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Technische Universität Berlin

10 ARTICLES PUBLISHED IN JoVE

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. Tavraz¹, Susan Spiller¹, Thomas Friedrich¹

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.

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. Lorenz¹, Reyk Horland^1,2, Katharina M. S. Schimek¹, Mathias Busek³, Frank Sonntag³, Roland Lauster¹, 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.

JoVE Core

A Cost-effective and Reliable Method to Predict Mechanical Stress in Single-use and Standard Pumps

Ina Dittler¹, Wolfgang Dornfeld², Reto Schöb², Jared Cocke³, Jürgen Rojahn³, Matthias Kraume⁴, Dieter Eibl¹

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.

Biology

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

Emanuel G. Worst¹, Matthias P. Exner², Alessandro De Simone², Marc Schenkelberger¹, Vincent Noireaux³, Nediljko Budisa², Albrecht Ott¹

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.

Cancer Research

Study of Viral Vectors in a Three-dimensional Liver Model Repopulated with the Human Hepatocellular Carcinoma Cell Line HepG2

Thomas Hiller¹, Viola Röhrs¹, Eva-Maria Dehne², Anke Wagner^1,3, Henry Fechner¹, Roland Lauster², Jens Kurreck¹

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.

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 Hsu¹, John-Kevin Kracht¹, Laura Elisabeth Harder¹, Kerstin Rudnik¹, Gerd Lindner², Katharina Schimek², Uwe Marx³, Ralf Pörtner¹

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.

Bioengineering

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Tobias Baumann¹, Franz-Josef Schmitt², Almut Pelzer¹, Vivian Jeanette Spiering³, Georg Johannes Freiherr von Sass¹, Thomas Friedrich², Nediljko Budisa¹

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).

Bioengineering

Antimicrobial Peptides Produced by Selective Pressure Incorporation of Non-canonical Amino Acids

Jessica H. Nickling^*1, Tobias Baumann^*1, Franz-Josef Schmitt², Maike Bartholomae³, Oscar P. Kuipers³, Thomas Friedrich², Nediljko Budisa¹

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.

Bioengineering

In Situ Microscopy for Real-time Determination of Single-cell Morphology in Bioprocesses

Anna Maria Marbà-Ardébol¹, Joern Emmerich², Michael Muthig², Peter Neubauer¹, Stefan Junne¹

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.

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 To¹, Vladimir Kubyshkin², Franz-Josef Schmitt³, Nediljko Budisa^1,2, Thomas Friedrich¹

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.