Light-induced gene expression with photocaged IPTG for induction profiling in a high-throughput screening system

Microbial Cell Factories

Research

Kyra Hoffmann¹  Jochen Büchs¹  Georg Wandrey¹  Claus Bier²  Jörg Pietruszka³  Dennis Binder⁴  Thomas Drepper⁴  Karl-Erich Jaeger⁵ 

[1] AVT-Biochemical Engineering, RWTH Aachen University, Worringerweg 1, 52074, Aachen, Germany;Institute of Bioorganic Chemistry, Heinrich-Heine-University Düsseldorf, Forschungszentrum Jülich, 52426, Jülich, Germany;Institute of Bioorganic Chemistry, Heinrich-Heine-University Düsseldorf, Forschungszentrum Jülich, 52426, Jülich, Germany;Institut für Bio- und Geowissenschaften (IBG-1: Biotechnologie), Forschungszentrum Jülich, 52428, Jülich, Germany;Institute of Molecular Enzyme Technology, Heinrich-Heine-University Düsseldorf, Forschungszentrum Jülich, 52426, Jülich, Germany;Institute of Molecular Enzyme Technology, Heinrich-Heine-University Düsseldorf, Forschungszentrum Jülich, 52426, Jülich, Germany;Institut für Bio- und Geowissenschaften (IBG-1: Biotechnologie), Forschungszentrum Jülich, 52428, Jülich, Germany;

关键词: Caged compounds;    Photocaged IPTG;    LED array;    Optical induction;    Automatization;    High-throughput screening;    Recombinant protein expression;    Escherichia coli;    FbFP;    Induction profiling;    BioLector;    Shaken microtiter plate;   

DOI  :  10.1186/s12934-016-0461-3

 received in 2015-12-04, accepted in 2016-04-13,  发布年份 2016

来源: Springer

【 摘 要 】

BackgroundInducible 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).ResultsA 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.ConclusionsOptical 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.

【 授权许可】

CC BY   
© Wandrey et al. 2016