Microbial Cell Factories for Biomanufacturing of Polysaccharides

Moradali, M. Fata; Rehm, Bernd H. A.

doi:10.1002/9783527818310.ch3

Cited by 5 publications

(2 citation statements)

References 193 publications

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“…Recent advances in other areas of synthetic biology and genome engineering have provided fundamental avenues to further our understanding of bacterial cellular biology, pathogenicity, and functional genomics 22 . These advances have enabled the use of bacteria as diagnostic and therapeutic agents targeting diverse diseases 23 , and as "biofactories" for industrial production of biofuels and beneficial small molecules 24 . Many of these applications require genomic insertion of customized DNA payloads (i.e.…”

Section: Introductionmentioning

confidence: 99%

Bacterial genome engineering using CRISPR RNA-guided transposases

Gelsinger

Klompe

et al. 2023

Preprint

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CRISPR-associated transposons (CASTs) have the potential to transform the technology landscape for kilobase-scale genome engineering, by virtue of their ability to integrate large genetic payloads with high accuracy, easy programmability, and no requirement for homologous recombination machinery. These transposons encode efficient, CRISPR RNA-guided transposases that execute genomic insertions in E. coli at efficiencies approaching ~100%, generate multiplexed edits when programmed with multiple guides, and function robustly in diverse Gram-negative bacterial species. Here we present a detailed protocol for engineering bacterial genomes using CAST systems, including guidelines on the available homologs and vectors, customization of guide RNAs and DNA payloads, selection of common delivery methods, and genotypic analysis of integration events. We further describe a computational crRNA design algorithm to avoid potential off-targets and CRISPR array cloning pipeline for DNA insertion multiplexing. Starting from available plasmid constructs, the isolation of clonal strains containing a novel genomic integration event-of-interest can be achieved in 1 week using standard molecular biology techniques.

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Section: Introductionmentioning

confidence: 99%

Bacterial genome engineering using CRISPR RNA-guided transposases

Gelsinger

Klompe

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…[47][48][49] The PHA display platform allows for simultaneous PHA particle formation, target protein/peptide production and immobilization in a one-pot manner. The platform has been used in research studies to create biomaterials for industrial biotechnology and environmental remediation applications, [50][51][52][53][54] but its application in creating antimicrobial biomaterials for water disinfection has not been explored.…”

Section: Introductionmentioning

confidence: 99%