Light-induced gene expression with photocaged IPTG for induction profiling in a high-throughput screening system
| العنوان: | Light-induced gene expression with photocaged IPTG for induction profiling in a high-throughput screening system |
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| المؤلفون: | Jörg Pietruszka, Georg Wandrey, Karl-Erich Jaeger, Claus Bier, Thomas Drepper, Kyra Hoffmann, Jochen Büchs, Dennis Binder |
| المصدر: | Microbial Cell Factories Microbial cell factories 15, 63 (2016). doi:10.1186/s12934-016-0461-3 Microbial cell factories 15(63), 16 Seiten (2016). doi:10.1186/s12934-016-0461-3 |
| بيانات النشر: | BioMed Central, 2016. |
| سنة النشر: | 2016 |
| مصطلحات موضوعية: | 0106 biological sciences, 0301 basic medicine, Isopropyl Thiogalactoside, Light, High-throughput screening, Heterologous, lac operon, Bioengineering, Biology, Recombinant protein expression, medicine.disease_cause, Shaken microtiter plate, 01 natural sciences, Applied Microbiology and Biotechnology, Automatization, 03 medical and health sciences, Microtiter plate, Industrial Microbiology, Viral Proteins, 010608 biotechnology, Gene expression, Optical induction, medicine, Protein biosynthesis, Escherichia coli, Inducer, BioLector, ddc:610, Photocaged IPTG, FbFP, Bacteriological Techniques, Research, Gene Expression Profiling, DNA-Directed RNA Polymerases, Gene Expression Regulation, Bacterial, Molecular biology, Caged compounds, High-Throughput Screening Assays, carbohydrates (lipids), 030104 developmental biology, Induction profiling, Biophysics, Caged compounds, Photocaged IPTG, LED array, Optical induction, Automatization, High-throughput screening, Recombinant protein expression, Escherichia coli, FbFP, Induction profiling, BioLector, Shaken microtiter plates, Biotechnology, LED array |
| الوصف: | Background Inducible expression systems are frequently used for the production of heterologous proteins. Achieving maximum product concentrations requires induction profiling, namely the optimization of induction time and inducer concentration. However, the respective experiments can be very laborious and time-consuming. In this work, a new approach for induction profiling is presented where induction in a microtiter plate based cultivation system (BioLector) is achieved by light using photocaged isopropyl β-d-1-thiogalactopyranoside (cIPTG). Results A flavin mononucleotide-based fluorescent reporter protein (FbFP) was expressed using a T7-RNA-polymerase dependent E. coli expression system which required IPTG as inducer. High power UV-A irradiation was directed into a microtiter plate by light-emitting diodes placed above each well of a 48-well plate. Upon UV irradiation, IPTG is released (uncaged) and induces product formation. IPTG uncaging, formation of the fluorescent reporter protein and biomass growth were monitored simultaneously in up to four 48-well microtiter plates in parallel with an in-house constructed BioLector screening system. The amount of released IPTG can be gradually and individually controlled for each well by duration of UV-A exposure, irradiance and concentration of photocaged IPTG added at the start of the cultivation. A comparison of experiments with either optical or conventional IPTG induction shows that product formation and growth are equivalent. Detailed induction profiles revealed that for the strain and conditions used maximum product formation is reached for very early induction times and with just 6–8 s of UV-A irradiation or 60–80 µM IPTG. Conclusions Optical induction and online monitoring were successfully combined in a high-throughput screening system and the effect of optical induction with photocaged IPTG was shown to be equivalent to conventional induction with IPTG. In contrast to conventional induction, optical induction is less costly to parallelize, easy to automate, non-invasive and without risk of contamination. Therefore, light-induced gene expression with photocaged IPTG is a highly advantageous method for the efficient optimization of heterologous protein production and has the potential to replace conventional induction with IPTG. Electronic supplementary material The online version of this article (doi:10.1186/s12934-016-0461-3) contains supplementary material, which is available to authorized users. |
| اللغة: | English |
| تدمد: | 1475-2859 |
| DOI: | 10.1186/s12934-016-0461-3 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::c73919b7c2233cbfd00996f18252edbd http://europepmc.org/articles/PMC4842301 |
| Rights: | OPEN |
| رقم الانضمام: | edsair.doi.dedup.....c73919b7c2233cbfd00996f18252edbd |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 14752859 |
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| DOI: | 10.1186/s12934-016-0461-3 |