Showing papers by "Michael Krogh Jensen published in 2015"
TL;DR: The development and successful application of a multiplex CRISPR/Cas9 system for genome engineering of up to 5 different genomic loci in one transformation step in baker's yeast Saccharomyces cerevisiae is reported.
365 citations
TL;DR: Recent progress in biosensor development and their applications in a metabolic engineering context are described and examples of how biosensors can be integrated with synthetic circuits to exert feedback regulation on the metabolism for improved performance of cell factories are highlighted.
157 citations
TL;DR: This work presents a method for marker-free multiloci integration of in vivo assembled DNA parts using CRISPR/Cas9-mediated one-step double-strand breaks at single, double and triple integration sites and reports the successful in vivo assembly and chromosomal integration of DNA parts.
Abstract: Homologous recombination (HR) in Saccharomyces cerevisiae has been harnessed for both plasmid construction and chromosomal integration of foreign DNA. Still, native HR machinery is not efficient enough for complex and marker-free genome engineering required for modern metabolic engineering. Here, we present a method for marker-free multiloci integration of in vivo assembled DNA parts. By the use of CRISPR/Cas9-mediated one-step double-strand breaks at single, double and triple integration sites we report the successful in vivo assembly and chromosomal integration of DNA parts. We call our method CasEMBLR and validate its applicability for genome engineering and cell factory development in two ways: (i) introduction of the carotenoid pathway from 15 DNA parts into three targeted loci, and (ii) creation of a tyrosine production strain using ten parts into two loci, simultaneously knocking out two genes. This method complements and improves the current set of tools available for genome engineering in S. cere...
145 citations
TL;DR: A novel method, CrEdit (CRISPR/Cas9 mediated genome Editing), which utilizes targeted double strand breaks caused by CRISPR or Cas9 to significantly increase the efficiency of homologous integration in order to edit and manipulate genomic DNA.
Abstract: Background
One of the bottlenecks in production of biochemicals and pharmaceuticals in Saccharomyces cerevisiae is stable and homogeneous expression of pathway genes. Integration of genes into the genome of the production organism is often a preferred option when compared to expression from episomal vectors. Existing approaches for achieving stable simultaneous genome integrations of multiple DNA fragments often result in relatively low integration efficiencies and furthermore rely on the use of selection markers.
131 citations
TL;DR: The genome size estimate and transcriptome analyses will contribute to future research on this fascinating, monotypic species and its heterotrophic adaptations.
Abstract: The insectivorous Venus flytrap (Dionaea muscipula) is renowned from Darwin’s studies of plant carnivory and the origins of species. To provide tools to analyze the evolution and functional genomics of D. muscipula, we sequenced a normalized cDNA library synthesized from mRNA isolated from D. muscipula flowers and traps. Using the Oases transcriptome assembler 79,165,657 quality trimmed reads were assembled into 80,806 cDNA contigs, with an average length of 679 bp and an N50 length of 1,051 bp. A total of 17,047 unique proteins were identified, and assigned to Gene Ontology (GO) and classified into functional categories. A total of 15,547 full-length cDNA sequences were identified, from which open reading frames were detected in 10,941. Comparative GO analyses revealed that D. muscipula is highly represented in molecular functions related to catalytic, antioxidant, and electron carrier activities. Also, using a single copy sequence PCR-based method, we estimated that the genome size of D. muscipula is approx. 3 Gb. Our genome size estimate and transcriptome analyses will contribute to future research on this fascinating, monotypic species and its heterotrophic adaptations.
16 citations