Design and assembly of the 117-kbPhaeodactylum tricornutumchloroplast genome

ABSTRACT 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 Sap I 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.