The unstructured C-terminus of the τ subunit of Escherichia coli DNA polymerase III holoenzyme is the site of interaction with the α subunit

The τ subunit of Escherichia coli DNA polymerase III holoenzyme interacts with the α subunit through its C-terminal Domain V, τC16. We show that the extreme C-terminal region of τC16 constitutes the site of interaction with α. The τC16 domain, but not a derivative of it with a C-terminal deletion of seven residues (τC16Δ7), forms an isolable complex with α. Surface plasmon resonance measurements were used to determine the dissociation constant (KD) of the α−τC16 complex to be ∼260 pM. Competition with immobilized τC16 by τC16 derivatives for binding to α gave values of KD of 7 μM for the α−τC16Δ7 complex. Low-level expression of the genes encoding τC16 and τC16▵7, but not τC16Δ11, is lethal to E. coli. Suppression of this lethal phenotype enabled selection of mutations in the 3′ end of the τC16 gene, that led to defects in α binding. The data suggest that the unstructured C-terminus of τ becomes folded into a helix–loop–helix in its complex with α. An N-terminally extended construct, τC24, was found to bind DNA in a salt-sensitive manner while no binding was observed for τC16, suggesting that the processivity switch of the replisome functionally involves Domain IV of τ

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Entwicklung von effizienten genetischen Werkzeugen für Vibrio natriegens

Aktuell werden in der Biotechnologie nur wenige gut erforschte Mikroorganismen eingesetzt, um beispielsweise Pharmazeutika herzustellen. Die verwendeten Organismen stellen daher nur einen verschwindend geringen Anteil der biologischen Vielfalt dar, was mögliche Anwendungsbereiche begrenzt. Aus diesem Grund ist die Etablierung von neuen Produktionsorganismen ein wichtiges Forschungsfeld

NT-CRISPR, combining natural transformation and CRISPR-Cas9 counterselection for markerless and scarless genome editing in Vibrio natriegens

The fast-growing bacterium Vibrio natriegens has recently gained increasing attention as a novel chassis organism for fundamental research and biotechnology. To fully harness the potential of this bacterium, highly efficient genome editing methods are indispensable to create strains tailored for specific applications. V. natriegens is able to take up free DNA and incorporate it into its genome by homologous recombination. This highly efficient natural transformation is able to mediate uptake of multiple DNA fragments, thereby allowing for multiple simultaneous edits. Here, we describe NT-CRISPR, a combination of natural transformation with CRISPR-Cas9 counterselection. In two temporally distinct steps, we first performed a genome edit by natural transformation and second, induced CRISPR-Cas9 targeting the wild type sequence, and thus leading to death of non-edited cells. Through cell killing with efficiencies of up to 99.999%, integration of antibiotic resistance markers became dispensable, enabling scarless and markerless edits with single-base precision. We used NT-CRISPR for deletions, integrations and single-base modifications with editing efficiencies of up to 100%. Further, we confirmed its applicability for simultaneous deletion of multiple chromosomal regions. Lastly, we showed that the near PAM-less Cas9 variant SpG Cas9 is compatible with NT-CRISPR and thereby broadens the target spectrum

Entwicklung von effizienten genetischen Werkzeugen für Vibrio natriegens

Aktuell werden in der Biotechnologie nur wenige gut erforschte Mikroorganismen eingesetzt, um beispielsweise Pharmazeutika herzustellen. Die verwendeten Organismen stellen daher nur einen verschwindend geringen Anteil der biologischen Vielfalt dar, was mögliche Anwendungsbereiche begrenzt. Aus diesem Grund ist die Etablierung von neuen Produktionsorganismen ein wichtiges Forschungsfeld

LVL0 cloning using annealed Oligos v1

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Establishing the fast-growing bacterium Vibrio natriegens as a next-generation chassis for synthetic biology

Vibrio natriegens is the fastest-growing organism known to date with a minimal doubling time of less than ten minutes under optimal conditions. Due to this exciting property and also because of its capability to use a wide range of carbon sources, it was proposed as a novel chassis for Synthetic Biology and Biotechnology. However, so far reports about successful applications of V. natriegens are scarce. This is likely due to the lack of efficient and reliable genetic tools. Furthermore, the knowledge about the biology of V. natriegens is still limited, which leads to unforeseeable challenges for its application. In combination, these two reasons have prevented the widespread application of V. natriegens in Synthetic Biology so far. In this cumulative thesis I describe my contribution to tackling these limitations. Within the scope of this thesis, two CRISPR-Cas9-based tools were added to the set of available methods for V. natriegens. The first tool is NT-CRISPR. This method was developed to improve our ability to perform genomic modifications in V. natriegens. NT-CRISPR relies on V. natriegens’ ability to take up free DNA from the environment in a process called natural transformation. This can be used to introduce a wide range of genomic modifications, such as deletions, integration of foreign DNA or the introduction of point mutations. In a subsequent step, CRISPR-Cas9 is activated to selectively kill non-modified cells, thereby drastically increasing the efficiency of genome modification by natural transformation. It was demonstrated that genome engineering can be performed with high efficiencies for various types of modifications, as well as for the simultaneous deletion of three sequences. In addition to NT-CRISPR, which leads to permanent genetic modifications, graded-CRISPRi was developed as a tool for the inducible repression of genes in V. natriegens. By using two different inducible promoters, a tightly regulated system was created, which does not show any activity in the absence of the inducers. Furthermore, libraries with gRNAs of different lengths and mismatches to the target sequence were used to generate graded knockdown strengths. This concept was applied to four different reporter genes to demonstrate that graded-CRISPRi can be used to generate various expression levels of multiple targets in V. natriegens. Lastly, graded-CRISPRi was targeted against native genes to study the relationship between protein abundance and growth behavior. In addition to developing genetic tools, some progress has been made regarding the understanding of the rapid growth of V. natriegens. Like almost all strains of the Vibrionaceae family, V. natriegens has a bipartite genome configuration. The two chromosomes were fused to generate the monopartite strain synSC1.0. A phenotypic characterization in comparison to the parental strain revealed that synSC1.0 did not show any major difference. This led to the conclusion that the bipartite genome configuration of V. natriegens is not a requirement for its rapid growth