Single Cell Characterization of a Synthetic Bacterial Clock with a Hybrid Feedback Loop Containing dCas9-sgRNA.

Genetic networks that generate oscillations in gene expression activity are found in a wide range of organisms throughout all kingdoms of life. Oscillatory dynamics facilitates the temporal orchestration of metabolic and growth processes inside cells and organisms, as well as the synchronization of such processes with periodically occurring changes in the environment. Synthetic oscillator gene circuits such as the "repressilator" can perform similar functions in bacteria. Until recently, such circuits were mainly based on a relatively small set of well-characterized transcriptional repressors and activators. A promising, sequence-programmable alternative for gene regulation is given by CRISPR interference (CRISPRi), which enables transcriptional repression of nearly arbitrary gene targets directed by short guide RNA molecules. In order to demonstrate the use of CRISPRi in the context of dynamic gene circuits, we here replaced one of the nodes of a repressilator circuit by the RNA-guided dCas9 protein. Using single cell experiments in microfluidic reactors we show that this system displays robust relaxation oscillations over multiple periods and over several days. With a period of ≈14 bacterial generations, our oscillator is similar in speed as previously reported oscillators. Using an information-theoretic approach for the analysis of the single cell data, the potential of the circuit to act as a synthetic pacemaker for cellular processes is evaluated. We also observe that the oscillator appears to affect cellular growth, leading to variations in growth rate with the oscillator's frequency.

Ex vivo characterization of pathologic fluids with quantitative phase-contrast computed tomography.

PURPOSE: X-ray phase-contrast imaging (PCI) provides additional information beyond absorption characteristics by detecting the phase shift of the X-ray beam passing through material. The grating-based system works with standard polychromatic X-ray sources, promising a possible clinical implementation. PCI has been shown to provide additional information in soft-tissue samples. The aim of this study was to determine if ex vivo quantitative phase-contrast computed tomography (PCCT) may differentiate between pathologic fluid collections. MATERIALS AND METHODS: PCCT was performed with the grating interferometry method. A protein serial dilution, human blood samples and 17 clinical samples of pathologic fluid retentions were imaged and correlated with clinical chemistry measurements. Conventional and phase-contrast tomography images were reconstructed. Phase-contrast Hounsfield Units (HUp) were used for quantitative analysis analogously to conventional HU. The imaging was analyzed using overall means, ROI values as well as whole-volume-histograms and vertical gradients. Contrast to noise ratios were calculated between different probes and between imaging methods. RESULTS: HUp showed a very good linear correlation with protein concentration in vitro. In clinical samples, HUp correlated rather well with cell count and triglyceride content. PCI was better than absorption imaging at differentiating protein concentrations in the protein samples as well as at differentiating blood plasma from cellular components. PCI also allowed for differentiation of watery samples (such as lymphoceles) from pus. CONCLUSION: Phase-contrast computed tomography is a promising tool for the differentiation of pathologic fluids that appear homogenous with conventional attenuation imaging.