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Technische Universität Berlin

10 ARTICLES PUBLISHED IN JoVE

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Biology

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays
Katharina L. Dürr 1,2, Neslihan N. Tavraz 1, Susan Spiller 1, Thomas Friedrich 1
1Institute of Chemistry, Technical University of Berlin, 2The Vollum Institute, Oregon Health & Science University

We describe a method to quantify the activity of K+-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb+ or Li+ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies.

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JoVE Journal

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture
Eva-Maria Materne *1, Ilka Maschmeyer *1,2, Alexandra K. Lorenz 1, Reyk Horland 1,2, Katharina M. S. Schimek 1, Mathias Busek 3, Frank Sonntag 3, Roland Lauster 1, Uwe Marx 1,2
1Medical Biotechnology, Technische Universität Berlin, 2TissUse GmbH, 3Fraunhofer IWS

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

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JoVE Core

A Cost-effective and Reliable Method to Predict Mechanical Stress in Single-use and Standard Pumps
Ina Dittler 1, Wolfgang Dornfeld 2, Reto Schöb 2, Jared Cocke 3, Jürgen Rojahn 3, Matthias Kraume 4, Dieter Eibl 1
1Institute of Biotechnology, School of Life Sciences and Facility Management, Zurich University of Applied Sciences, 2Levitronix Ltd., 3SOPAT Ltd., 4Institute of Chemical and Process Engineering, Technische Universität Berlin

Shear stress investigations on an oil-water emulsion system result in drop breakup over the experimental time. To count drop sizes in pumping processes, the suitability of inline endoscopy was successfully demonstrated in this protocol.

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Biology

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System
Emanuel G. Worst 1, Matthias P. Exner 2, Alessandro De Simone 2, Marc Schenkelberger 1, Vincent Noireaux 3, Nediljko Budisa 2, Albrecht Ott 1
1Department of Experimental Physics, Saarland University, 2Institute of Chemistry, Technische Universität Berlin, 3School of Physics and Astronomy, University of Minnesota

An easy-to-use, cell-free expression protocol for the residue-specific incorporation of noncanonical amino acid analogs into proteins, including downstream analysis, is presented for medical, pharmaceutic, structural and functional studies.

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Cancer Research

Study of Viral Vectors in a Three-dimensional Liver Model Repopulated with the Human Hepatocellular Carcinoma Cell Line HepG2
Thomas Hiller 1, Viola Röhrs 1, Eva-Maria Dehne 2, Anke Wagner 1,3, Henry Fechner 1, Roland Lauster 2, Jens Kurreck 1
1Department of Applied Biochemistry, Institute of Biotechnology, Berlin University of Technology, 2Department of Medical Biotechnology, Institute of Biotechnology, Berlin University of Technology, 3Department of Bioprocess Engineering, Institute of Biotechnology, Berlin University of Technology

The recellularized extracellular matrix of a decellularized rat liver can be used as a humanized, three-dimensional ex vivo model to study the distribution and transgene expression of a virus or viral vector.

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Bioengineering

A Method for Determination and Simulation of Permeability and Diffusion in a 3D Tissue Model in a Membrane Insert System for Multi-well Plates
Hao-Hsiang Hsu 1, John-Kevin Kracht 1, Laura Elisabeth Harder 1, Kerstin Rudnik 1, Gerd Lindner 2, Katharina Schimek 2, Uwe Marx 3, Ralf Pörtner 1
1Institute of Bioprocess and Biosystems Engineering, Hamburg University of Technology, 2Institute of Biotechnology, Department Medical Biotechnology, Technische Universität Berlin, 3TissUse GmbH

A method for determination of permeability in a membrane insert system for multi-well plates and in silico parameter optimization for the calculation of diffusion coefficients using simulation are presented.

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Bioengineering

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence
Tobias Baumann 1, Franz-Josef Schmitt 2, Almut Pelzer 1, Vivian Jeanette Spiering 3, Georg Johannes Freiherr von Sass 1, Thomas Friedrich 2, Nediljko Budisa 1
1Institute of Chemistry L 1, Department of Biocatalysis, Technical University of Berlin, 2Institute of Chemistry PC 14, Department of Bioenergetics, Technical University of Berlin, 3Institute of Chemistry TC 7, Department of Physical Chemistry/Molecular Material Sciences, Technical University of Berlin

Synthetic biology enables the engineering of proteins with unprecedented properties using the co-translational insertion of non-canonical amino acids. Here, we presented how a spectrally red-shifted variant of a GFP-type fluorophore with novel fluorescence spectroscopic properties, termed "gold" fluorescent protein (GdFP), is produced in E. coli via selective pressure incorporation (SPI).

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Bioengineering

Antimicrobial Peptides Produced by Selective Pressure Incorporation of Non-canonical Amino Acids
Jessica H. Nickling *1, Tobias Baumann *1, Franz-Josef Schmitt 2, Maike Bartholomae 3, Oscar P. Kuipers 3, Thomas Friedrich 2, Nediljko Budisa 1
1Department of Biocatalysis, Institute of Chemistry, Technische Universität Berlin, 2Department of Bioenergetics, Institute of Chemistry, Technische Universität Berlin, 3Molecular Genetics Group, Groningen Biomolecular Sciences and Biotechnology Institute, Department of Molecular Genetics, University of Groningen

The protocol presents the Escherichia coli-based selective pressure incorporation of non-canonical amino acids (ncAAs) into the lactococcal antimicrobial peptide nisin. Its properties can be changed during recombinant expression via substitution with desired ncAAs in defined growth media. Resulting changes in bioactivity are mapped by growth inhibition assays and fluorescence microscopy.

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Bioengineering

In Situ Microscopy for Real-time Determination of Single-cell Morphology in Bioprocesses
Anna Maria Marbà-Ardébol 1, Joern Emmerich 2, Michael Muthig 2, Peter Neubauer 1, Stefan Junne 1
1Chair of Bioprocess Engineering, Department of Biotechnology, Technische Universität Berlin, 2SOPAT GmbH

A photo-optical in situ microscopy device was developed to monitor the size of single cells directly in the cell suspension. The real-time measurement is conducted by coupling the photo-optical sterilizable probe to an automated image analysis. Morphological changes appear with dependence on the growth state and cultivation conditions.

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Bioengineering

Residue-Specific Exchange of Proline by Proline Analogs in Fluorescent Proteins: How "Molecular Surgery" of the Backbone Affects Folding and Stability
Tuyet Mai Thi To 1, Vladimir Kubyshkin 2, Franz-Josef Schmitt 3, Nediljko Budisa 1,2, Thomas Friedrich 1
1Institute of Chemistry, Technische Universität Berlin, 2Department of Chemistry, University of Manitoba, 3Institute of Physics, Martin-Luther-Universität Halle-Wittenberg

To overcome the limitations of classical site-directed mutagenesis, proline analogs with specific modifications were incorporated into several fluorescent proteins. We show how the replacement of hydrogen by fluorine or of the single by double bonds in proline residues ("molecular surgery") affects fundamental protein properties, including their folding and interaction with light.

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