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

London / Biomed Central (2016) [Journal Article]

Microbial cell factories
Volume: 15
Issue: 63
Page(s): 16 Seiten

Abstract

Background: Inducible expression systems are frequently used for the production of heterologous proteins. Achievingmaximum product concentrations requires induction profiling, namely the optimization of induction time andinducer 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-polymerasedependent E. coli expression system which required IPTG as inducer. High power UV-A irradiation was directedinto a microtiter plate by light-emitting diodes placed above each well of a 48-well plate. Upon UV irradiation, IPTG isreleased (uncaged) and induces product formation. IPTG uncaging, formation of the fluorescent reporter protein andbiomass growth were monitored simultaneously in up to four 48-well microtiter plates in parallel with an in-houseconstructed BioLector screening system. The amount of released IPTG can be gradually and individually controlledfor each well by duration of UV-A exposure, irradiance and concentration of photocaged IPTG added at the start ofthe cultivation. A comparison of experiments with either optical or conventional IPTG induction shows that productformation and growth are equivalent. Detailed induction profiles revealed that for the strain and conditions usedmaximum product formation is reached for very early induction times and with just 6–8 s of UV-A irradiation or60–80 μM IPTG.Conclusions: Optical induction and online monitoring were successfully combined in a high-throughput screeningsystem and the effect of optical induction with photocaged IPTG was shown to be equivalent to conventional inductionwith 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 ahighly advantageous method for the efficient optimization of heterologous protein production and has the potentialto replace conventional induction with IPTG.$$leng

Authors

Selected Authors

Wandrey, Georg Benjamin
Bier, Claus
Binder, Dennis
Hoffmann, Kyra
Jaeger, Karl-Erich

Other Authors

Pietruszka, Jörg
Drepper, Thomas
Büchs, Jochen

Identifier