Here we report the contributions made by our team towards the iGEM competition. During our project we developed a shuttle vector for Komagataella phaffii, a protocol for zymosan labelling, a protocol for a binding assay and two scripts for the binding site prediction tool P2Rank. Additionally, we also designed two protocols for school visits focusing on restriction enzymes and pamphlets regarding tips for the inclusion of visually impaired persons.
Traditionally, most iGEM teams decide to work with Escherichia coli in their projects. Despite all its advantages it has serious shortcomings for heterologous protein expression and purification [1]. Therefore, eukaryotic expression systems, such as yeast have received more and more attention [2]. However, most iGEM teams still rely on E. coli as the sole chassis within their project. A reasonable explanation for this is the fact that a lack in iGEM teams working with yeasts also results in a low number of parts in the iGEM registry. And even if basic parts like plasmid backbones are available, they are often only designed for model organisms such as Saccharomyces cerevisiae. As we wanted to expand the scope of organisms utilized by iGEM teams within their projects, we developed an RFC[1000] compatible shuttle vector system for the non-model yeast Komagataella phaffii which is an excellent expression system for recombinant protein production, especially in terms of posttranslational modifications [3]. The system is also well suited for correct folding and secretion of the desired protein [3] and we regard it as a great contribution for future iGEM projects. We believe with our adapted vector; more teams can work with such a versatile expression system.
To this end, we build upon the pSB3CY yeast shuttle vector, designed by the iGEM Teams Münster and Dresden 2022, the iGEM Teams of WWU Münster and TU Dresden 2022 designed a level 2 shuttle vector for Saccharomyces cerevisiae, the so called pSB3CY_aeBlue (BBa_K4188001), which is a further development of the iGEM standard part pSB301 (Fig. 1). Due to the design of the plasmid the color of the transformed E. coli colonies changes, depending on the presence of a selection marker. If a selection marker gene is present, the colony shows a red color, and if not, the colonies appear in a dark blue color. The selection marker is interchangeable using the BsmBI type II restriction enzyme. The expression cassette can be inserted in the shuttle vector using standard iGEM parts and is compatible with the MoClo system.