142 research outputs found
Development of the Model Marine Diatom Phaeodactylum tricornutum for Synthetic Biology Applications
Harnessing organisms for protein and chemical production is useful to the scientific community and has applications in the fuel, food, and pharmaceutical industries. Biological systems commonly used for industrial chemical production include yeast and bacteria due to their fast growth rates and potential for high product yields. However, biologically active proteins, such as for human therapeutics, usually require production in mammalian and insect systems that are prohibitively expensive to grow at scale. Recently, photoautotrophic microalgae have emerged as promising platforms, as some species can be grown quickly and inexpensively at large scales and have the potential to produce biologically active proteins that mimic those produced in mammalian systems. Thus, they combine advantages from several traditional biological systems, but require further genetic and biotechnological development for their full potential to be unlocked.
Ideal biological production systems generally possess a variety of genetic tools to enable foreign gene expression, and genome editing systems to create desired genetic modifications. Here I present a robust gene editing system, novel genetic tools, and valuable strains for the microalga Phaeodactylum tricornutum. First, I identify novel endogenous regulatory elements that can drive expression of foreign genes in P. tricornutum, and design a plasmid-based Cas9 gene editing system for this species. Next, I demonstrate the utility of these tools with the generation of auxotrophic P. tricornutum strains through the genetic knockout of key enzymes in the uracil and histidine biosynthesis pathways. I complement the phenotype of the auxotrophs by introducing intact versions of these genes on replicating plasmids and demonstrate that these genes function as selective markers for transformation of their respective auxotrophic strain. Finally, I highlight the potential of these tools by creating a P. tricornutum expression system for the production of SARS-CoV-2 antigens. This will potentially address the need for a cheap, scalable source of serologically-active antigens for population-wide serological testing to combat the SARS-CoV-2 pandemic, and this system can be rapidly adapted to tackle future pandemics. I hope that these novel tools and strains will broaden the potential applications of P. tricornutum for industrial production of high-value products, and further the study of diatom biology
Telomere-to-telomere genome assembly of Phaeodactylum tricornutum
Phaeodactylum tricornutum is a marine diatom with a growing genetic toolbox available and is being used in many synthetic biology applications. While most of the genome has been assembled, the currently available genome assembly is not a completed telomere-to-telomere assembly. Here, we used Oxford Nanopore long reads to build a telomere-to-telomere genome for Phaeodactylum tricornutum. We developed a graph-based approach to extract all unique telomeres, and used this information to manually correct assembly errors. In total, we found 25 nuclear chromosomes that comprise all previously assembled fragments, in addition to the chloroplast and mitochondrial genomes. We found that chromosome 19 has filtered long-read coverage and a quality estimate that suggests significantly less haplotype sequence variation than the other chromosomes. This work improves upon the previous genome assembly and provides new opportunities for genetic engineering of this species, including creating designer synthetic chromosomes
An Expanded Plasmid-Based Genetic Toolbox Enables Cas9 Genome Editing and Stable Maintenance of Synthetic Pathways in <i>Phaeodactylum tricornutum</i>
With
the completion of the genome sequence, and development of
an efficient conjugation-based transformation system allowing the
introduction of stable episomes, <i>Phaeodactylum tricornutum</i> has become an ideal platform for the study of diatom biology and
synthetic biology applications. The development of plasmid-based genetic
tools is the next step to improve manipulation of this species. Here,
we report the identification of endogenous <i>P. tricornutum</i> promoters and terminators allowing selective expression of antibiotic
resistance markers from stably replicating plasmids in <i>P. tricornutum</i>. Significantly, we developed a protocol for sequential conjugation
of plasmids from <i>Escherichia coli</i> to <i>P. tricornutum</i> and demonstrated simultaneous replication of two plasmids in <i>P. tricornutum</i>. We developed a simple and robust conjugative
system for Cas9 editing that yielded up to 60% editing efficiency
of the urease gene. Finally, we constructed a plasmid encoding eight
genes involved in vanillin biosynthesis that was propagated in <i>P. tricornutum</i> over four months with no evidence of
rearrangements, with whole-plasmid sequencing indicating that the
majority of mutations occurred after plasmid assembly and initial
conjugation rather than during long-term propagation. The plasmid-based
tools described here will facilitate investigation of the basic biology
of <i>P. tricornutum</i> and enable synthetic biology
applications
Lysosomal Degradation of CD44 Mediates Ceramide Nanoliposome-induced Anoikis and Diminished Extravasation in Metastatic Carcinoma Cells
Intratympanic injection of dexamethasone 4âmg/mL versus 10âmg/mL for management of idiopathic sudden sensorineural hearing loss
Analysis of shared common genetic risk between amyotrophic lateral sclerosis and epilepsy
Because hyper-excitability has been shown to be a shared pathophysiological mechanism, we used the latest and largest genome-wide studies in amyotrophic lateral sclerosis (n = 36,052) and epilepsy (n = 38,349) to determine genetic overlap between these conditions. First, we showed no significant genetic correlation, also when binned on minor allele frequency. Second, we confirmed the absence of polygenic overlap using genomic risk score analysis. Finally, we did not identify pleiotropic variants in meta-analyses of the 2 diseases. Our findings indicate that amyotrophic lateral sclerosis and epilepsy do not share common genetic risk, showing that hyper-excitability in both disorders has distinct origins
ApoE deficiency promotes colon inflammation and enhances the inflammatory potential of oxidized-LDL and TNF-α in primary colon epithelial cells
Potentially Functional SNPs (pfSNPs) as Novel Genomic Predictors of 5-FU Response in Metastatic Colorectal Cancer Patients
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