A machine vision system for automated non-invasive assessment of cell viability via dark field microscopy, wavelet feature selection and classification

Wei N, Flaschel E, Friehs K, Nattkemper TW. A machine vision system for automated non-invasive assessment of cell viability via dark field microscopy, wavelet feature selection and classification. BMC Bioinformatics. 2008;9(1):449.Background: Cell viability is one of the basic properties indicating the physiological state of the cell, thus, it has long been one of the major considerations in biotechnological applications. Conventional methods for extracting information about cell viability usually need reagents to be applied on the targeted cells. These reagent-based techniques are reliable and versatile, however, some of them might be invasive and even toxic to the target cells. In support of automated noninvasive assessment of cell viability, a machine vision system has been developed. Results: This system is based on supervised learning technique. It learns from images of certain kinds of cell populations and trains some classifiers. These trained classifiers are then employed to evaluate the images of given cell populations obtained via dark field microscopy. Wavelet decomposition is performed on the cell images. Energy and entropy are computed for each wavelet subimage as features. A feature selection algorithm is implemented to achieve better performance. Correlation between the results from the machine vision system and commonly accepted gold standards becomes stronger if wavelet features are utilized. The best performance is achieved with a selected subset of wavelet features. Conclusion: The machine vision system based on dark field microscopy in conjugation with supervised machine learning and wavelet feature selection automates the cell viability assessment, and yields comparable results to commonly accepted methods. Wavelet features are found to be suitable to describe the discriminative properties of the live and dead cells in viability classification. According to the analysis, live cells exhibit morphologically more details and are intracellularly more organized than dead ones, which display more homogeneous and diffuse gray values throughout the cells. Feature selection increases the system's performance. The reason lies in the fact that feature selection plays a role of excluding redundant or misleading information that may be contained in the raw data, and leads to better results

Perceiving molecular evolution processes in Escherichia coli by comprehensive metabolite and gene expression profiling

Transcript and metabolite abundance changes were analyzed in evolved and ancestor strains of Escherichia coli in three different evolutionary condition

Antibiotic-free segregational plasmid stabilization in Escherichia coli owing to the knockout of triosephosphate isomerase (tpiA)

Selvamani RSV, Telaar M, Friehs K, Flaschel E. Antibiotic-free segregational plasmid stabilization in Escherichia coli owing to the knockout of triosephosphate isomerase (tpiA). Microbial Cell Factories. 2014;13(1): 58.Background: Segregational stability of plasmids is of major concern for recombinant bacterial production strains. One of the best strategies to counteract plasmid loss is the use of auxotrophic mutants which are complemented with the lacking gene along with the product-relevant ones. However, these knockout mutants often show unwanted growth in complex standard media or no growth at all under uncomplemented conditions. This led to the choice of a gene for knockout that only connects two essential arms of an essential metabolic pathway - the glycolysis. Results: Triosephosphate isomerase was chosen because its knockout will have a tremendous effect on growth on glucose as well as on glycerol. On glycerol the effect is almost absolute whereas on glucose growth is still possible, but with considerably lower rate than usual. This feature is essential because it may render cloning easier. This enzymatic activity was successfully tested as an alternative to antibiotic-based plasmid selection. Expression of a model recombinant beta-glucanase in continuous cultivation was possible with stable maintenance of the plasmid. In addition, the complementation of tpiA knockout strains by the corresponding plasmids and their growth characteristics were tested on a series of complex and synthetic media. The accumulation of methylglyoxal during the growth of tpiA-deficient strains was shown to be a possible cause for the growth disadvantage of these strains in comparison to the parent strain for the Keio Collection strain or the complemented knock-out strain. Conclusion: Through the use of this new auxotrophic complementation system, antibiotic-free cloning and selection of recombinant plasmid were possible. Continuous cultivation and recombinant protein expression with high segregational stability over an extended time period was also demonstrated

Multiple model approach to modelling of Escherichia coli fed-batch cultivation extracellular production of bacterial phytase

The paper presents the implementation of multiple model approach to modelling of Escherichia coli BL21(DE3)pPhyt109 fed-batch cultivation processes for an extracellular production of bacterial phytase. Due to the complex metabolic pathways of microorganisms, the accurate modelling of bioprocesses is rather difficult. Multiple model approach is an alternative concept which helps in modelling and control of complex processes. The main idea is the development of a model based on simple submodels for the purposes of further high quality process control. The presented simulations of E. coli fed-batch cultivation show how the process could be divided into different functional states and how the model parameters could be obtained easily using genetic algorithms. The obtained results and model verification demonstrate the effectiveness of the applied concept of multiple model approach and of the proposed identification scheme. © 2007 by Pontificia Universidad Católica de Valparaíso

Non-canonical integration events in Pichia pastoris encountered during standard transformation analysed with genome sequencing

Schwarzhans JP, Wibberg D, Winkler A, Luttermann T, Kalinowski J, Friehs K. Non-canonical integration events in Pichia pastoris encountered during standard transformation analysed with genome sequencing. Scientific Reports. 2016;6(1): 38952.The non-conventional yeast Pichia pastoris is a popular host for recombinant protein production in scientific research and industry. Typically, the expression cassette is integrated into the genome via homologous recombination. Due to unknown integration events, a large clonal variability is often encountered consisting of clones with different productivities as well as aberrant morphological or growth characteristics. In this study, we analysed several clones with abnormal colony morphology and discovered unpredicted integration events via whole genome sequencing. These include (i) the relocation of the locus targeted for replacement to another chromosome (ii) co-integration of DNA from the E. coli plasmid host and (iii) the disruption of untargeted genes affecting colony morphology. Most of these events have not been reported so far in literature and present challenges for genetic engineering approaches in this yeast. Especially, the presence and independent activity of E. coli DNA elements in P. pastoris is of concern. In our study, we provide a deeper insight into these events and their potential origins. Steps preventing or reducing the risk for these phenomena are proposed and will help scientists working on genetic engineering of P. pastoris or similar non-conventional yeast to better understand and control clonal variability

A Mitochondrial Autonomously Replicating Sequence from Pichia pastoris for Uniform High Level Recombinant Protein Production

Friehs K, Schwarzhans JP, Luttermann T, et al. A Mitochondrial Autonomously Replicating Sequence from Pichia pastoris for Uniform High Level Recombinant Protein Production. Frontiers in Microbiology. 2017;8: 780.Pichia pastoris is a non-conventional methylotrophic yeast that is widely used for recombinant protein production, typically by stably integrating the target gene into the genome as part of an expression cassette. However, the comparatively high clonal variability associated with this approach usually necessitates a time intense screening step in order to find strains with the desired productivity. Some of the factors causing this clonal variability can be overcome using episomal vectors containing an autonomously replicating sequence (ARS). Here, we report on the discovery, characterization, and application of a fragment of mitochondrial DNA from P. pastoris for use as an ARS. First encountered as an off-target event in an experiment aiming for genomic integration, the newly created circular plasmid named “pMito” consists of the expression cassette and a fragment of mitochondrial DNA. Multiple matches to known ARS consensus sequence motifs, but no exact match to known chromosomal ARS from P. pastoris were detected on the fragment, indicating the presence of a novel ARS element. Different variants of pMito were successfully used for transformation and their productivity characteristics were assayed. All analyzed clones displayed a highly uniform expression level, exceeding by up to fourfold that of a reference with a single copy integrated in its genome. Expressed GFP could be localized exclusively to the cytoplasm via super-resolution fluorescence microscopy, indicating that pMito is present in the nucleus. While expression levels were homogenous among pMito clones, an apparent upper limit of expression was visible that could not be explained based on the gene dosage. Further investigation is necessary to fully understand the bottle-neck hindering this and other ARS vectors in P. pastoris from reaching their full capability. Lastly, we could demonstrate that the mitochondrial ARS from P. pastoris is also suitable for episomal vector transformation in Saccharomyces cerevisiae, widening the potential for biotechnological application. pMito displayed strong potential to reduce clonal variability in experiments targeting recombinant protein production. These findings also showcase the as of yet largely untapped potential of mitochondrial ARS from different yeasts for biotechnological applications

Multiple model approach to modelling of Escherichia coli fed-batch cultivation extracellular production of bacterial phytase

The paper presents the implementation of multiple model approach to modelling of Escherichia coli BL21(DE3)pPhyt109 fed-batch cultivation processes for an extracellular production of bacterial phytase. Due to the complex metabolic pathways of microorganisms, the accurate modelling of bioprocesses is rather difficult. Multiple model approach is an alternative concept which helps in modelling and control of complex processes. The main idea is the development of a model based on simple submodels for the purposes of further high quality process control. The presented simulations of E. coli fed-batch cultivation show how the process could be divided into different functional states and how the model parameters could be obtained easily using genetic algorithms. The obtained results and model verification demonstrate the effectiveness of the applied concept of multiple model approach and of the proposed identification scheme

Maßnahmen zur Verbesserung der Produktion von rekombinanten Proteinen und Plasmid-DNS

Friehs K. Maßnahmen zur Verbesserung der Produktion von rekombinanten Proteinen und Plasmid-DNS. Bielefeld (Germany): Bielefeld University; 1999.Die vorliegende Arbeit umfasst neben einer kurzen Einführung, in der es um Begriffsdefinitionen und Ziele geht, und einer ebenso kurzen Schlussbetrachtung, die einen Vergleich von Expressionssystemen und einen kurzen Blick in die Zukunft enthält, einen theoretischen Teil und einen experimentellen Teil. Die theoretischen Grundlagen beginnen im ersten Kapitel mit einem Abriss der Gentechnik als Methodensammlung zur Optimierung von Mikroorganismen. In fünf weiteren Kapiteln werden Maßnahmen beschrieben, mit denen bestimmte Optimierungsziele der Stammentwicklung erreicht werden können. Diese fünf Kapitel folgen dem theoretischen Ablauf der Produktion von rekombinanten Proteinen. Beginnend mit Betrachtungen der DNS-Ebene, folgt die Ebene der Transkription/Translation, die Protein-Ebene und die Frage nach der Lokalisierung rekombinanter Produkte. Das letzte Kapitel behandelt Aspekte der Aufarbeitung. Die Einteilung des experimentellen Teils erfolgt zwar in Anlehnung an die Gliederung des theoretischen Teils, sie wird jedoch durch spezifische Schwerpunkte der durchgeführten Forschung geprägt. Eigene Untersuchungen und Ergebnisse werden dargestellt und diskutiert. Dabei nehmen Fragen der DNS-Ebene, die sich vor allem auf Plasmide beziehen, eine wichtige Rolle ein und umfassen die ersten drei Kapitel. Experimente auf der Ebene der Transkription/Translation auf dem Gebiet regulierbarer Promotoren beinhaltet Kapitel vier. Die beiden sich anschließenden Kapitel umfassen Experimente zu Fragen der Lokalisierung und Aufarbeitung rekombinanter Proteine. Die Entsorgung von rekombinanter und nicht rekombinanter Biomasse ist Thema des letzten experimentellen Kapitels. Die meisten der präsentierten Daten wurden unter Verwendung von Escherichia coli gewonnen, einige mit zwei anderen GRAM-negativen Bakterien, Alcaligenes eutrophus (Ralstonia eutrophus) und Klebsiella planticola. Die Studien zur Verwertung von mikrobieller Biomasse wurden neben Escherichia coli mit dem GRAM-positiven Bakterium Bacillus licheniformis durchgeführt

Proteinsekretion und -aufarbeitung

Risse JM, Friehs K. Sekretion und Affinitätschromatografie rekombinanter Proteine. BIOspektrum. 2010;5:550-552

Rapid determination of plasmid copy number

Schmidt T, Friehs K, Flaschel E. Rapid determination of plasmid copy number. JOURNAL OF BIOTECHNOLOGY. 1996;49(1-3):219-229.Plasmid copy number, the number of expression vectors per host cell, is a key variable in recombinant microbial cultivation. Therefore, it would be very helpful, if the plasmid copy number could be determined during the operating process period. A rapid quantification of this important process variable would even open the possibility of its use in process control. However, current assays like gel electrophoresis, CsCl-gradient centrifugation, HPLC and other methods are time consuming and difficult to quantify. Indirect methods: like the correlation of copy number with, e.g. the activity of an enzyme, coded on the plasmid, are prone to errors due to the production kinetics, turnover rate and protein denaturation. Here, a method is presented, which enables the plasmid copy number to be determined in less than 30 min. This novel procedure based on plasmid isolation by means of a commercial DNA-isolation kit and quantification by capillary electrophoresis, should allow the copy number to be used in process control