Cochrane, R. R. | bioRxiv Science

bioRxiv2023

Design and assembly of the 117-kb Phaeodactylum tricornutum chloroplast genome

There is a growing impetus to expand the repository of chassis available to synthetic biologists. The chloroplast genome presents a unique chassis for engineering photosynthetic eukaryotes by virtue of its compact size, lack of epigenetic regulation, and containment within the secluded lipid bilayers of the organelle. The development of the chloroplast as a synthetic biology chassis, however, has been limited by a lack of efficient techniques for whole genome cloning and engineering. Here, we demonstrate two approaches for cloning the 117 kb Phaeodactylum tricornutum chloroplast genome that have 90 to 100% efficiency when screening as few as ten Saccharomyces cerevisiae colonies following yeast assembly. The first method directly uses PCR-amplified fragments of the genome for yeast assembly, whereas the second method relies upon the pre-cloning of eight overlapping genomic regions into individual plasmids that they can later be released from. The cloned genome can be stably maintained and propagated within Escherichia coli, which provides an exciting opportunity for engineering a novel delivery mechanism for bringing DNA directly to the algal chloroplast. As well, one of the cloned genomes was designed to contain a single SapI site within the yeast URA3 open-reading frame, which can be used to linearize the genome and integrate designer cassettes via golden-gate cloning or further iterations of yeast assembly.

synthetic biology↗

bioRxiv2019

Rapid method for generating designer algal mitochondrial genomes

With synthetic biology, we can turn algae into bio-factories that produce high-value molecules (e.g. medicines or biofuels) or tackle global challenges (e.g. malnutrition and climate change). This realization has provoked rapid progress towards the creation of genetic tools for multiple algal species, notably Phaeodactylum tricornutum. The power of synthetic biology to generate more useful or productive organisms is contingent on the ability to produce diverse DNA molecules and rapidly screen them for beneficial variants. However, it is still relatively expensive to synthesize DNA, and delivering large DNA (>50 kbp) to eukaryotic cellular compartments remains challenging. In this study, we establish a robust system for building algal mitochondrial genomes as a practical alternative to DNA synthesis. Our approach permits an inexpensive and rapid generation of mitochondrial derivatives designed for testing targeted DNA delivery. First, we cloned the mitochondrial genome of P. tricornutum into the eukaryotic host organism S. cerevisiae using two different techniques: transformation-associated recombination; and PCR-based cloning. Next, we analyzed the cloned genomes by multiplex PCR, and correct genomes were transferred to prokaryotic host E. coli. Then these genomes were again analyzed by multiplex PCR, followed by diagnostic digest and complete-plasmid sequencing to evaluate the fidelity of each cloning method. Finally, we assessed the burden on eukaryotic and prokaryotic hosts to propagate the cloned genomes. We conclude that our system can reliably generate variants for genome-level engineering of algal mitochondria. HIGHLIGHTSO_LITAR-cloned mitochondrial genome of P. tricornutum in yeast C_LIO_LIDeveloped PCR-based cloning method to create designer algal mitochondrial genomes C_LIO_LIStably propagated algal mitochondrial genomes in S. cerevisiae and E. coli hosts C_LI

synthetic biology↗