, they need to be able to utilize the product of PETase as carbon source. This way, in an otherwise carbon-depraved culture medium, the mutants expressing the best PETase should also be the fastest growers. This creates a situation where the fittest species will also make up the biggest portion of the population. Since PETase is secreted into the medium in order to degrade the plastic film, this might not show as readily, with less efficient mutants benefitting from more efficient mutants in their proximity. However, as long as some form of selection pressure is upheld, the D.I.V.E.R.T. method should yield improved mutants.

PETase hydrolyzes PET into MHET, mainly. MHET is then degraded into TPA and ethylene glycol by the enzyme MHETase. E. coli is most likely not able to utilize MHET as carbon source, however, some mutant strains of E. coli are able to to grow on media containing ethylene glycol as their sole carbon source.¹

The E. coli strain may have to be modified further, to be able to effectively grow on- and degrade PET-film. E. coli can be made “sticky” to hydrophobic surfaces, like PET-film, by having them display an adhesive catecholamine moiety on their cell surface. These proteins are inspired by mussel adhesive proteins (with mussels being able to stick to almost any surface in an aquatic environment) and have been developed by J. P. Park et al. in 2014. One disadvantage in the application of these proteins is that they require the addition of tyrosinase to the media, in order to be adhesive.²

Accordingly, three genes have to be inserted into the E. coli chassis, additionally to the D.I.V.E.R.T. cassette: PETase, MHETase and adhesive proteins. Such a mutant E. coli strain should be capable of growing on- and degrading PET-plastic films.

Step 2 - Cell separation.

For the screening process, mutant cells have to be separated first, in order to screen for the most efficient mutant. Separation can theoretically be done using a flow cytometric cell sorter. In Flow Cytometry, cells in suspension are passed single file across a laser interrogation point. In the case of our application, light scattering signals from the laser could be measured, which correlates with cell morphology, to identify single cells and divert them from the fluid stream.³ This way, single cells can be sorted and assembled into microwell-plates with up to 100.000 wells.⁴ These wells can then theoretically be directly used for the screening process. One big downside of simply singling out cells into microwell plates is that they cannot incorporate a whole library created with D.I.V.E.R.T. In other applications it may be possible to classify the cells during the cell sorting process, but this is hardly possible with catalytic enzymes.

Step 3 - Cell screening.

In 2016 M. T. Zumstein reported on their high-throughput screening method for enzymatic hydrolysis of biodegradable polyesters, utilizing the co-hydrolysis of a fluorogenic ester for real time activity measurement, which seems ideal for screening of mutated PETase, in concert with the D.I.V.E.R.T. method.⁵ The authors embedded a fluorogenic ester, called fluorescein dilaurate, in thin polyester films. As the hydrolytic enzymes degrade the polyester matrix, they incidentally hydrolyse the embedded fluorogenic compound, making it fluorescent and releasing it into the aquatic environment. This means that, as the enzymes degrade the polyester-film, a proportional increase in fluorescence can be measured. What makes this method ideal, is that it can be conducted in microtiter plates, by coating the well with polyester film.

The method, as it is established by Zumstein et al., could potentially be adapted to be fit for PETase screening using D.I.V.E.R.T. Microtiter plates, coated with PET-film⁶, could be inoculated from aforementioned pre-cultures and then measured in plate readers, for real time analysis of hydrolysis activity of PETase. As negative control and blank for quantification, E. coli carrying only adhesive mussel proteins, but no PETase, could be inoculated in parallel. Finally, the most active clone can be picked from the well plates, for another round of mutation.

Mutation of a target gene can be conducted in a simple shaker-culture, induced with arabinose. According to S. Yoshida et al. (2016), I. sakaiensis, the organism originally carrying our gene of interest, encoding PETase, can be grown on PET film immersed in YSV medium, shaken at 300 strokes/ min at 30 °C.⁷

[1]: A. Boronat, E. Caballero, J. Aguilar. Experimental evolution of a metabolic pathway for ethylene glycol utilization by Escherichia coli. Journal of Bacteriology, 153(1): 134-9 (1983)

[2]: J. P. Park, M. J. Choi, S. H. Kim, S. H. Lee, H. Lee. Preparation of Sticky Escherichia coli through Surface Display of an Adhesive Catecholamine Moiety, Applied Environmental Microbiology, 80(1): 43-53 (2014)

[3]: A. Kumar, I. Y. Galaev, B. Mattiasson, Cell Separation: Fundamentals, Analytical and Preparative Methods. Advances in Biochemical Engineering/ Biotechnology 106, Springer-Verlag Berlin Heidelberg (2007)

[4]: S. Lindström, H. Andersson-Svahn. Single-Cell Analysis: Methods and Protocols. Methods in Molecular Biology, 853, Humana Press (2012)

[5]: M. T. Zumstein, H.P. E. Kohler, K. McNeill. M. Sander. High-Throughput Analysis of Enzymatic Hydrolysis of Biodegradeable Polyesters by Monitoring Cohydrolysis of a Polyester-Embedded Fluorogenic Probe. Environmental Science and Technology, 51 (8), 4358-4367 (2017)

[6]: M. T. Zumstein, H.P. E. Kohler, K. McNeill, M. Sander. Enzymatic Hydrolysis of Polyester Thin Films: Real-Time Analysis of Film Mass Changes and Dissipation Dynamics. Environmental Science and Technology, 50, 197-206 (2016)

[7]: S. Yoshida, K. Hiraga, T. Takehana, I. Taniguchi, H. Yamaji, Y. Maeda, K. Toyohara, K. Miyamoto, Y. Kimura, K. Oda. Supplementary Materials for A bacterium that degrades and assimilates poly(ethylene terephtalate). Science, 351, 1196 (2016)