Currently, only two selectable markers sh-ble and nat are predominantly used to select transgenic events in P. tricornutum was shown to be insensitive to a wide range of commonly used antibiotics and herbicides 31. tricornutum hinders the prospect of simultaneous or tandem delivery of multiple traits for gene stacking or engineering complex biosynthetic pathways. Limited availability of efficient and cost-effective selection schemes for nuclear transformation in P. These tools depend on an efficient selectable marker for the selection of transgenic events. Numerous genetic engineering tools, such as gene delivery 21, 22, 23, 24, genome editing 15, 25, 26, 27, 28, 29 and gene silencing 30, have been established to explore the genome information and empower the biotechnological exploitation of P. The production of triterpenoid compounds 14, reconstruction of vanillin biosynthesis 15, therapeutic peptide production 16, 17, 18, and trophic conversion 19, 20 have been achieved in this industrially relevant diatom. This diatom is an emerging synthetic biology chassis, enabling synthesis of a variety of compounds, and a proven host for recombinant protein production. The pennate oleaginous diatom Phaeodactylum tricornutum has been engineered for enhanced fatty acid biosynthesis 6, 7 and storage lipid accumulation 8, 9, 10 and improved LC-PUFA production 11, 12, 13. As many diatoms are oleaginous species that are able to accumulate high amounts of storage lipids, they have attracted significant attention as potent biofuel organisms. The capability of diatoms to use photosynthetic energy for rapid growth makes them excellent target organisms for microalgal biotechnology and genetic engineering 3, 4, 5. In marine environments, photosynthetic diatoms display rapid growth by assimilating available nutrients, resulting in blooms. Additionally, the ease of in vivo UV-mutagenesis may be employed as a strategy to create PDS-norflurazon-based selectable markers for other diatoms.ĭiatoms are an extremely morphologically diverse group of photosynthetic microalgae that inhabit nearly all aquatic environments and contribute approximately 20 to 40% of atmospheric O 2 1, 2. An optimized biolistic transformation system is reported which allowed the isolation of positive transgenic events at the rate of 96.7%. The plasmid carrying the mutated PDS1 as a selection marker and eGFP as a reporter was created. We report the characterization of PtPDS1 mutants and the development of the first endogenous selectable marker in diatoms suitable for industrial strain development, with the added benefit of biocontainment. Homology-based structural modeling of mutated PDS1, over a resolved crystallographic model of rice PDS1 complexed with norflurazon, suggests steric hindrance by bulkier residue substitution may confer herbicide resistance. These mutants revealed novel point mutations at a conserved residue Gly290 to Ser/Arg. Two mutants displaying high tolerance to norflurazon and carrying unique mutations in PtPDS1 (PHATRDRAFT_45735) were selected. tricornutum to obtain mutations in the phytoene desaturase (PDS) gene, conferring resistance to the bleaching herbicide norflurazon. An alternative cost-effective and preferably endogenous selectable marker would facilitate gene stacking efforts through successive transformation or conjugation. Till now, these tools have mainly relied on two selectable markers of bacterial origin which confer resistance to antibiotics Zeocin and nourseothricin. Share: export a plasmid map as an image, or export an annotated DNA sequence to GenBank format and freely share data with your colleagues or customers using the universally accessible SnapGene format.Phaeodactylum tricornutum is a well-developed model diatom for both marine ecology and microalgal biotechnology, which has been enabled by the sequenced genome and the availability of gene delivery tools, such as biolistic transformation and E. Annotate: automatically annotate common features, or manually annotate coding sequences and other features, design and annotate primers for PCR, sequencing, or mutagenesis, and identify open reading frames (ORFs) with a single mouse click. Search a DNA sequence to match either a DNA query, or a protein translation, or an annotation. Browse or print a DNA sequence and its annotations using customizable Map, Sequence, Enzymes, Features, Primers, and History views. Features include visualize: Create a DNA sequence file by either entering a sequence, or importing a record from GenBank, or opening an annotated sequence stored in one of many common file formats.
#Solid v dotted line in map snapgene viewer software#
SnapGene Viewer is software that allows molecular biologists to create, browse, and share richly annotated DNA sequence files up to 1 GB in length.
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