Please use this identifier to cite or link to this item: http://dx.doi.org/10.14279/depositonce-6250
Main Title: On-chip automation of cell-free protein synthesis: new opportunities due to a novel reaction mode
Author(s): Georgi, V.
Georgi, Leopold
Blechert, Martin
Bergmeister, Merlin
Zwanzig, Michael
Wüstenhagen, Doreen A.
Bier, Frank F.
Jung, Erik
Kubick, Stefan
Type: Article
Language Code: en
Abstract: Many pharmaceuticals are proteins or their development is based on proteins. Cell-free protein synthesis (CFPS) is an innovative alternative to conventional cell based systems which enables the production of proteins with complex and even new characteristics. However, the short lifetime, low protein production and expensive reagent costs are still limitations of CFPS. Novel automated microfluidic systems might allow continuous, controllable and resource conserving CFPS. The presented microfluidic TRITT platform (TRITT for Transcription - RNA Immobilization & Transfer - Translation) addresses the individual biochemical requirements of the transcription and the translation step of CFPS in separate compartments, and combines the reaction steps by quasi-continuous transfer of RNA templates to enable automated CFPS. In detail, specific RNA templates with 5' and 3' hairpin structures for stabilization against nucleases were immobilized during in vitro transcription by newly designed and optimized hybridization oligonucleotides coupled to magnetizable particles. Transcription compatibility and reusability for immobilization of these functionalized particles was successfully proven. mRNA transfer was realized on-chip by magnetic actuated particle transfer, RNA elution and fluid flow to the in vitro translation compartment. The applicability of the microfluidic TRITT platform for the production of the cytotoxic protein Pierisin with simultaneous incorporation of a non-canonical amino acid for fluorescence labeling was demonstrated. The new reaction mode (TRITT mode) is a modified linked mode that fulfills the precondition for an automated modular reactor system. By continual transfer of new mRNA, the novel procedure overcomes problems caused by nuclease digestion and hydrolysis of mRNA during TL in standard CFPS reactions.
URI: https://depositonce.tu-berlin.de//handle/11303/6911
http://dx.doi.org/10.14279/depositonce-6250
Issue Date: 2016
Date Available: 24-Oct-2017
DDC Class: 004 Informatik
570 Biowissenschaften, Biologie
540 Chemie und zugeordnete Wissenschaften
Sponsor/Funder: BMBF, 0312039, Nachwuchsgruppe- Biotechnologie 2020+: Chip-basierter Automat für die zellfreie Proteinsynthese
BMBF, 0315942, Zellfreie Bioproduktion - Etablierung einer Bioproduktionsanlage für die zellfreie Proteinsynthese mit integrierter Energieversorgung - Biomoleküle vom Band
Creative Commons License: https://creativecommons.org/licenses/by-nc/3.0/
Journal Title: Lab on a chip : miniaturisation for chemistry and biology
Publisher: Royal Society of Chemistry
Publisher Place: Cambridge
Volume: 16
Issue: 2
Publisher DOI: 10.1039/c5lc00700c
Page Start: 269
Page End: 281
EISSN: 1473-0189
ISSN: 1473-0197
Appears in Collections:Fachgebiet Nano Interconnect Technologies » Publications

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