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

Abstract

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0461-3) contains supplementary material, which is available to authorized users.

Prediction of recombinant protein production by Escherichia coli derived online from indicators of metabolic burden

“…The induction took place with different IPTG concentrations after 7 h (indicated by black arrow in Figure ). As already described by previous studies, the OTR and ScL pattern changes with increasing inducer concentration . The OTR and ScL curves as well as the dScL/dt curves are plotted in Figure .…”

“…Induction profiling is an approach to investigate the impact of different inducer concentrations and induction times on the production performance and is described in detail elsewhere . A complete induction profiling was conducted during the main cultivation in one 48‐well MTP using up to 48 different induction conditions.…”

“…Induction profiling is an approach to investigate the impact of different inducer concentrations and induction times on the production performance and is described in detail elsewhere. 45,46 A complete induction profiling was conducted during the main cultivation in one 48-well MTP using up to 48 different induction conditions. The induction was performed automatically by a liquid handling robot for all Flowerplate cultivations with the so-called RoboLector platform 42,45 using a previously presented method.…”

Scale‐up of a Type I secretion system in E. coli using a defined mineral medium

“…Time of induction and inducer concentration is known to have a high impact on recombinant protein expression . Since secretion of HlyA1 is well controlled by addition of arabinose, time of induction and inducer concentration were varied to study the effect on the respiration behavior of E. coli and the HlyA1 concentration in the supernatant.…”

Illuminating pathogen–host intimacy through optogenetics

“…As depicted in Fig 1A and [13,14], cGMP [15,16], or c-di-GMP [42,43]. It is also possible to develop light-responsive expression systems using photocaged effectors, such as doxycycline [44] and IPTG (isopropyl β-D-1-thiogalactopyranoside) [45] or proteins [46,47], which are released or activated upon illumination to control the gene expression or protein activity. Just recently, the LOV2-ODC-degron system has been reported, which targets the conjugated protein of interest to light-dependent proteasomal degradation, thereby controlling the protein stability [48] (Fig 1B).…”