Team:Heidelberg/Human Practice/Experts

As a student team, we highly appreciated recommendations and feedback from experts of different scientific areas. Especially for a scientific project like the iGEM competition, it was valuable to unclose new perspecives and assess feasibility of our project ideas. Following, short protocols of the meetings and conversations are listed. At this point we would like to thank all individuals, that have supported us with inspiring discussions, provision of constructs or material.

Contents 1 Experts on Inteins 1.1 Prof. Dr. Henning D. Mootz (WWU Münster, Department of Biochemistry and Biotechnology) 1.2 PD Dr. Matthias Mayer (University Heidelberg, DKFZ-ZMBH alliance) 2 Experts on DNMT1 2.1 Prof. Dr. A. Jeltsch and Dr. P. Bashtrykov (Stuttgart University, Department of Biochemistry) 2.2 Prof. Dr. C. Plass and PD Dr. Dieter Weichenhan (DKFZ, Epigenomics and Cancer Risk Factors)

Experts on Inteins

Prof. Dr. Henning D. Mootz (WWU Münster, Department of Biochemistry and Biotechnology)

When we started our literature research on the use of inteins for posttranslational processing we realized the great contributions of Prof. Dr. Henning D. Mootz to this field of research. Dr. Henning Mootz holds a professorship in Biochemistry and Biotechnology at the Institute of Biochemistry WWU Münster (Germany). In our conversation he gave a nice overview about the most prominent inteins that are used in biotechnology or research and highlighted their special features. We further discussed about the use of inteins for our aim of circularization and appreciated his offer to send us appropriate intein constructs very much. Prof. Dr. Henning Mootz highlighted that until now the induction of inteins cannot be regulated by an in vivo expressible biological mechanism. Therefore, he strongly recommended to pursue this very interesting approach. Moreover, we talked with him about the oligomerization of multidomain proteins to allow expression of their components in E.coli. When talking about the circularization of large proteins, Prof. Dr. Mootz emphasized that efficiency of this approach cannot be predicted due to the complex nature of protein folding. Nevertheless, he commented that proteins have to be imagined as dynamic entities that can potentially be circularized. Nevertheless, efficiency and preservation of the protein function would have to be part of our experiment.

At this point we would again like to thank Prof. Dr. Henning D. Mootz for providing his constructs and sharing his great expertise with us.

PD Dr. Matthias Mayer (University Heidelberg, DKFZ-ZMBH alliance)

The iGEM team Heidelberg met Dr Matthias Mayer, group leader in the center for molecular biology in Heidelberg (ZMBH), for expert counsel. Dr Meyer already knew about intein splicing and various applications and advantages that were proved effective in literature. Together we philosophized about new ideas of intein utilization and our DNMT1 project. He thinks that circularizing DNMT1 would be a risky project, since the increase of thermo stability cannot be predicted. Anyhow if the stability of DNMT1 increases, it would be a break- through not only in terms of a new intein application, but also regarding the opportunities that arise with a heat-stable DNMT1. Matthias Mayer was also fascinated about the idea of a linker software, that calculates the best linker to connect the ends of a protein and evaluates its rigidity to further increase thermo stability. If we manage to set up this software, he would definitely use it!

Experts on DNMT1

Prof. Dr. A. Jeltsch and Dr. P. Bashtrykov (Stuttgart University, Department of Biochemistry)

The group of Prof. Dr. Jeltsch is known for excellent work on characterization of DNMT1. We had the great chance to talk to Dr. Bashtrykov, who recently published several papers on the specificity and function of DNMT1. Together, we discussed the possible ways to express DNMT1 using a baculovirus system in Sf21. Dr. Bashtrykov gave us the decisive hint that there exists an active truncated version of DNMT1 which can be expressed in E.coli and could be used for our purposes of circularization. We appreciated his offer to send us the corresponding construct after approval by Dr. J. Song and Dr. D. Patel very much and would like to thank him for his great support. Moreover, Dr. Bashtrykov provided detailed protocols and information for expression and purification of the protein, including for example the DNA-PAGE Gel composition and staining, which have been adapted and included into our protocols.

Prof. Dr. C. Plass and PD Dr. Dieter Weichenhan (DKFZ, Epigenomics and Cancer Risk Factors)

Prof. Dr. Plass is heading the division Epigenomics and Cancer Risk Factors at the German Cancer Research Center (DKFZ). In our meeting we discussed about the feasibility of a methylation maintaining PCR. Even though his work does not include characterization of DNMT1, he had the idea of improving DNMT1 function in vitro by adding associated proteins. He raised the object that the DNMT1 cofactor SAM could be instable at high temperatures. Overall he emphasized the great impact of a methylation maintaining PCR2.0 on the field of epigenetic.

Dr. Weichenhan is known for his great work on characterization of methylation patterns that occur in the process of disease establishment. Together we discussed the possibility to express DNMT1 using a Baculovirus based system and Drosophia S2 cells. He emphasized that one great advantage of using such a system is the direct secretion of recombinant proteins into the medium. Moreover, he suggested that the use of a eukaryotic expression system might increase active yields of DNMT1 due to proper folding and modifications. We would like to thank him for the great offer to join his lab in case we would have used this eukaryotic expression system.