Cloning a Chloroplast Genome in Saccharomyces cerevisiae and Escherichia coli.

Abstract

Chloroplast genomes present an alternative strategy for large-scale engineering of photosynthetic eukaryotes. Prior to our work, the chloroplast genomes of Chlamydomonas reinhardtii (204 kb) and Zea mays (140 kb) had been cloned using bacterial and yeast artificial chromosome (BAC/YAC) libraries, respectively. These methods lack design flexibility as they are reliant upon the random capture of genomic fragments during BAC/YAC library creation; additionally, both demonstrated a low efficiency (≤ 10%) for correct assembly of the genome in yeast. With this in mind, we sought to create a highly flexible and efficient approach for assembling the 117 kb chloroplast genome of Phaeodactylum tricornutum, a photosynthetic marine diatom. Our original article demonstrated a PCR-based approach for cloning the P. tricornutum chloroplast genome that had 90%-100% efficiency when screening as few as 10 yeast colonies following assembly. In this article, we will discuss this approach in greater depth as we believe this technique could be extrapolated to other species, particularly those with a similar chloroplast genome size and architecture. Key features • Large fragments of the chloroplast genome can be readily amplified through PCR from total algal DNA isolate. • Assembly protocol can be completed within a day, and yeast colonies harboring chloroplast genomes can be obtained in as few as 4-5 days. • Cloned genomes isolated from yeast transformants can be moved to Escherichia coli through electroporation.