Using 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 organisms depends on the ability to produce diverse DNA molecules and rapidly screen them for beneficial variants in chosen hosts. 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 designer algal mitochondrial genomes ranging in sizes from approximately 60 to 95 kbp as a practical alternative to DNA synthesis. Our approach permits the 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 Saccharomyces cerevisiae using two different techniques: transformation-associated recombination; and PCR-based cloning. Next, we screened the cloned genomes by multiplex PCR, and transformed correct genomes into the prokaryotic host organism Escherichia coli. 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.

利用合成生物学，我们可以将藻类转变为生产高价值分子的生物工厂（例如，药物或生物燃料）或应对全球挑战（例如，营养不良和气候变化）。这种认识促使在为多种藻类，尤其是三角角线藻（Phaeodactylum tricornutum）的遗传工具的创建方面迅速发展。。合成生物学产生更多有用生物的能力取决于产生各种DNA分子并快速筛选出所选宿主中有益变体的能力。然而，合成DNA仍然相对昂贵，并且将大DNA（> 50kbp）递送至真核细胞区室仍然具有挑战性。在这项研究中，我们建立了一个健壮的系统来构建藻类线粒体设计者基因组，其大小范围从大约60到95 kbp，可以替代DNA合成。我们的方法允许设计用于测试目标DNA传递的线粒体衍生物的廉价，快速生成。首先，我们将三角假单胞菌的线粒体基因组克隆到了真核宿主酿酒酵母中使用两种不同的技术：与转化相关的重组；和基于PCR的克隆。接下来，我们通过多重PCR筛选了克隆的基因组，并将正确的基因组转化为原核宿主生物大肠杆菌。再次通过多重PCR分析这些基因组，然后进行诊断性消化和完整的质粒测序，以评估每种克隆方法的保真度。最后，我们评估了真核和原核宿主繁殖克隆基因组的负担。我们得出的结论是，我们的系统可以可靠地生成用于藻类线粒体基因组水平工程的变体。