Design principles for nuclease-deficient CRISPR-based transcriptional regulators

The engineering of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated proteins (Cas) continues to expand the toolkit available for genome editing, reprogramming gene regulation, genome visualization, and epigenetic studies of living organisms. In this review the emerging design principles on the use of nuclease-deficient CRISPR-based reprogramming of gene expression will be presented. The review will focus on the designs implemented in yeast both at the level of CRISPR proteins and gRNA, but will lend due credits to the seminal studies performed in other species where relevant. In addition to design principles, this review also highlights applications benefitting from the use of CRISPR-mediated transcriptional regulation and discuss the future directions to further expand the toolkit for nuclease-deficient reprogramming of genomes. As such this review should be of general interest for experimentalists to get familiarised with the parameters underlying the power of reprogramming genomic functions by use of nuclease-deficient CRISPR technologies

Genetic biosensor enables in vivo glycosyltransferase screening

Glycosylation of natural products can alter their solubility and bioavailability, among other properties, which makes glycosyltransferases useful tools for increasing the production and/or generating novel compounds in microbial cell factories. However, the discovery and screening of new enzymes and engineered variants is often a low-throughput endeavor due to the need for over-expression and purification prior to in vitro experiments, which do not necessarily represent the in vivo activities of the enzyme. Therefore, a genetic biosensor controlling GFP expression was developed based on the flavonoid responsive transcriptional-repressor QdoR and expressed in E. coli. Due to the induced fluorescent response upon feeding the flavonoids Quercetin and Kaempferol, but not to their glucosides, the activity of UDP-dependent glycosyltransferases (UGTs) could be screened in vivo. Furthermore, a variant of QdoR was generated by directed evolution that showed greater dose-responsiveness and proved to allow greater discrimination of cellular populations and was thus more useful for in vivo UGT screening. The designed biosensor-based method will greatly increase the throughput of glycosyltransferase discovery and engineering. Please click Additional Files below to see the full abstract

Polyphosphate granule biogenesis is temporally and functionally tied to cell cycle exit during starvation in Pseudomonas aeruginosa

Polyphosphate (polyP) granule biogenesis is an ancient and ubiquitous starvation response in bacteria. Although the ability to make polyP is important for survival during quiescence and resistance to diverse environmental stresses, granule genesis is poorly understood. Using quantitative microscopy at high spatial and temporal resolution, we show that granule genesis in Pseudomonas aeruginosa is tightly organized under nitrogen starvation. Following nucleation as many microgranules throughout the nucleoid, polyP granules consolidate and become transiently spatially organized during cell cycle exit. Between 1 and 3 h after nitrogen starvation, a minority of cells have divided, yet the total granule number per cell decreases, total granule volume per cell dramatically increases, and individual granules grow to occupy diameters as large as ∼200 nm. At their peak, mature granules constitute ∼2% of the total cell volume and are evenly spaced along the long cell axis. Following cell cycle exit, granules initially retain a tight spatial organization, yet their size distribution and spacing relax deeper into starvation. Mutant cells lacking polyP elongate during starvation and contain more than one origin. PolyP promotes cell cycle exit by functioning at a step after DNA replication initiation. Together with the universal starvation alarmone (p)ppGpp, polyP has an additive effect on nucleoid dynamics and organization during starvation. Notably, cell cycle exit is temporally coupled to a net increase in polyP granule biomass, suggesting that net synthesis, rather than consumption of the polymer, is important for the mechanism by which polyP promotes completion of cell cycle exit during starvation

Optimizations of Pt/SiC and W/Si multilayers for the Nuclear Spectroscopic Telescope Array

The Nuclear Spectroscopic Telescope Array, NuSTAR, is a NASA funded Small Explorer Mission, SMEX, scheduled for launch in mid 2011. The spacecraft will fly two co-aligned conical approximation Wolter-I optics with a focal length of 10 meters. The mirrors will be deposited with Pt/SiC and W/Si multilayers to provide a broad band reflectivity from 6 keV up to 78.4 keV. To optimize the mirror coating we use a Figure of Merit procedure developed for gazing incidence optics, which averages the effective area over the energy range, and combines an energy weighting function with an angular weighting function to control the shape of the desired effective area. The NuSTAR multilayers are depth graded with a power-law, d_i = a/(b + i)^c, and we optimize over the total number of bi-layers, N, c, and the maximum bi-layer thickness, d_(max). The result is a 10 mirror group design optimized for a flat even energy response both on and off-axis

The effect of aging on cervical parameters in a normative North American population

Study Design: Retrospective cohort study. Objectives: To investigate age-based changes in cervical alignment parameters in an asymptomatic population. Methods: Retrospective review of a prospective study of 118 asymptomatic subjects who underwent biplanar imaging with 3-dimensional capabilities. Demographic and health-related quality of life data was collected prior to imaging. Patients were stratified into 5 age groups: &lt;35 years, 35-44 years, 45-54 years, 55-64 years, and ≥65 years. Radiographic measurements of the cervical spine and spinopelvic parameters were compared between age groups. The normal distribution of parameters was assessed followed by analysis of variance for comparison of variance between age groups. Results: C2-C7 lordosis, C0-C7 lordosis, and T1 slope demonstrated significant increases with age. C0-C7 lordosis was significantly less in subjects &lt;35 years compared with ≥55 years. Significant differences in T1 slope were identified in patients &lt;35 versus ≥65, 35-44 versus ≥65, and 45-54 versus ≥65 years. T1 slope demonstrated a positive correlation with age. Horizontal gaze parameters did not change linearly with age and mean averages of all age groups were within 10° of one another. Cervical kyphosis was present in approximately half of subjects who were &lt;55 compared with approximately 10% of subjects ≥55 years. Differences in pelvic tilt, pelvic incidence-lumbar lordosis, and C7-S1 sagittal vertical axis were identified with age. Conclusions: C0-C7 lordosis, C2-C7 lordosis, and T1 slope demonstrate age-based changes while other cervical and horizontal gaze parameters remain relatively constant with age. </jats:sec

Minimizing Unsatisfaction in Colourful Neighbourhoods

Colouring sparse graphs under various restrictions is a theoretical problem of significant practical relevance. Here we consider the problem of maximizing the number of different colours available at the nodes and their neighbourhoods, given a predetermined number of colours. In the analytical framework of a tree approximation, carried out at both zero and finite temperatures, solutions obtained by population dynamics give rise to estimates of the threshold connectivity for the incomplete to complete transition, which are consistent with those of existing algorithms. The nature of the transition as well as the validity of the tree approximation are investigated.Comment: 28 pages, 12 figures, substantially revised with additional explanatio

Small-molecule biosensors for high-throughput metabolic engineering

Allosteric transcription factors (aTFs) have proven widely applicable for biotechnology and synthetic biology as ligand-specific biosensors enabling real-time monitoring, selection and regulation of cellular metabolism. However, both the biosensor specificity and the correlation between ligand concentration and biosensor output signal, also known as the transfer function, often needs to be optimized before meeting application needs. In this presentation we outline a versatile and high-throughput method to evolve and functionalize prokaryotic aTF ligand specificity and transfer functions in a eukaryote chassis, namely baker’s yeast Saccharomyces cerevisiae. From a single round of directed evolution of the aTF ligand-binding domain coupled with various toggled selection regimes, we robustly select aTF variants evolved for change in ligand specificity, increased dynamic output range, shifts in operational range, and a complete inversion of function from activation to repression. Importantly, by targeting only the ligand-binding domain, the evolved biosensors display DNA-binding affinities similar to parental aTFs and are functional when ported back into a non-native prokaryote chassis. The developed platform technology thus leverages aTF evolvability for the development of new biosensors with user-defined small-molecule specificities and transfer functions. Finally, the presentation will highlight examples on biosensor applications for high-throughput metabolic engineering. Please click Additional Files below to see the full abstract