On-chip automation of cell-free protein synthesis: new opportunities due to a novel reaction mode

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dc.contributor.authorGeorgi, V.
dc.contributor.authorGeorgi, Leopold
dc.contributor.authorBlechert, Martin
dc.contributor.authorBergmeister, Merlin
dc.contributor.authorZwanzig, Michael
dc.contributor.authorWüstenhagen, Doreen A.
dc.contributor.authorBier, Frank F.
dc.contributor.authorJung, Erik
dc.contributor.authorKubick, Stefan
dc.date.accessioned2017-10-24T07:23:00Z
dc.date.available2017-10-24T07:23:00Z
dc.date.issued2016
dc.description.abstractMany 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.en
dc.description.sponsorshipBMBF, 0312039, Nachwuchsgruppe- Biotechnologie 2020+: Chip-basierter Automat für die zellfreie Proteinsyntheseen
dc.description.sponsorshipBMBF, 0315942, Zellfreie Bioproduktion - Etablierung einer Bioproduktionsanlage für die zellfreie Proteinsynthese mit integrierter Energieversorgung - Biomoleküle vom Banden
dc.identifier.eissn1473-0189
dc.identifier.issn1473-0197
dc.identifier.pmid26554896
dc.identifier.urihttps://depositonce.tu-berlin.de/handle/11303/6911
dc.identifier.urihttp://dx.doi.org/10.14279/depositonce-6250
dc.language.isoen
dc.rights.urihttps://creativecommons.org/licenses/by-nc/3.0/
dc.subject.ddc004 Datenverarbeitung; Informatikde
dc.subject.ddc570 Biowissenschaften; Biologiede
dc.subject.ddc540 Chemie und zugeordnete Wissenschaftende
dc.titleOn-chip automation of cell-free protein synthesis: new opportunities due to a novel reaction modeen
dc.typeArticleen
dc.type.versionpublishedVersionen
dcterms.bibliographicCitation.doi10.1039/c5lc00700c
dcterms.bibliographicCitation.issue2
dcterms.bibliographicCitation.journaltitleLab on a chip : miniaturisation for chemistry and biologyen
dcterms.bibliographicCitation.originalpublishernameRoyal Society of Chemistryde
dcterms.bibliographicCitation.originalpublisherplaceCambridgede
dcterms.bibliographicCitation.pageend281
dcterms.bibliographicCitation.pagestart269
dcterms.bibliographicCitation.volume16
tub.accessrights.dnbfree
tub.affiliationFak. 4 Elektrotechnik und Informatik::Inst. Hochfrequenz- und Halbleiter-Systemtechnologien::FG Nano Interconnect Technologiesde
tub.affiliation.facultyFak. 4 Elektrotechnik und Informatikde
tub.affiliation.groupFG Nano Interconnect Technologiesde
tub.affiliation.instituteInst. Hochfrequenz- und Halbleiter-Systemtechnologiende
tub.publisher.universityorinstitutionTechnische Universität Berlin

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