Automated seamless DNA co-transformation cloning with direct expression vectors applying positive or negative insert selection

<p>Abstract</p> <p>Background</p> <p>Molecular DNA cloning is crucial to many experiments and with the trend to higher throughput of modern approaches automated techniques are urgently required. We have established an automated, fast and flexible low-cost expression cloning approach requiring only vector and insert amplification by PCR and co-transformation of the products.</p> <p>Results</p> <p>Our vectors apply positive selection for the insert or negative selection against empty vector molecules and drive strong expression of target proteins in <it>E.coli </it>cells. Variable tags are available both in N-terminal or C-terminal position. A newly developed β-lactamase (ΔW290) selection cassette contains a segment inside the β-lactamase open reading frame encoding a stretch of hydrophilic amino acids that result in a T7 promoter when back-translated. This position of the promoter permits positive selection and attenuated expression of fusion proteins with C-terminal tags. We have tested eight vectors by inserting six target sequences of variable length, provenience and function. The target proteins were cloned, expressed and detected using an automated Tecan Freedom Evo II liquid handling work station. Only two colonies had to be picked to score with 85% correct inserts while 80% of those were positive in expression tests.</p> <p>Conclusions</p> <p>Our results establish co-transformation and positive/negative selection cloning in conjunction with the provided vectors and selection cassettes as an automatable alternative to commercialized high-throughput cloning systems like Gateway^®or ligase-independent cloning (LIC) <b/>.</p

WDR90 is a centriolar microtubule wall protein important for centriole architecture integrity

Centrioles are characterized by a nine-fold arrangement of microtubule triplets held together by an inner protein scaffold. These structurally robust organelles experience strenuous cellular processes such as cell division or ciliary beating while performing their function. However, the molecular mechanisms underlying the stability of microtubule triplets, as well as centriole architectural integrity remain poorly understood. Here, using ultrastructure expansion microscopy for nanoscale protein mapping, we reveal that POC16 and its human homolog WDR90 are components of the microtubule wall along the central core region of the centriole. We further found that WDR90 is an evolutionary microtubule associated protein. Finally, we demonstrate that WDR90 depletion impairs the localization of inner scaffold components, leading to centriole structural abnormalities in human cells. Altogether, this work highlights that WDR90 is an evolutionary conserved molecular player participating in centriole architecture integrity.</jats:p

Structural insights into the mechanism of the membrane integral N-acyltransferase step in bacterial lipoprotein synthesis

Lipoproteins serve essential roles in the bacterial cell envelope. The posttranslational modification pathway leading to lipoprotein synthesis involves three enzymes. All are potential targets for the development of new antibiotics. Here we report the crystal structure of the last enzyme in the pathway, apolipoprotein N-acyltransferase, Lnt, responsible for adding a third acyl chain to the lipoprotein’s invariant diacylated N-terminal cysteine. Structures of Lnt from Pseudomonas aeruginosa and Escherichia coli have been solved; they are remarkably similar. Both consist of a membrane domain on which sits a globular periplasmic domain. The active site resides above the membrane interface where the domains meet facing into the periplasm. The structures are consistent with the proposed ping-pong reaction mechanism and suggest plausible routes by which substrates and products enter and leave the active site. While Lnt may present challenges for antibiotic development, the structures described should facilitate design of therapeutics with reduced off-target effects

Crystal structure of undecaprenyl-pyrophosphate phosphatase and its role in peptidoglycan biosynthesis

As a protective envelope surrounding the bacterial cell, the peptidoglycan sacculus is a site of vulnerability and an antibiotic target. Peptidoglycan components, assembled in the cytoplasm, are shuttled across the membrane in a cycle that uses undecaprenyl-phosphate. A product of peptidoglycan synthesis, undecaprenyl-pyrophosphate, is converted to undecaprenyl-phosphate for reuse in the cycle by the membrane integral pyrophosphatase, BacA. To understand how BacA functions, we determine its crystal structure at 2.6 Å resolution. The enzyme is open to the periplasm and to the periplasmic leaflet via a pocket that extends into the membrane. Conserved residues map to the pocket where pyrophosphorolysis occurs. BacA incorporates an interdigitated inverted topology repeat, a topology type thus far only reported in transporters and channels. This unique topology raises issues regarding the ancestry of BacA, the possibility that BacA has alternate active sites on either side of the membrane and its possible function as a flippase

High-throughput full-automatic synchrotron-based tomographic microscopy

The automatization tools for high-throughput tomographic microscopy developed at the TOMCAT beamline of the Swiss Light Source are describe

Engineering Dion-Jacobson Perovskites in Polariton Waveguides

Hybrid two-dimensional perovskites hold considerable promise as semiconductors for a wide range of optoelectronic applications. Many efforts are addressed to exploit the potential of these materials by tailoring their characteristics. In this work, the optical properties and electronic band structure in three new Dion-Jacobson (DJ) perovskites (PVKs) are engineered by modulating their structural distortion. Two different interlayer cations: 1-6, Hexamethylendiammonium, HE, and 3-(Dimethylamino)-1-propylammonium, DMPA, have been selected to investigate the role of the cation length and the ammonium binding group on the crystalline structure. This study provides new insights into the understanding of the structure-property relationship in DJ perovskites and demonstrates that exciton characteristics can be easily modulated with the judicious design of the organic cations. DJ PVKs developed in this work were also grown as size-controlled single crystal microwires through a microfluidic-assisted synthesis technique and integrated in a nanophotonic device. The DJ PVK microwire acts as a waveguide exhibiting strong light-matter coupling between the crystal optical modes and DJ PVK exciton. Through the investigation of these polariton waveguides, the nature of the double peak emission, which is often observed in these materials and whose nature is largely debated in the literature, is demonstrated originating from the hybrid polariton state

Compounds binding to the bacterial beta ring

The present invention relates to compounds which bind to the hydrophobic pocket of the B clamp, i.e., to the surface of the Bring with which said protein interacts with other proteins of the bacterial replication complex during DNA replication. These compounds are derived from the acetylated peptide AcOLDLF (P6) to improve their affinity to their target

The mechanism of kinesin inhibition by kinesin binding protein

Subcellular compartmentalisation is necessary for eukaryotic cell function. Spatial and temporal regulation of kinesin activity is essential for building these local environments via control of intracellular cargo distribution. Kinesin binding protein (KBP) interacts with a subset of kinesins via their motor domains, inhibits their microtubule (MT) attachment and blocks their cellular function. However, its mechanisms of inhibition and selectivity have been unclear. Here we use cryo-electron microscopy to reveal the structure of KBP and of a KBP-kinesin motor domain complex. KBP is a TPR-containing, right-handed α-solenoid that sequesters the kinesin motor domain’s tubulin-binding surface, structurally distorting the motor domain and sterically blocking its MT attachment. KBP uses its α-solenoid concave face and edge loops to bind the kinesin motor domain, and selected structure-guided mutations disrupt KBP inhibition of kinesin transport in cells. The KBP-interacting motor domain surface contains motifs exclusively conserved in KBP-interacting kinesins, suggesting a basis for kinesin selectivity

A simple and versatile microfluidic device for efficient biomacromolecule crystallization and structural analysis by serial crystallography

Determining optimal conditions for the production of well diffracting crystals is a key step in every biocrystallography project. Here, a microfluidic device is described that enables the production of crystals by counter-diffusion and their direct on-chip analysis by serial crystallography at room temperature. Nine ‘nonmodel’ and diverse biomacromolecules, including seven soluble proteins, a membrane protein and an RNA duplex, were crystallized and treated on-chip with a variety of standard techniques including micro-seeding, crystal soaking with ligands and crystal detection by fluorescence. Furthermore, the crystal structures of four proteins and an RNA were determined based on serial data collected on four synchrotron beamlines, demonstrating the general applicability of this multipurpose chip conceptThe following funding is acknowledged: Agence Nationale de la Recherche (contract No. ANR-11-LABX- 0057_MITOCROSS to Claude Sauter, Bernard Lorber; contract No. ANR-10-LABX-0036_NETRN to Claude Sauter, Bernard Lorber; contract No. ANR-13-BS07-0007-01 to Eric Girard, Sylvain Engilberge); Ministère des Affaires Etrangères (contract No. PROCOPE Hubert Curien to Claude Sauter, Mario Mörl); Deutsche Forschungsgemeinschaft (contract No. Mo 634/10-1 to Mario Mörl, Heike Betat); Université de Strasbourg [grant No. Initiative d’excellence (IDEX) to Claude Sauter, Raphaël de Wijn]; Centre National de la Recherche Scientifique (grant No. MRCT- 2012_PTI_UPR9002 to Claude Sauter)