Cellulolytic RoboLector – towards an automated high-throughput screening platform for recombinant cellulase expression

Mühlmann, Martina; Kunze, Martin; Joaquim, Ribeiro; Geinitz, Bertram; Lehmann, Christian W.; Schwaneberg, Ulrich; Commandeur, Ulrich; Büchs, Jochen

doi:10.1186/s13036-016-0043-2

Cited by 47 publications

(59 citation statements)

References 40 publications

(51 reference statements)

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“…Wild type represents the clone expressing the original enzyme. Detailed information about the preparation of the clone bank is described elsewhere …”

Section: Methodsmentioning

confidence: 99%

“…Precultures and main cultures were cultivated in modified Wilms–3‐( N ‐morpholino)propanesulfonic acid (MOPS) mineral medium . The exact composition is described elsewhere . For batch experiments (preculture and main culture), an initial glucose concentration of 20 g L −1 was applied.…”

Section: Methodsmentioning

confidence: 99%

“…With well‐known biomass concentrations, each culture can be inoculated with a customized preculture volume to achieve equal biomass starting conditions. This biomass‐specific inoculation of the main culture, ideally with viable and exponentially growing cells, compensates for variations in growth characteristics during preculture . Alternatively, an automated method to maintain cells at high growth rates until all cultures reach a predetermined biomass concentration is described .…”

Section: Introductionmentioning

confidence: 99%

“…This enables contemporaneous inoculation of the main culture with an equal number of cells in similar metabolic states. If these approaches are to be applied in high‐throughput procedures, automated liquid‐handling systems are unavoidable . Unfortunately, these systems require considerable investment, and integration into an existing high‐throughput process may be challenging.…”

Section: Introductionmentioning

confidence: 99%

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Precultures Grown under Fed‐Batch Conditions Increase the Reliability and Reproducibility of High‐Throughput Screening Results

Keil

Landenberger

Dittrich

et al. 2019

Biotechnology Journal

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One essential task in bioprocess development is strain selection. A common screening procedure consists of three steps: first, the picking of colonies; second, the execution of a batch preculture and main culture, e.g., in microtiter plates (MTPs); and third, the evaluation of product formation. Especially during the picking step, unintended variations occur due to undefined amounts and varying viability of transferred cells. The aim of this study is to demonstrate that the application of polymer‐based controlled‐release fed‐batch MTPs during preculture eliminates these variations. The concept of equalizing growth through fed‐batch conditions during preculture is theoretically discussed and then tested in a model system, namely, a cellulase‐producing Escherichia coli clone bank containing 32 strains. Preculture is conducted once in the batch mode and once in the fed‐batch mode. By applying the fed‐batch mode, equalized growth is observed in the subsequent main culture. Furthermore, the standard deviation of cellulase activity is reduced compared to that observed in the conventional approach. Compared with the strains in the batch preculture process, the first‐ranked strain in the fed‐batch preculture process is the superior cellulase producer. These findings recommend the application of the fed‐batch MTPs during preculture in high‐throughput screening processes to achieve accurate and reliable results.

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“…Wild type represents the clone expressing the original enzyme. Detailed information about the preparation of the clone bank is described elsewhere …”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Precultures Grown under Fed‐Batch Conditions Increase the Reliability and Reproducibility of High‐Throughput Screening Results

Keil

Landenberger

Dittrich

et al. 2019

Biotechnology Journal

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“…Small changes, as reported by Mühlmann et al [17] were applied. The 4-MUC assay was conducted after reaching the stationary phase (indicated by the scattered light signal) and, thus, the time-point of analysis varied due to different culture conditions between 10 and 24 h.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2017

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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.

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Microbioreactor Systems for Accelerated Bioprocess Development

Hemmerich

Noack

Wiechert

et al. 2018

Biotechnology Journal

134

143

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In recent years, microbioreactor (MBR) systems have evolved towards versatile bioprocess engineering tools. They provide a unique solution to combine higher experimental throughput with extensive bioprocess monitoring and control, which is indispensable to develop economically and ecologically competitive bioproduction processes. MBR systems are based either on down-scaled stirred tank reactors or on advanced shaken microtiter plate cultivation devices. Importantly, MBR systems make use of optical measurements for non-invasive, online monitoring of important process variables like biomass concentration, dissolved oxygen, pH, and fluorescence. The application range of MBR systems can be further increased by integration into liquid handling robots, enabling automatization and, thus standardization, of various handling and operation procedures. Finally, the tight integration of quantitative strain phenotyping with bioprocess development under industrially relevant conditions greatly increases the probability of finding the right combination of producer strain and bioprocess control strategy. This review will discuss the current state of the art in the field of MBR systems and we can readily conclude that their importance for industrial biotechnology will further increase in the near future.

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Cellulolytic RoboLector – towards an automated high-throughput screening platform for recombinant cellulase expression

Cited by 47 publications

References 40 publications

Precultures Grown under Fed‐Batch Conditions Increase the Reliability and Reproducibility of High‐Throughput Screening Results

Precultures Grown under Fed‐Batch Conditions Increase the Reliability and Reproducibility of High‐Throughput Screening Results

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

Microbioreactor Systems for Accelerated Bioprocess Development

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