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Creators/Authors contains: "Dueber, John E."

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  1. Abstract

    Long single-stranded DNA (ssDNA) is a versatile molecular reagent with applications including RNA-guided genome engineering and DNA nanotechnology, yet its production is typically resource-intensive. We introduce a novel method utilizing an engineered Escherichia coli ‘helper’ strain and phagemid system that simplifies long ssDNA generation to a straightforward transformation and purification procedure. Our method obviates the need for helper plasmids and their associated contamination by integrating M13mp18 genes directly into the E. coli chromosome. We achieved ssDNA lengths ranging from 504 to 20 724 nt with titers up to 250 μg/l following alkaline lysis purification. The efficacy of our system was confirmed through its application in primary T-cell genome modifications and DNA origami folding. The reliability, scalability and ease of our approach promise to unlock new experimental applications requiring large quantities of long ssDNA.

     
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  4. Abstract

    Optimizing microbial hosts for the large-scale production of valuable metabolites often requires multiple mutations and modifications to the host’s genome. We describe a three-round screen for increased L-DOPA production inS. cerevisiaeusing FACS enrichment of an enzyme-coupled biosensor for L-DOPA. Multiple rounds of screening were enabled by a single build of a barcodedin vitrotransposon-mediated disruption library. New background strains for screening were built for each iteration using results from previous iterations. The samein vitrotransposon-mediated disruption library was integrated by homologous recombination into new background strains in each round of screening. Compared with creating new transposon insertions in each round, this method takes less time and saves the cost of additional sequencing to characterize transposon insertion sites. In the first two rounds of screening, we identified deletions that improved biosensor compartmentalization and, consequently, improved our ability to screen for L-DOPA production. In a final round, we discovered that deletion of heme oxygenase (HMX1) increases total heme concentration and increases L-DOPA production, using dopamine measurement as a proxy. We further demonstrated that deleting HMX1 may represent a general strategy for P450 function improvement by improving activity of a second P450 enzyme, BM3, which performs a distinct reaction.

     
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  5. Abstract

    We describe herein formal syntheses of the indole alkaloidscis‐trikentrin A and herbindole B from a commonmeso‐hydroquinone intermediate prepared by a ruthenium‐catalyzed [2+2+1+1] cycloaddition that has not been used previously in natural product synthesis. Key steps include a sterically demanding Buchwald–Hartwig amination as well as a unique C(sp3)−H amination/indole formation. Studies toward a selective desymmetrization of themeso‐hydroquinone are also reported.

     
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  6. Abstract

    We describe herein formal syntheses of the indole alkaloidscis‐trikentrin A and herbindole B from a commonmeso‐hydroquinone intermediate prepared by a ruthenium‐catalyzed [2+2+1+1] cycloaddition that has not been used previously in natural product synthesis. Key steps include a sterically demanding Buchwald–Hartwig amination as well as a unique C(sp3)−H amination/indole formation. Studies toward a selective desymmetrization of themeso‐hydroquinone are also reported.

     
    more » « less