Strategies to Improve Resistance and Production Phenotypes of E. coli

Many of the microbial phenotypes of interest to metabolic engineers are complex in that multiple genes, pathways, and regulatory networks are involved in generating the targeted behavior. A priori prediction of additional changes that will further improve phenotypes can therefore prove difficult due to our incomplete understanding of the functions and connectivity of gene products far removed from the pathway of interest. One complement to rational approaches is to exploit the strength of mutation and selection or screening to obtain strains capable of improved resistance to pretreatment growth improved production titers in the case of production phenotypes. Towards these goals, we are employing and refining methods that rely on natural or augmented mutation rates or on directed protein evolution to improve E. coli phenotypes. Our three major avenues of investigation include selection for inhibitor resistance by continuous culture in chemostats, development of inducible and temperature sensitive mutator plasmids, and generation and screening plasmid libraries of mutated gene regulators for enhanced phenotypic behavior. We are interested in both the genetic and regulatory alterations that underlie phenotypic improvements

Dissecting heterogeneous cell populations across drug and disease conditions with PopAlign

Single-cell measurement techniques can now probe gene expression in heterogeneous cell populations from the human body across a range of environmental and physiological conditions. However, new mathematical and computational methods are required to represent and analyze gene expression changes that occur in complex mixtures of single cells as they respond to signals, drugs, or disease states. Here, we introduce a mathematical modeling platform, PopAlign, that automatically identifies subpopulations of cells within a heterogeneous mixture, and tracks gene expression and cell abundance changes across subpopulations by constructing and comparing probabilistic models. We apply PopAlign to analyze the impact of 42 different immunomodulatory compounds on a heterogeneous population of donor-derived human immune cells as well as patient-specific disease signatures in multiple myeloma. PopAlign scales to comparisons involving tens to hundreds of samples, enabling large-scale studies of natural and engineered cell populations as they respond to drugs, signals or physiological change

Droplet Microfluidic Platforms for Single-Cell Sequencing

DNA sequencin

Preliminary Investigations Into the Etiology and Pathogenesis of Equine Leucoencephalomalacia

181 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1982.Equine leucoencephalomalacis (ELEM) is a fungal-related (Fusarium moniliforme) disease which tends to occur in sporadic seasonal outbreaks in the United States. ELEM is most frequently seen between November and March following corn growing seasons marked by an early to mid-seasonal drought followed by wet and humid conditions accompanied by moderate to cool fluctuating temperatures prior to harvest. The naturally occurring disease syndrome is usually manifested by an acute neurologic disorder, death, and leucomalacia in one or both of the cerebral hemispheres.In this project numerous experiments were conducted with donkeys, ponies, horses, pigs, rabbits, and ducklings in an attempt to characterize the etiology and pathogenesis of ELEM.The signs and lesions characteristic of those typically seen in naturally occurring cases of ELEM were seen in 1 donkey that was fed corn that had been inoculated with an isolate of F. moniliforme recovered from a field outbreak of ELEM. A hepatotoxic form of ELEM, only reported in one other experimental study, was seen in 2 donkeys and 1 pony also given the experimentally inoculated corn. The hepatotoxic syndrome was manifested by an acute and rapidly progressive illness characterized by anorexia, jaundice, lethargy, ataxia, diaphoresis, stupor, recumbency, coma, and death. Centrilobular hepatocellular necrosis and periportal fibrosis were the principal lesions seen in the equids that died from hepatotoxic ELEM.Moniliformin, one of two known mycotoxins produced by F. moniliforme, was shown to be highly toxic to equids and ducklings when given orally or intravenously. The clinical signs and lesions seen in the moniliformin poisoned animals included acute death with no premonitory signs and multifocal myocardial degeneration and early necrosis and marked pulmonary edema and congestion. Two other chemical metabolites produced by F. moniliforme, fusaric acid and 2-methoxy,4-ethyl carbolic acid, were studied in equids and neither appeared to be highly toxic.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

The diplomatic policy of Mr. Madison unveiled; in strictures upon the late correspondence between Mr. Smith and Mr. Jackson ...

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Droplet barcoding for massively parallel single-molecule deep sequencing.

The ability to accurately sequence long DNA molecules is important across biology, but existing sequencers are limited in read length and accuracy. Here, we demonstrate a method to leverage short-read sequencing to obtain long and accurate reads. Using droplet microfluidics, we isolate, amplify, fragment and barcode single DNA molecules in aqueous picolitre droplets, allowing the full-length molecules to be sequenced with multi-fold coverage using short-read sequencing. We show that this approach can provide accurate sequences of up to 10 kb, allowing us to identify rare mutations below the detection limit of conventional sequencing and directly link them into haplotypes. This barcoding methodology can be a powerful tool in sequencing heterogeneous populations such as viruses

Droplet barcoding for massively parallel single-molecule deep sequencing.

The ability to accurately sequence long DNA molecules is important across biology, but existing sequencers are limited in read length and accuracy. Here, we demonstrate a method to leverage short-read sequencing to obtain long and accurate reads. Using droplet microfluidics, we isolate, amplify, fragment and barcode single DNA molecules in aqueous picolitre droplets, allowing the full-length molecules to be sequenced with multi-fold coverage using short-read sequencing. We show that this approach can provide accurate sequences of up to 10 kb, allowing us to identify rare mutations below the detection limit of conventional sequencing and directly link them into haplotypes. This barcoding methodology can be a powerful tool in sequencing heterogeneous populations such as viruses