Biology subjects

Bracharz, F.

Publications and source records attributed to Bracharz, F..

3 recordsLinked to original sources

bioRxiv2019

Characterization of three Yarrowia lipolytica strains in respect to different cultivation temperatures and metabolite secretion

Despite the increasing relevance, ranging from academic research to industrial applications, only a limited number of nonconventional, oleaginous Yarrowia lipolytica strains are characterized in detail. Therefore, we analyzed three strains in regard to their metabolic and physiological properties and in respect to important characteristics of a production strains. A flow cytometry method was set up to evaluate their fitness in a rapid manner. By investigating different cultivation conditions and media compositions, similarities and differences between the distinct strain backgrounds could be derived. Especially sugar alcohol production, as well as a agglomeration of cells were found to be connected with growth at high temperatures. In addition, sugar alcohol production was independent of high substrate concentrations under these conditions. To investigate particular traits, including growth characteristics and metabolite concentrations, genomic analysis were performed. We found sequence variations for one third of the annotated proteins but no obvious link to all phenotypic features.

microbiology↗

bioRxiv2019

Quantification and Isolation of Bacillus subtilis Spores using Cell Sorting and automated Gating.

The Gram-positive bacterium Bacillus subtilis is able to form endospores which have a variety of biotechnological applications. Due to this ability, B. subtilis is as well a model organism for cellular differentiation processes. Sporulating cultures of Bacillus subtilis form sub-populations which include vegetative cells, spore forming cells and spores. In order to readily and rapidly quantify spore formation we employed flow cytometric and fluorescence activated cell sorting techniques in combination with nucleic acid fluorescent staining in order to investigate the distribution of sporulating cultures on a single cell level. Moreover we tested different fluorescent dyes as well as different conditions in order to develop a method for optimal separation of distinct populations during sporulation. Automated gating procedures using k-means clustering and thresholding by gaussian mixture modeling were employed to avoid subjective gating and allow for the simultaneous measurement of controls. We utilized the presented method for monitoring sporulation over time in strains harboring different genome modifications. We identified the different subpopulations formed during sporulation by employing sorting and microscopy. Finally, we employed the technique to show that a double knock-out mutant of the phosphatase gene encoding Spo0E and of the spore killing factor SkfA results in faster spore formation.

microbiology↗

bioRxiv2018

CopySwitch - in vivo optimization of gene copy numbers for heterologous gene expression in Bacillus subtilis

1.The Gram-positive bacterium Bacillus subtilis has long been used as a host for production and secretion of industrially relevant enzymes like amylases and proteases. It is imperative for optimal efficiency, to balance protein yield and correct folding. Gene copy numbers are an important tuning valve for the optimization of heterologous gene expression. While some genes are best expressed from many gene copies, for other genes, medium or even single copy numbers are the only way to avoid formation of inclusion bodies, toxic gene dosage effects or achieve desired levels for metabolic engineering. In order to provide a simple and robust method to address above-mentioned issues in the Gram-positive bacterium Bacillus subtilis, we have developed an automatable system for the tuning of heterologous gene expression based on the hosts intrinsic natural competence and homologous recombination capabilities. By supplying our reporter strains with a linearized, low copy number plasmid containing homology regions left and right of the reporter genes and an antibiotic resistance marker, we could show an up to 3.6-fold higher gfp (green fluorescent protein) expression and up to 1.3-fold higher mPLC (mature phospholipase C) expression after successful recombination and thus circularization of our plasmid. Furthermore, the plasmid-borne gfp expression seems to be more stable, since over the whole cultivation period the share of fluorescent cells compared to all measured cells is consistently higher.

bioengineering↗