Browsing by Author "Koehler, Stephan A."

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Artifact-free quantification and sequencing of rare recombinant viruses by using drop-based microfluidics
(2015-10) Tao, Ye; Rotem, Assaf; Zhang, Huidan; Cockrell, Shelley K.; Koehler, Stephan A.; Chang, Connie B.; Ung, Lloyd W.; Cantalupo, Paul G.; Ren, Yukun; Lin, Jeffrey S.; Feldman, Andrew B.; Wobus, Christiane E.; Pipas, James M.; Weitz, David A.
Recombination is an important driver in the evolution of viruses and thus is key to understanding viral epidemics and improving strategies to prevent future outbreaks. Characterization of rare recombinant subpopulations remains technically challenging because of artifacts such as artificial recombinants, known as chimeras, and amplification bias. To overcome this, we have developed a high-throughput microfluidic technique with a second verification step in order to amplify and sequence single recombinant viruses with high fidelity in picoliter drops. We obtained the first artifact-free estimate of in vitro recombination rate between murine norovirus strains MNV-1 and WU20 co-infecting a cell (P(rec) = 3.3 x 10(-4) ± 2 x 10(-5) ) for a 1205 nt region. Our approach represents a time- and cost-effective improvement over current methods, and can be adapted for genomic studies requiring artifact- and bias-free selective amplification, such as microbial pathogens, or rare cancer cells.
Probing phenotypic growth in expanding Bacillus subtilis biofilms
(2016-05) Wang, Xiaoling; Koehler, Stephan A.; Wilking, James N.; Sinha, Naveen N.; Cabeen, Matthew T.; Srinivasan, Siddarth; Seminara, Agnesen; Sun, Qingping; Brenner, Michael P.; Weitz, David A.
We develop an optical imaging technique for spatially and temporally tracking biofilm growth and the distribution of the main phenotypes of a Bacillus subtilis strain with a triple-fluorescent reporter for motility, matrix production, and sporulation. We develop a calibration procedure for determining the biofilm thickness from the transmission images, which is based on Beer-Lambert’s law and involves cross-sectioning of biofilms. To obtain the phenotype distribution, we assume a linear relationship between the number of cells and their fluorescence and determine the best combination of calibration coefficients that matches the total number of cells for all three phenotypes and with the total number of cells from the transmission images. Based on this analysis, we resolve the composition of the biofilm in terms of motile, matrix-producing, sporulating cells and low-fluorescent materials which includes matrix and cells that are dead or have low fluorescent gene expression. We take advantage of the circular growth to make kymograph plots of all three phenotypes and the dominant phenotype in terms of radial distance and time. To visualize the nonlocal character of biofilm growth, we also make kymographs using the local colonization time. Our technique is suitable for real-time, noninvasive, quantitative studies of the growth and phenotype distribution of biofilms which are either exposed to different conditions such as biocides, nutrient depletion, dehydration, or waste accumulation.