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Cloning and transfer of long-stranded DNA in the size of a bacterial whole genome has become possible by recent advancements in synthetic biology. For the whole genome cloning and whole genome transplantation, bacteria with small genomes have been mainly used, such as mycoplasmas and related species. The key benefits of whole genome cloning include the effective maintenance and preservation of an organism's complete genome within a yeast host, the capability to modify these genome sequences through yeast-based genetic engineering systems, and the subsequent use of these cloned genomes for further experiments. This approach provides a versatile platform for in-depth genomic studies and applications in synthetic biology. Here, we cloned an entire genome of an insect-associated bacterium, Spiroplasma chrysopicola, in yeast. The 1.12 Mbp whole genome was successfully cloned in yeast, and sequences of several clones were confirmed by Illumina sequencing. The cloning efficiency was high, and the clones contained only a few mutations, averaging 1.2 nucleotides per clone with a mutation rate of 4 ×10-6. The cloned genomes could be distributed and used for further research. This study serves as an initial step in the synthetic biology approach to Spiroplasma. [ABSTRACT FROM AUTHOR]

Abstract: An improved bacmid technology for cloning complete genomes of large dsDNA viruses with circular genomes has been developed and tested. The system, termed EZ::BAC, is based on Escherichia coli F factor replicon, a chloramphenicol resistant marker gene with the mosaic ends recognized specifically by the transposase of the Tn5. In vitro transposition was carried out for the baculovirus shuttle vector pMON14272 (136kb) and the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) genome (134kb) as target DNAs. Transposon EZ::BAC was inserted randomly into the target DNAs, leading to 9bp duplication of the flanking end at the insertion site. One of the obtained AcMNPV::BACs replicated in Sf21 cells after transfection. The random in vitro generation of viral bacmids using EZ::BAC facilitates the host-independent propagation of intact and functional viral genomes in E. coli cells and does not require sequence information of the target DNA as is necessary for the generation of bacmids in conventional systems. [Copyright &y& Elsevier]

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. Unlabelled Image • Used transformation-associated recombination (TAR) method to clone mitochondrial genome of Phaeodactylum tricornutum in yeast • Developed PCR-based cloning method to create designer algal mitochondrial genomes • Stably propagated algal mitochondrial genomes in Saccharomyces cerevisiae and Escherichia coli hosts [ABSTRACT FROM AUTHOR]