TY - JOUR A1 - Grunzel, Petra A1 - Pilarek, Maciej A1 - Steinbrueck, Doerte A1 - Neubauer, Antje A1 - Brand, Eva A1 - Kumke, Michael Uwe A1 - Neubauer, Peter A1 - Krause, Mirja T1 - Mini-scale cultivation method enables expeditious plasmid production in Escherichia coli JF - Biotechnology journal : systems & synthetic biology, nanobiotech, medicine N2 - The standard procedure in the lab for plasmid isolation usually involves a 2-mL, 16 h over-night cultivation in 15-mL bioreaction tubes in LB medium. This is time consuming, and not suitable for high-throughput applications. This study shows that it is possible to produce plasmid DNA (pDNA) in a 1.5-mL microcentrifuge tube with only 100 L cultivation volume in less than 7 h with a simple protocol. Compared with the standard LB cultivation for pDNA production reaching a final pDNA concentration range of 1.5-4 mu g mL(-1), a 6- to 10-fold increase in plasmid concentration (from 10 up to 25 mu g mL(-1) cultivation volume) is achieved using an optimized medium with an internal substrate delivery system (EnBase (R)). Different strains, plasmids, and the applicability of different inoculation tools (i.e. different starting ODs) were compared, demonstrating the robustness of the system. Additionally, dissolved oxygen was monitored in real time online, indicating that under optimized conditions oxygen limitation can be avoided. We developed a simple protocol with a significantly decreased procedure time, enabling simultaneous handling of more samples, while a consistent quality and a higher final pDNA concentration are ensured. KW - Escherichia coli KW - High-cell-density culture KW - Miniaturized cultivations KW - Optical oxygen sensor KW - Plasmid DNA production Y1 - 2014 U6 - https://doi.org/10.1002/biot.201300177 SN - 1860-6768 SN - 1860-7314 VL - 9 IS - 1 SP - 128 EP - 136 PB - Wiley-VCH CY - Weinheim ER - TY - JOUR A1 - Dechtrirat, Decha A1 - Gajovic-Eichelmann, Nenad A1 - Wojcik, Felix A1 - Hartmann, Laura A1 - Bier, Frank Fabian A1 - Scheller, Frieder W. T1 - Electrochemical displacement sensor based on ferrocene boronic acid tracer and immobilized glycan for saccharide binding proteins and E. coli JF - Biosensors and bioelectronics : the principal international journal devoted to research, design development and application of biosensors and bioelectronics N2 - Pathogens such as viruses and bacteria use their envelope proteins and their adhesin lectins to recognize the glycan residues presented on the cell surface of the target tissues. This principle of recognition is used in a new electrochemical displacement sensor for the protein concanavalin A (ConA). A gold electrode was first modified with a self-assembled monolayer of a thiolated mannose/OEG conjugate and a ferrocene boroxol derivative was pre-assembled as reporter molecule onto the mannose surface. The novel tracer molecule based on a 2-hydroxymethyl phenyl boronic acid derivative binds even at neutral pH to the saccharides which could expand the application towards biological samples (i.e., urine and feces). Upon the binding of ConA, the tracer was displaced and washed away from the sensor surface leading to a decrease in the electrochemical signal. Using square wave voltammetry (SWV), the concentration of ConA in the sample solution could be determined in the dynamic concentration range established from 38 nmol L-1 to 5.76 mu mol L-1 with a reproducible detection limit of 1 mu g mL(-1) (38 nmol L-1) based on the signal-to-noise ratio (S/N=3) with fast response of 15 min. The new reporter molecule showed a reduced non-specific displacement by BSA and ribonuclease A. The sensor was also successfully transferred to the first proof of principle for the detection of Escherichia coli exhibiting a detection limit of approximately 6 x 102 cells/mL Specificity of the displacement by target protein ConA and E. coli was demonstrated since the control proteins (i.e., BSA and RNaseA) and the control E. coli strain, which lack of type 1 fimbriae, were ineffective. (C) 2014 Elsevier B.V. All rights reserved. KW - Ferrocene benzoboroxol biosensor KW - Concanavalin A KW - Displacement KW - Escherichia coli KW - Ferrocene boronic acid KW - Self-assembled monolayer Y1 - 2014 U6 - https://doi.org/10.1016/j.bios.2014.02.028 SN - 0956-5663 SN - 1873-4235 VL - 58 SP - 1 EP - 8 PB - Elsevier CY - Oxford ER -