Team:RUBochum/Contribution

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Structural measures to establish iGEM sustainably at the Ruhr-University Bochum

We have achieved our goal of paving the way for future iGEM teams.
As the first team of our university, we had to cope with organizational challenges, especially in the first months, which teams that are already established at their institute do not have and which the future iGEM team of the Ruhr-University Bochum does not have to deal with either.
The contacts we have made in companies and organizations this year, as well as the reputation we have built up, will make it easier for future teams to find external help and financial support.
Work materials have been accumulated which will be passed on to the next teams and the faculty has been informed that an annual recurring use of a workspace will be necessary. Future iGEM teams will thus have the opportunity to start lab-based work earlier. This will give them more time to produce results and lay experimental foundations which other, national and international, iGEM teams can build on. But not only the Faculty of Biology and Biotechnology, but also all other faculties of the University and the Rectorate were informed about the iGEM competition and this thus contributed to the interdisciplinary fusion of the different fields.
By being the first iGEM team to compete, we managed to give more and more people, of all fields of study at the university, the chance to get in touch with synthetic biology. Last but not least, we have also introduced a course on synthetic biology related to iGEM to the faculty, through which it is possible to recruit members for future iGEM teams.

Figure 1: Accumulated devices during the first iGEM season

The Yeast Libary

One big part of our project was the transformation and cultivation of Pichia pastoris, where we improved protocols to establish high transformation.
In the course of our project we noticed that it was difficult to find protocols that created good results.
That is how we got the idea to create a Yeast-Protocol-Library, not only for Pichia pastoris but also to expand our collect of best practice protocols to Saccharomyces cerevisiae, so future iGEM Teams will have easy access to reliable Yeast-Protocols.

Introduction to Yeast
Yeast are eukaryotic microbes that are able to perform post translational modifications and grow on complex or defined media.
Saccharomyces cerevisiae is one of the most commonly used yeast not only in the production of many foods and drinks that are fermented, like beer, bread and wine. But also in the laboratory as a model organism and for the production of recombinant proteins. S. cerevisiae is one of the most studied eukaryotic model organisms and reproduces by budding.
P. pastoris is a methylotrophic yeast that has a lot of similarities to S. cerevisiae, but can also process proteins with larger molecular weight and is able to secret those.

Advantages and disadvantages between Yeast and Bacteria as host organisms for recombinant protein production
Escherichia coli is the most popular host for the production of recombinant proteins. But there are a few key differences between the production in yeast versus in E. coli.
E. coli is a fast and cheap way to produce recombinant proteins. It also has a very short doubling time with around 20 min. In contrast S. cerevisiae and P. pastoris both have doubling times well over one hour. Prokaryotes are generally easier to modify genetically, but eucaryotes like yeast offer more regulation options. Yeast are also non-pathogenic and if your recombinant protein requires post translational modifications like Phosphorylation or Methylation, yeast are a good pick as a host organism.

How to decide between S. cerevisiae and P. pastoris?
If your Team isn't sure which yeast is the best organism for your project, here are the biggest differences between the two: both are non-pathogenic for humans and are able to perform post translational modifications

P. pastoris

• is able to secret the recombinant proteins
• has higher density cell growth and thus higher secretion protein yields
• Doubling time 1 to 3 h at 30 °C
• thrives best in continuous stirred-tank reactor (CSTR), thus may need more time and equipment

S. cerevisiae

• has a larger range of strains and expression vectors
• is more studied and has more extensive literature
• Doubling time 1,5 to 2,5 h at 30 °C

Tips and Tricks for working with Yeast in the lab

• doubling time is often longer in practice than written in the literature
• if you have plates with colonies and are not sure if its yeast or something else, both S. cerevisiae and P. pastoris smell like typical baking yeast
• working with multiple strains as controls, for example with GS P.pastoris
• YNB minimal medium is better for high expression of recombinant proteins

The Libary

Picha pastoris Protcols

Saccharomyces cerevisiaes Protcols

References

Pichia pastoris

Protocol for P. pastoris preparation of competent cells and transformation

our Electroporations worked best when we raised the added template from 50 to 120 ng

Saccharomyces cerevisiae

Protocol for S. cerevisiae competent cells and transformation
Transformation with Electroporation
Competent cells