Controlling cellular functions by light allows simple triggering of biological processes in a non-invasive fashion with high spatiotemporal resolution. In this context, light-regulated gene expression has enormous potential for achieving optogenetic control over almost any cellular process. Here, we report on two novel one-step cleavable photocaged arabinose compounds, which were applied as light-sensitive inducers of transcription in bacteria. Exposure of caged arabinose to UV-A light resulted in rapid activation of protein production, as demonstrated for GFP and the complete violacein biosynthetic pathway. Moreover, single-cell analysis revealed that intrinsic heterogeneity of arabinose-mediated induction of gene expression was overcome when using photocaged arabinose. We have thus established a novel phototrigger for synthetic bio(techno)logy applications that enables precise and homogeneous control of bacterial target gene expression.
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.
Background Escherichia coli is often used for recombinant protein production. The expression of recombinant proteins negatively affects the microbial growth, thus, a balance between protein expression and biomass formation is preferable to reach high product- and space-time-yield. Already in screening experiments, suboptimal conditions causing too weak or too strong induction must be avoided. High-throughput screening devices such as the BioLector are often applied for screening experiments. The BioLector allows optical online monitoring of each well in a continuously orbitally shaken microtiter plate via scattered light and fluorescence measurements. This technique enables a fast identification of promising clones. However, to determine the expression performance of non-fluorescent products elaborated offline analysis is often required.MethodsA mathematical method is developed to distinguish between cultures, which are insufficiently, optimally or too strongly induced. Therefore, just the temporal development of the scattered light intensity signal is investigated. It is found that discrimination between the different intensities of induction is possible via principal component analysis. By fitting an extended sigmoidal function to the trajectory of the scattered light over time, two characteristic parameters are found. These are used in an empirical model to predict the expression performance.ResultsThe method was established for a wide range of culture conditions based on 625 E. coli cultures. Three E. coli host strains (Tuner(DE3), BL21(DE3), and BL21-Gold(DE3)) expressing either flavin-mononucleotide-based fluorescent protein (FbFP) or Cellulase celA2 were investigated. Cultures were conducted in two different types of microtiter plates (48- and 96-wells), in two online measurement devices at four temperatures (28 °C, 30 °C, 34 °C, and 37 °C). More than 95% of the predicted values are in agreement with the offline measured expression performances with a satisfying accuracy of ±30%.ConclusionsThe properties of cultures studied can be represented by only two characteristic parameters (slope at and time of the inflection point) received from fitting an extended sigmoidal function to the respective scattered light trajectory. Based on these two characteristic parameters, predictions of the standardized expression performance are possible and for a first screen elaborated offline analysis can be avoided. To the best of our knowledge, this is the first work presenting a method for the general prediction of expression performance of E. coli based solely on the temporal development of scattered light signals.Electronic supplementary materialThe online version of this article (doi:10.1186/s13036-017-0064-5) contains supplementary material, which is available to authorized users.
Background Ustilago maydis is known for its natural potential to produce a broad range of valuable chemicals, such as itaconate, from both industrial carbon waste streams and renewable biomass. Production of itaconate, and many other secondary metabolites, is induced by nitrogen limitation in U. maydis. The clustered genes responsible for itaconate production have recently been identified, enabling the development of new expression tools that are compatible with biotechnological processes.ResultsHere we report on the investigation of two of the native promoters, Ptad1 and Pmtt1, from the itaconate cluster of U. maydis MB215. For both promoters the specific activation upon nitrogen limitation, which is known to be the trigger for itaconate production in Ustilago, could be demonstrated by gfp expression. The promoters cover a broad range of expression levels, especially when combined with the possibility to create single- and multicopy construct integration events. In addition, these reporter constructs enable a functional characterization of gene induction patterns associated with itaconate production.ConclusionsThe promoters are well suited to induce gene expression in response to nitrogen limitation, coupled to the itaconate production phase, which contributes towards the further improvement of organic acid production with Ustilago.
Shake flasks are still the most relevant experimental tool in the development of viscous fermentation processes. The phase number, which defines the onset of the unfavorable out‐of‐phase (OP) phenomenon in shake flasks, was previously defined via specific power input measurements. In the OP state, the bulk liquid no longer follows the orbital movement of the imposed centrifugal force, which is for example, detrimental to oxygen transfer. In this study, an optical fluorescence technique was used to measure the three‐dimensional liquid distribution in shake flasks. Four new optically derived evaluation criteria for the phase transition between the in‐phase and OP condition were established: (a) thickness of the liquid film left on the glass wall by the rotating bulk liquid, (b) relative slope of the leading edge of bulk liquid (LB) lines, (c) trend of the angular position of LB, and (d) very high angular position of the leading edge. In contrast to the previously applied power input measurements, the new optical evaluation criteria describe the phase transition in greater detailed. Instead of Ph = 1.26, a less conservative value of Ph = 0.91 is now suggested for the phase transfer, which implies a broader operating window for shake flask cultivations with higher viscosities.
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