Subdiffusion of loci and cytoplasmic particles are different in compressed Escherichia coli cells.

The complex physical nature of the bacterial intracellular environment remains largely unknown, and has relevance for key biochemical and biological processes of the cell. Although recent work has addressed the role of non-equilibrium sources of activity and crowding, the consequences of mechanical perturbations are relatively less explored. Here we use a microfabricated valve system to track both fluorescently labeled chromosomal loci and cytoplasmic particles in Escherichia coli cells shortly after applying a compressive force, observing the response on time scales that are too sudden to allow for biochemical response from the cell. Cytoplasmic diffusion slows markedly on compression but the exponent governing the growth of the ensemble-averaged mean-squared displacement of cytoplasmic particles is unaffected. In contrast, the corresponding exponent for DNA loci changes significantly. These results suggest that DNA elasticity and nucleoid organization play a more important role in loci subdiffusion than cytoplasmic viscoelasticity under such short time scales.

Role of growth rate on the orientational alignment of Escherichia coli in a slit.

We present experimental data on the nematic alignment of Escherichia coli bacteria confined in a slit, with an emphasis on the effect of growth rate and corresponding changes in cell aspect ratio. Global alignment with the channel walls arises from the combination of local nematic ordering of nearby cells, induced by cell division and the elongated shape of the cells, and the preferential orientation of cells proximate to the side walls of the slit. Decreasing the growth rate leads to a decrease in alignment with the walls, which is attributed primarily to effects of changing cell aspect ratio rather than changes in the variance in cell area. Decreasing confinement also reduces the degree of alignment by a similar amount as a decrease in the growth rate, but the distribution of the degree of alignment differs. The onset of alignment with the channel walls is coincident with the slits reaching their steady-state occupancy and connected to the re-orientation of locally aligned regions with respect to the walls during density fluctuations.

Modeling the relaxation of internal DNA segments during genome mapping in nanochannels.

We have developed a multi-scale model describing the dynamics of internal segments of DNA in nanochannels used for genome mapping. In addition to the channel geometry, the model takes as its inputs the DNA properties in free solution (persistence length, effective width, molecular weight, and segmental hydrodynamic radius) and buffer properties (temperature and viscosity). Using pruned-enriched Rosenbluth simulations of a discrete wormlike chain model with circa 10 base pair resolution and a numerical solution for the hydrodynamic interactions in confinement, we convert these experimentally available inputs into the necessary parameters for a one-dimensional, Rouse-like model of the confined chain. The resulting coarse-grained model resolves the DNA at a length scale of approximately 6 kilobase pairs in the absence of any global hairpin folds, and is readily studied using a normal-mode analysis or Brownian dynamics simulations. The Rouse-like model successfully reproduces both the trends and order of magnitude of the relaxation time of the distance between labeled segments of DNA obtained in experiments. The model also provides insights that are not readily accessible from experiments, such as the role of the molecular weight of the DNA and location of the labeled segments that impact the statistical models used to construct genome maps from data acquired in nanochannels. The multi-scale approach used here, while focused towards a technologically relevant scenario, is readily adapted to other channel sizes and polymers.

Distribution of distances between DNA barcode labels in nanochannels close to the persistence length.

We obtained experimental extension data for barcoded E. coli genomic DNA molecules confined in nanochannels from 40 nm to 51 nm in width. The resulting data set consists of 1 627 779 measurements of the distance between fluorescent probes on 25 407 individual molecules. The probability density for the extension between labels is negatively skewed, and the magnitude of the skewness is relatively insensitive to the distance between labels. The two Odijk theories for DNA confinement bracket the mean extension and its variance, consistent with the scaling arguments underlying the theories. We also find that a harmonic approximation to the free energy, obtained directly from the probability density for the distance between barcode labels, leads to substantial quantitative error in the variance of the extension data. These results suggest that a theory for DNA confinement in such channels must account for the anharmonic nature of the free energy as a function of chain extension.

Measurements of DNA barcode label separations in nanochannels from time-series data.

We analyzed time-series data for fluctuations of intramolecular segments of barcoded E. coli genomic DNA molecules confined in nanochannels with sizes near the persistence length of DNA. These dynamic data allowed us to measure the probability distribution governing the distance between labels on the DNA backbone, which is a key input into the alignment methods used for genome mapping in nanochannels. Importantly, this dynamic method does not require alignment of the barcode to the reference genome, thereby removing a source of potential systematic error in a previous study of this type. The results thus obtained support previous evidence for a left-skewed probability density for the distance between labels, albeit at a lower magnitude of skewness. We further show that the majority of large fluctuations between labels are short-lived events, which sheds further light upon the success of the linearized DNA genome mapping technique. This time-resolved data analysis will improve existing genome map alignment algorithms, and the overall idea of using dynamic data could potentially improve the accuracy of genome mapping, especially for complex heterogeneous samples such as cancer cells.

Engineered antifouling microtopographies: surface pattern effects on cell distribution.

Microtopography has been observed to lead to altered attachment behavior for marine fouling organisms; however, quantification of this phenomenon is lacking in the scientific literature. Here, we present quantitative measurement of the disruption of normal attachment behavior of the fouling algae Ulva linza by antifouling microtopographies. The distribution of the diatom Navicula incerta was shown to be unaffected by the presence of topography. The radial distribution function was calculated for both individual zoospores and cells as well as aggregates of zoospores from attachment data for a variety topographic configurations and at a number of different attachment densities. Additionally, the screening distance and maximum values were mapped according to the location of zoospore aggregates within a single unit cell. We found that engineered topographies decreased the distance between spore aggregates compared to that for a smooth control surface; however, the distributions for individual spores were unchanged. We also found that the local attachment site geometry affected the screening distance for aggregates of zoospores, with certain geometries decreasing screening distance and others having no measurable effect. The distribution mapping techniques developed and explored in this article have yielded important insight into the design parameters for antifouling microtopographies that can be implemented in the next generation of antifouling surfaces.

Mixed confinement regimes during equilibrium confinement spectroscopy of DNA.

We have used a combination of fluorescence microscopy experiments and Pruned Enriched Rosenbluth Method simulations of a discrete wormlike chain model to measure the mean extension and the variance in the mean extension of λ-DNA in 100 nm deep nanochannels with widths ranging from 100 nm to 1000 nm in discrete 100 nm steps. The mean extension is only weakly affected by the channel aspect ratio. In contrast, the fluctuations of the chain extension qualitatively differ between rectangular channels and square channels with the same cross-sectional area, owing to the "mixing" of different confinement regimes in the rectangular channels. The agreement between experiment and simulation is very good, using the extension due to intercalation as the only adjustable parameter.