CH391L/S14/Genome Synthesis
Synthetic Genomes A synthetic genome a chemically synthesized piece of DNA that contains all the information needed to maintain cellular life. Currently, few examples of synthetic genomes exist, and existing works have been proofs of concepts, and improvements on previously established laboratory techniques. The full utility of synthetic genomes is not yet known but the future of synthetic genome work could lead to synthesis of new and novel life forms, with unique properties, or to replacement parts to existing multicellular organisms. Current efforts are also focusing on establishing a minimal genome.
History The J. Craig Venter Institute (JCVI) has been a leader in genome synthesis. Daniel G. Gibson from JVCI has authored the first three fully synthetic genomes constructed and assembled from chemically synthesized DNA. The first was the Mycoplasma genitalium genome, published in Science in 2008. The second was also Mycoplasma genitalium in PNAS in 2008. More recently, the Mycoplasma mycoides genome was synthesized and used to create the first functioning, reproducing synthetic cell.
Technology The synthesis of an entire genome is limited by a number of factors. First and foremost is the ability to chemically synthesize fragments of DNA from individual nucleotides. The larger the starting blocks, the fewer assembly steps need to occur. JCVI used M. genetalium for early genome synthesis work due to it's small genome size. In total, about 10,000 individual fragments were assembled, each with an approximate length of 50 base-pairs. As DNA synthesis technology advances, entire genome synthesis will become simpler, cheaper, and less time consuming. Assembly of the individual fragments is the second major factor in genome synthesis, though this becomes less of a factor as the DNA synthesis lengths increase. Current genome synthesis examples use a combination of assembly techniques. PCR can be used to assembly smaller fragments into the 10's kb range, and after that point, fragments are propagated in host cells using either bacterial artificial chromosomes (BACs) or yeast artificial chromosomes (YACs). These fragments are purified, and further assembled and returned to BACs until the entire genome is assembled. At JCVI, assembly of genomic fragments has driven advances in cloning, specifically the invention of Gibson assembly. Furthermore, the products of future synthetic synthes, specifically synthetic cells, may lead to leaps in technology and innovation. The cells created through synthetic genomes can be designer cells, with the properties desired by specific researchers.
"""Early Work""" Itaya et al. published in PNAS in 2005 a conglomerate genome created by combining the genomes of Synechocystis PCC6803 and Bacillus subtilis into a single genome. The composite genome itself is not composed of synthetic DNA, but demonstrates that two nearly complete genomes can coexist in a single organism simultaneously, an essential technique for future attempts. This work established other information essential for following work. Of note was the fact that ribosome operons had to be deleted from the Synechoocystis genome for achieving cellular viability. It is hypothesized that Synechocystis rRNA may have translated genes from the Synechocystis genome which may have been detrimental to cell viability.
Gibson et al, from JCVI, published the first fully synthesized genome in Science in 2008. The work involved assembling 10,000 individual fragments of DNA into a complete copy of the genome of M. genitalium, with the addition of several watermarks to allow identification. The full length of the genome after assembly was 580 kilobase pairs (kb), significantly longer than the previously reported longest synthetic DNA construct of 32 kb. To get to the full genome, JCVI outsourced assembly of 50 bp oligonucleotides into 5-7 kb cassettes. The cassettes were then assembled into ~24 kb fragments using in vitro recombination in a manner similar to gibson cloning, of 4 adjacent cassettes. These large, assembled fragments were cloned into bacterial artificial chromosomes (BACs). A total of twenty five 24 kb fragments were required to cover the genome. These were then further assembled by combining three 24 kb fragments, resulting in 8 fragments of 72 kb. This was repeated once more, with two fragments at a time, resulting in four quarters of the full genome in indiviual BACs. The assembly into halves and the full genome was done in saccharomyces cerevisiae. S. cerevisiae was transformed with the four quarters of the genome, one of which was cleaved in half, and a YAC/BAC chromosome backbone. The yeast assembled the full genome. The result of this was a M. genetalium genome complete, with a BACYAC backbone included. This was sequenced, and accurate, full genomes were identified.
In late 2008, the JCVI published in PNAS another Mycoplasma genitalium genome assembled in yeast. In this second iteration, 25 overlapping DNA fragments were transformed into S. cerevisiae and assembled in vivo. This reduced the number of steps to get to a full genome by about half of the assembly methods, and particularly got rid of the more difficult cloning steps.
Synthia A Bacteria Controlled By A Chemically Synthesized Genome
In 2010, the JVCI team reported in Science the first synthetic genome used in a cell. The work this time was done with M. Mycoides. A similar scheme was followed to produce the genome as was done in the 2008 articles. Cassettes were assembled into larger fragments using Gibson assembly, and maintained as YACs. The genome fragments assembled were 10,000 bps. They were assembled into eleven 100,000 bp fragments, which in turn were assembled into a 1.08 Mbp genome. This was purified from yeast, and transformed into M. capricolum cell with its genome removed. The cell began to behave like M. Mycoides, was viable, and was capable of reproduction. This was hailed as the first synthetic cell by the JVCI.
"""Other Notable "Synthetic Genomes"""" There are other notable genomes which are not derived from entirely from chemically synthesized DNA, but are highly modified organisms.
- Amberless E. coli
In 2013, Farren Isaacs group created and published the first organism with a codon removed from all ORFs in the genome. The amber stop codon was removed at all 321 natural sites, and the release factor 1 gene was removed. These modifications served two purposes; first, the strain is far more resistant to some bacteriophage, including T7 phage, and second, the amber codon can now be used for new purposes, including introducing unnatural amino acids.
"""Future""" Daniel J. Gibson, a lead author on the JVCI synthetic genome papers, has stated that the main focus of synthetic genome work at JCVI is "synthesizing a minimal cell containing only the genes necessary to sustain life in its simplest form. This will help us better understand how cells work." The JCVI has termed this Mycoplasma Laboritorium. This Mycoplasma laboritorium would be the first synthetic organism to be brought to life, if successful.
"""iGEM""" There have been a variety of projects which have focused on minimal genomes and essential genome features. The 2009 Johns-Hopkins University iGEM team worked on a yeast minimal genome, focusing largely on the role of tRNAs and genome stability. The 2012 CBNU-Korea iGEM team worked towards creating software that will assist in designing minimal genomes. The 2009 University of Alberta team worked towards a minimal genome through step-by-step replacement of the original genome with a minimal genome equivalent of the same genome segment. For example, if a segment of the genome encoded genes A, B, C, and D, and it was though only genes A and C were essential, the genome segment ABCD would be replaced with AC.
