Prediction of Escherichia coli expression performance in microtiter plates by analyzing only the temporal development of scattered light during culture

Abstract

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.

Online measurement of the respiratory activity in shake flasks enables the identification of cultivation phases and patterns indicating recombinant protein production in various Escherichia coli host strains

“…This observation combined with the fact that the OTR is on a very low level and almost no increase in the recombinant protein ratio can be seen, leads to the conclusion that this strain might have been induced too early and thus has been induced too strongly. This phenomenon has recently also been observed for E. coli cultures that had been induced too early with IPTG . The strains BL21(DE3) and Rosetta(DE3) show good initial growth and a phase of protein production and are, therefore, qualified best (of the strains investigated in this work) for recombinant BSLA and HBD expression in mineral Wilms‐MOPS autoinduction medium under the applied conditions.…”

“…After the preselection of suitable host strains has been carried out with our method, the next step in process development would be the quantitative analysis of the product formation and process optimization. Ladner et al recently presented a method for the prediction of the expression performance of E. coli based on scattered light measurements . This method might be suited for a quantitative analysis.…”

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 .…”

“…Accordingly, the ScL curve reveals a disturbed biomass growth after induction with a long phase of linear increase (~12–20 h). This was already described as indicative for high product formation . As it is difficult for humans to quantitatively interpret variations in slopes within graphs, it is easier to have a look on the ScL derivative.…”

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

“…Furthermore, additional plasmid systems (e.g., T7‐system) will be tested. A mathematical method was recently developed to predict the expression performance of different E. coli strains cultured under different conditions, based on the course of the scattered light signal over time . This approach might in the future be used to evaluate the secretion performance of different products (e.g., enzymes) in the presented secretion system.…”