Doktorand am Lehrstuhl für Fermentationstechnik
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Jan-Philipp Schwarzhans, Master of Science der Molekularen Biotechnologie
Tel +49 (0)5 21 - 106-5287 (Büro)
Fax +49 (0)5 21 - 106-6475 (Sekretariat)
Email: jsc@fermtech.techfak.uni-bielefeld.de
Curriculum vitae:
06/2006 Abitur, Waldörfer Gymnasium, Hamburg
10/2007-09/2010 Bachelor of Science in "Molecular Biotechnology", Bielefeld University
10/2010-4/2013 Master of Science in "Molecular Biotechnology", Bielefeld University
Practical specializations:
04/2011-11/2011 Part of the student team "Bielefeld University" in the iGEM 2011competition held by the MIT, Boston
04/2012-03/2013 Master thesis in the research group "Fermentation engineering" on the topic "Production of various high value products with Euglena gracilis and Galdieria sulphuraria in the context of a biorefinery concept"
seit 05/2013 PhD student in the research group "Fermentation engineering" as a scholar of the CLIB-Graduate Cluster Industrial Biotechnology CLIB-Graduate Cluster Industrial Biotechnology, Bielefeld University
My main research:
Multiple and directed integration of recombinant protein expression cassettes into the genome of Pichia pastoris
Pichia pastoris is an industrially established producer for recombinant proteins. The expression cassette, together with e.g. the resistance against Zeocin for selection, is integrated via recombination into the genome. The locus of integration is determined through the flanking 5'UTR and 3'UTR of the cassette. An often used locus is the AOX1 site. After successful integration, the AOX1 gene should be replaced, leading to a very low methanol consumption (mutS-type). But often clones show the selection phenotype and still have a functional AOX1 (mut+-type). That means, the integration happened in a random manner. Sometimes the cassette is integrated more than once, leading to a higher copy number of cassettes and occasionally to a higher productivity of recombinant proteins.
The aim of the project is to analyze the random integration and to enhance the copy number of cassettes in the genome. The integration locus and the copy number of a sufficient number of mut+- and mutS-types are analyzed via inside-out-PCR cloning and sequencing and qRT-PCR. Are there favored loci? Does the length of the flanking 5'UTR and 3'UTR have an influence on the loci? The copy number of expression cassettes in the genome could be increased by multiple transformations. To use the same selection marker and expression cassette, the integrated markers have to be eliminated. For such a knock out, the lox/Cre function can be used with the Zeocin cassette, flanked by the lox sequences. After the knock out, other specific distinct loci can be chosen, like the first enzyme in the gylcosylation pathway or a protease. This should allow getting clones with a very high copy number and hopefully high productivity of recombinant proteins. Sequencing the genome of real high producers clones could answer the questions of loci and copy numbers at once.