Biology subjects

Gynna, A. H.

Publications and source records attributed to Gynna, A. H..

3 recordsLinked to original sources

bioRxiv2024

Assessment of DNA quality for whole genome library preparation

In recent years, more sophisticated DNA technologies for genotyping have enabled considerable progress in various fields such as clinical genetics, archaeogenetics and forensic genetics. DNA samples previously rejected as too challenging to analyze due to low amounts of degraded DNA can now provide useful information. To increase the chances of success with the new methodologies, it is crucial to know the fragment size of the template DNA molecules, and whether the DNA in a sample is mostly single or double stranded. With this knowledge, an appropriate library preparation method can be chosen, and the DNA shearing parameters of the protocol can be adjusted to the DNA fragment size in the sample. In this study, we first developed and evaluated a user-friendly fluorometry-based protocol for estimation of DNA strandedness. We also evaluated different capillary electrophoresis methods for estimation of DNA fragmentation levels. Next, we applied the developed methodologies to a broad variety of DNA samples processed with different DNA extraction protocols. Our findings show that both the applied DNA extraction method and the sample type affect the DNA strandedness and fragmentation. The established protocols and the gained knowledge will be applicable for future sequencing-based high-density SNP genotyping in various fields.

molecular biology↗

bioRxiv2024

The energy metabolism of Cupriavidus necator in different trophic conditions

The knallgas bacterium Cupriavidus necator is attracting interest for biotechnological applications due to its extremely versatile metabolism. C. necator can use hydrogen or formic acid as an energy source, fixes CO2 via the Calvin-Benson-Bassham (CBB) cycle, and also grows well on various other organic acids and sugars. Its tri-partite genome is notable for its size and manifold duplications of key genes (CBB cycle, hydrogenases, nitrate reductases). Comparatively little is known about which of these (iso-) enzymes and their cofactors are actually utilized for growth on different energy sources. Here, we investigated the energy metabolism of C. necator H16 by growing a barcoded transposon knockout library on various substrates including succinate, fructose, hydrogen (H2/CO2) and formic acid. The fitness contribution of each gene was determined from enrichment or depletion of the corresponding mutants. Fitness analysis revealed that 1) some, but not all, molybdenum cofactor biosynthesis genes were essential for growth on formate and nitrate respiration. 2) Soluble formate dehydrogenase (FDH) was the dominant enzyme for formate oxidation, not membrane-bound FDH. 3) For hydrogenases, both soluble and membrane-bound enzymes were beneficial for lithoautotrophic growth. 4) Of the six terminal respiratory complexes in C. necator H16, only some are utilized and utilization depends on the energy source. 5) Deletion of hydrogenase-related genes boosted heterotrophic growth, and we show that the relief from associated protein cost is responsible for this phenomenon. This study evaluates the contribution of each of C. necators genes to fitness in biotechnologically relevant growth regimes. Our results illustrate the genomic redundancy of this generalist bacterium, and may inspire future strategies for strain engineering. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/582058v2_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@1aac357org.highwire.dtl.DTLVardef@1fe970corg.highwire.dtl.DTLVardef@16ea53dorg.highwire.dtl.DTLVardef@122498d_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIa barcoded transposon library was used to assess gene fitness for hydrogenase, formate dehydrogenase and electron transport chain complexes C_LIO_LIthe utilization of terminal respiratory complexes is substrate specific C_LIO_LIsoluble formate dehydrogenase is more important than the membrane-bound enzyme C_LIO_LIsoluble hydrogenase and membrane-bound hydrogenase are equally utilized C_LIO_LIinactivation of hydrogenase and its accessory genes leads to faster heterotrophic growth C_LIO_LIwe demonstrate that faster growth is caused by the relief of protein synthesis burden C_LIO_LIfitness data is available in an interactive app at https://m-jahn.shinyapps.io/ShinyLib/ C_LI

microbiology↗

bioRxiv2020

RecA mediated homology search finds segregated sister locus in minutes after a double stranded break

Homologous recombination (HR) is essential for the accurate repair of double-stranded DNA breaks (DSBs); it begins when the RecBCD2 complex resects the ends of the DSB into 3' single-stranded DNA (ssDNA) on which a RecA filament assembles. HR depends on the ability of this RecA-ssDNA filament to locate the homologous repair template on the sister chromosome. The mechanism by which the homology is located among vast amounts of heterologous DNA is not yet understood, despite a long history of research. Here, we directly visualize the repair of DSBs in hundreds of individual cells, using high-throughput microfluidics and fluorescence microscopy. We find that in E. coli, DSB repair is completed in 15 minutes without fitness loss. We further show that the search takes less than 10 minutes and is mediated by a thin, highly dynamic RecA filament that stretches throughout the cell. We propose a model in which the architecture of the RecA filament effectively reduces search dimensionality to two dimensions. The model is corroborated by the observation that the search time does not depend on the length of the cell or the amount of DNA, and also predicts a search time that is consistent with our measurement. Since the RecA family proteins are conserved in all organisms, our results also translate to other systems that rely on homologous recombination.

microbiology↗