| Summary: | Algae are attractive organisms for biotechnology applications such as the production of biofuels, medicines, and other high-value compounds due to their genetic diversity, varied physical characteristics, and metabolic processes. As new species are being domesticated, rapid nuclear and organelle genome engineering methods need to be developed or optimized. To that end, we have previously demonstrated that the mitochondrial genome of microalgae <i>Phaeodactylum tricornutum</i> can be cloned and engineered in <i>Saccharomyces cerevisiae</i> and <i>Escherichia coli</i>. Here, we show that the same approach can be used to clone mitochondrial genomes of another microalga, <i>Thalassiosira pseudonana.</i> We have demonstrated that these genomes can be cloned in <i>S. cerevisiae</i> as easily as those of <i>P. tricornutum</i>, but they are less stable when propagated in <i>E. coli</i>. Specifically, after approximately 60 generations of propagation in <i>E. coli</i>, 17% of cloned <i>T. pseudonana</i> mitochondrial genomes contained deletions compared to 0% of previously cloned <i>P. tricornutum</i> mitochondrial genomes. This genome instability is potentially due to the lower G+C DNA content of <i>T. pseudonana</i> (30%) compared to <i>P. tricornutum</i> (35%). Consequently, the previously established method can be applied to clone <i>T. pseudonana</i>’s mitochondrial genome, however, more frequent analyses of genome integrity will be required following propagation in <i>E. coli</i> prior to use in downstream applications.
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