Light-induced gene expression with photocaged IPTG for induction profiling in a high-throughput screening system.
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.
AVT-Biochemical Engineering, RWTH Aachen University, Worringerweg 1, Aachen, 52074, Germany.
Georg
G
Wandrey
Institute of Bioorganic Chemistry, Heinrich-Heine-University Düsseldorf, Forschungszentrum Jülich, Jülich, 52426, Germany.
Claus
C
Bier
Institute of Molecular Enzyme Technology, Heinrich-Heine-University Düsseldorf, Forschungszentrum Jülich, Jülich, 52426, Germany.
Dennis
D
Binder
AVT-Biochemical Engineering, RWTH Aachen University, Worringerweg 1, Aachen, 52074, Germany.
Kyra
K
Hoffmann
Institute of Molecular Enzyme Technology, Heinrich-Heine-University Düsseldorf, Forschungszentrum Jülich, Jülich, 52426, Germany.|Institut für Bio- und Geowissenschaften (IBG-1: Biotechnologie), Forschungszentrum Jülich, Jülich, 52428, Germany.
Karl-Erich
KE
Jaeger
Institute of Bioorganic Chemistry, Heinrich-Heine-University Düsseldorf, Forschungszentrum Jülich, Jülich, 52426, Germany.|Institut für Bio- und Geowissenschaften (IBG-1: Biotechnologie), Forschungszentrum Jülich, Jülich, 52428, Germany.
Jörg
J
Pietruszka
Institute of Molecular Enzyme Technology, Heinrich-Heine-University Düsseldorf, Forschungszentrum Jülich, Jülich, 52426, Germany.
Thomas
T
Drepper
AVT-Biochemical Engineering, RWTH Aachen University, Worringerweg 1, Aachen, 52074, Germany. jochen.buechs@avt.rwth-aachen.de.
Jochen
J
Büchs
D001431:Bacteriological Techniques / Q000379:methods; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D020869:Gene Expression Profiling / Q000379:methods; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects / Q000528:radiation effects; D057166:High-Throughput Screening Assays / Q000379:methods*; D007218:Industrial Microbiology / Q000379:methods*; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology*; D008027:Light; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
D014764:Viral Proteins; D007544:Isopropyl Thiogalactoside; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases
Automatization; BioLector; Caged compounds; Escherichia coli; FbFP; High-throughput screening; Induction profiling; LED array; Optical induction; Photocaged IPTG; Recombinant protein expression; Shaken microtiter plate