Systematic investigation of CRISPR-Cas9 configurations for flexible and efficient genome editing in Corynebacterium glutamicum NRRL-B11474.

This study details a reliable and efficient method for CRISPR-Cas9 genome engineering in the high amino acid-producing strain of Corynebacterium glutamicum, NRRL-B11474. Our investigation demonstrates that a plasmid-encoded single-guide RNA paired with different edit-encoding fragments is sufficient to generate edits without the addition of an exogenous recombinase. This approach leverages a genome-integrated copy of the cas9 gene for reduced toxicity, in combination with a single plasmid carrying the targeting guide RNA and matching edit fragment.

Low-Cost, High-Throughput Sequencing of DNA Assemblies Using a Highly Multiplexed Nextera Process.

In recent years, next-generation sequencing (NGS) technology has greatly reduced the cost of sequencing whole genomes, whereas the cost of sequence verification of plasmids via Sanger sequencing has remained high. Consequently, industrial-scale strain engineers either limit the number of designs or take short cuts in quality control. Here, we show that over 4000 plasmids can be completely sequenced in one Illumina MiSeq run for less than $3 each (15× coverage), which is a 20-fold reduction over using Sanger sequencing (2× coverage).

Rapid and reliable DNA assembly via ligase cycling reaction.

Assembly of DNA parts into DNA constructs is a foundational technology in the emerging field of synthetic biology. An efficient DNA assembly method is particularly important for high-throughput, automated DNA assembly in biofabrication facilities and therefore we investigated one-step, scarless DNA assembly via ligase cycling reaction (LCR). LCR assembly uses single-stranded bridging oligos complementary to the ends of neighboring DNA parts, a thermostable ligase to join DNA backbones, and multiple denaturation-annealing-ligation temperature cycles to assemble complex DNA constructs.

A mechanism for the evolution of phosphorylation sites.

Protein phosphorylation provides a mechanism for the rapid, reversible control of protein function. Phosphorylation adds negative charge to amino acid side chains, and negatively charged amino acids (Asp/Glu) can sometimes mimic the phosphorylated state of a protein. Using a comparative genomics approach, we show that nature also employs this trick in reverse by evolving serine, threonine, and tyrosine phosphorylation sites from Asp/Glu residues.

Tuning the activation threshold of a kinase network by nested feedback loops.

Determining proper responsiveness to incoming signals is fundamental to all biological systems. We demonstrate that intracellular signaling nodes can tune a signaling network's response threshold away from the basal median effective concentration established by ligand-receptor interactions. Focusing on the bistable kinase network that governs progesterone-induced meiotic entry in Xenopus oocytes, we characterized glycogen synthase kinase-3beta (GSK-3beta) as a dampener of progesterone responsiveness.

Investigating macromolecules inside cultured and injected cells by in-cell NMR spectroscopy.

The noninvasive character of NMR spectroscopy, combined with the sensitivity of the chemical shift, makes it ideally suited to investigate the conformation, binding events and dynamics of macromolecules inside living cells. These 'in-cell NMR' experiments involve labeling the macromolecule of interest with a nonradioactive but NMR-active isotope (15N or 13C). Cellular samples are prepared either by selectively overexpressing the protein in suitable cells (e.

Tuning bulk electrostatics to regulate protein function.

Cyclin-dependent kinase activation can prevent yeast cells from responding to mating pheromone. Strickfaden et al. (2007) now show that this block arises from the multisite phosphorylation of Ste5.

Quantitative NMR analysis of the protein G B1 domain in Xenopus laevis egg extracts and intact oocytes.

We introduce a eukaryotic cellular system, the Xenopus laevis oocyte, for in-cell NMR analyses of biomolecules at high resolution and delineate the experimental reference conditions for successful implementations of in vivo NMR measurements in this cell type. This approach enables quantitative NMR experiments at defined intracellular concentrations of exogenous proteins, which is exemplified by the description of in-cell NMR properties of the protein G B1 domain (GB1). Additional experiments in Xenopus egg extracts and artificially crowded in vitro solutions suggest that for this biologically inert protein domain, intracellular viscosity and macromolecular crowding dictate its in vivo behavior.

In-cell NMR spectroscopy.

The role of a protein inside a cell is determined by both its location and its conformational state. Although fluorescence techniques are widely used to determine the cellular localization of proteins in vivo, these approaches cannot provide detailed information about a protein's three-dimensional state. This gap, however, can be filled by NMR spectroscopy, which can be used to investigate both the conformation as well as the dynamics of proteins inside living cells.

Methyl groups as probes for proteins and complexes in in-cell NMR experiments.

Studying protein components of large intracellular complexes by in-cell NMR has so far been impossible because the backbone resonances are unobservable due to their slow tumbling rates. We describe a methodology that overcomes this difficulty through selective labeling of methyl groups, which possess more favorable relaxation behavior. Comparison of different in-cell labeling schemes with three different proteins, calmodulin, NmerA, and FKBP, shows that selective labeling with [(13)C]methyl groups on methionine and alanine provides excellent sensitivity with low background levels at very low costs.

Quantitative identification of the protonation state of histidines in vitro and in vivo.

The C[bond]N coupling constants centered at the C(epsilon 1) and C(delta 2) carbons in histidine residues depend on the protonation state and tautomeric form of the imidazole ring, making them excellent indicators of pH or pK(a), and the ratio of the tautomeric states. In this paper, we demonstrate that the intensity ratios for the C(epsilon 1)-H and C(delta 2)-H cross-peaks measured with a constant time HSQC experiment without and with J(C[bond]N) amplitude modulation are determined by the ratios of the protonated and deprotonated forms and tautomeric states. This allows one to investigate the tautomeric state of histidines as well as their pK(a) in situations where changing the pH value by titration is difficult, for example, for in-cell NMR experiments.

A C-terminal inhibitory domain controls the activity of p63 by an intramolecular mechanism.

The human genome is far smaller than originally estimated, and one explanation is that alternative splicing creates greater proteomic complexity than a simple count of open reading frames would suggest. The p53 homologue p63, for example, is a tetrameric transcription factor implicated in epithelial development and expressed as at least six isoforms with widely differing transactivation potential. In particular, p63alpha isoforms contain a 27-kDa C-terminal region that drastically reduces their activity and is of clear biological importance, since patients with deletions in this C terminus have phenotypes very similar to patients with mutations in the DNA-binding domain.