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No two clones are alike: characterization of heterologous subpopulations in a transgenic cell line of the model diatom Phaeodactylum tricornutum
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No two clones are alike: characterization of heterologous subpopulations in a transgenic cell line of the model diatom Phaeodactylum tricornutum
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No two clones are alike: characterization of heterologous subpopulations in a transgenic cell line of the model diatom Phaeodactylum tricornutum
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Journal Article
No two clones are alike: characterization of heterologous subpopulations in a transgenic cell line of the model diatom Phaeodactylum tricornutum
2024
Overview
Background
Conjugation-based episome delivery is a highly efficient method used to transfer DNA into the diatom
Phaeodactylum tricornutum
, facilitating the production of recombinant proteins and high-value metabolites. However, previous reports have indicated phenotypic heterogeneity among individual cells from clonally propagated exconjugant cell lines, potentially affecting the stability of recombinant protein production in the diatom.
Results
Here, we characterized the differences between subpopulations with distinct fluorescence intensity phenotypes derived from a single exconjugant colony of
P. tricornutum
expressing the enhanced green fluorescent protein (
eGFP
). We analyzed the expression cassette sequence integrity, plasmid copy number, and global gene expression. Our findings reveal that lower copy numbers and the deletion of the expression cassette in part of the population contributed to low transgene expression. Gene co-expression analysis identified a set of genes with similar expression pattern to
eGFP
including a gene encoding a putative Flp recombinase, which may be related to variations in fluorescence intensity. These genes thus present themselves as potential candidates for increasing recombinant proteins production in
P. tricornutum
episomal expression system.
Conclusions
Overall, our study elucidates genetic and transcriptomic differences between distinct subpopulations in a clonally propagated culture, contributes to a better understanding of heterogeneity in diatom expression systems for synthetic biology applications.
Publisher
BioMed Central,BioMed Central Ltd,BMC
Subject
/ Chemistry and Materials Science
/ Cloning
/ Control
/ Diatom
/ Diatoms
/ Genes
/ Green Fluorescent Proteins - genetics
/ Green Fluorescent Proteins - metabolism
/ Identification and classification
/ Methods
/ Microbial Genetics and Genomics
/ Recombinant Proteins - biosynthesis
/ Recombinant Proteins - genetics
