Difference between revisions of "CH391L/S14/Genome Synthesis"

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There are other notable genomes which are not derived from entirely from chemically synthesized DNA, but are highly modified organisms.   
 
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 <cite>Lajoie2013</cite>.  The amber stop codon was removed at all 321 natural sites, and the release factor 1 (''prf1'')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.   
+
*'''Amberless ''E.'' ''coli''''' - In 2013, Farren Isaacs group created and published the first organism with a codon removed from all ORFs in the genome <cite>Lajoie2013</cite>.  The [http://en.wikipedia.org/wiki/Stop_codon amber stop codon] was removed at all 321 natural sites in ''E coli'', and the release factor 1 (''prf1'')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 [http://en.wikipedia.org/wiki/Expanded_genetic_code unnatural amino acid incorporation].   
  
 
*'''Reduced Genome ''E.'' ''coli''''' - In 2003, a reduced ''E.'' ''coli'' genome was published which had been reduced by 8.1% in total genome size, and 9.3% in total gene number <cite>Kolisnychenko2002</cite>.  This heavily modified E. ''coli'' is designed for increased genome stability, with 24 of the 44 transposable elements of the ''E.'' ''coli'' genome removed.
 
*'''Reduced Genome ''E.'' ''coli''''' - In 2003, a reduced ''E.'' ''coli'' genome was published which had been reduced by 8.1% in total genome size, and 9.3% in total gene number <cite>Kolisnychenko2002</cite>.  This heavily modified E. ''coli'' is designed for increased genome stability, with 24 of the 44 transposable elements of the ''E.'' ''coli'' genome removed.

Latest revision as of 17:31, 10 February 2014

Contents

Synthetic Genomes

A synthetic genome is 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 for existing multicellular organisms. Current efforts are also focused on establishing a minimal genome, or a genome composed of only essential genes with all optional machinery removed. Minimal genomes are expected to help in understanding the origins, the mechanisms, and the requirements of life.

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. genatalium 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.

Assembling 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 assemble smaller fragments into the 10's kb range, and after that point, fragments are assembled using Chew Back and Anneal assembly, or Gibson assembly, and 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 genomes, 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.

There have been a few techniques that have developed specificity for this purpose, several pertain to the transformation of an entire genome into a different species. The first method developed was the modification of the recipient species, reducing restriction enzyme activity to prevent donor genome degradation, and the second is methylation of the donor genome by treating with crude cell lysate from the acceptor species [1].

Early Work

In 2002, it was reported in Science magazine that the polio virus genome could be synthesized from oligonucleotides and assembled [2]. After assembly, the genome could be transcribed and active virus formed. This demonstrated that a virus could be brought forth without an original viral source, but from a strand of synthesized DNA. It received significant press coverage and is still used as an example of the dangers of biotechnology. The paper demonstrated that an active genome could be synthesized without a natural template, and was used to showcase advances in science and technology, and at the same time demonstrating that there are potential dangers to advancing science, and was an example of dual use research.

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 [3]. 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 synthetic genome work. 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 [4]. 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.
Assembly of M. genatalium genome from cassettes to final assembly in yeast.
The initial starting material was 10,000 individual 50 bp fragments of DNA eventually assembled to create the full genome of M. genitalium. The procedure to assemble the entire genome was:
  1. The JCVI outsourced assembly of 50 bp oligonucleotides into 5-7 kb cassettes.
  2. The cassettes were assembled into ~24 kb fragments using in vitro recombination in a manner similar to gibson cloning. Cassettes were assembled as groups of four.
  3. The 24 kb assembled fragments were cloned into BACs. A total of twenty five 24 kb fragments were required to cover the genome.
  4. These large fragments were then further assembled by combining three 24 kb fragments, resulting in 8 fragments of 72 kb.
  5. This was repeated once more, with two fragments at a time, resulting in four quarters of the full genome in indiviual BACs, each containing approximately 144 kb of then ~580 kb genome.
  6. The assembly into halves and the full genome was done in vivo using 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 using natural recombination pathways.

The result of this was a M. genatalium genome complete, with a BAC/YAC 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 [5]. In this second iteration,
Figure from [5] demonstrating a speedier genome assembly method in vivo using yeast for recombination
25 overlapping DNA fragments were transformed into S. cerevisiae and assembled in vivo. This reduced the number of steps to get from 50 bp fragments to full genome by about half, and replaced some of the more difficult cloning steps.

At this point, full genomes had been synthesized. These genomes were carried in a host organism, S. cerevisiae, but were not contributing to the fitness of the cell.

Synthia: A Bacterial Cell Controlled By A Chemically Synthesized Genome

A visual representation of the genome assembly of M. mycoides, from The 2010 JCVI Science, paper[6].
In 2010, the JVCI team reported in Science the first synthetic genome used to control a cell. The work this time was done with the genome from M. mycoides. While larger than M. genitalium, M. mycoides grows faster, significantly reducing experiment length. After the genome was synthesized and transferred to the acceptor cell, the cell began to behave like M. mycoides, it was viable, and was capable of reproduction. This was hailed as the first synthetic cell by the JVCI and was widely reported by media outlets. 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.
  1. The JCVI began with 1078 fragments of synthesized DNA, each ~1000 bases long. These micro-fragments were assembled into 10,000 bp segments, and cloned into YACs.
  2. Fragments were further assembled into eleven 100,000 bp fragments using in vivo recombination in S. cerevisiae.
  3. S. cerevisiae was also used to assemble the final genome, with a total size of 1.08 Mb.
  4. Genome was purified from yeast, and treated with M. capricolum crude lysate to methylate the genome.
  5. Genome was transformed into M. capricolum cell which had it's genome removed.

This streamlined procedure essentially brought genome synthesis down to four steps, followed by purification and methylation. Transforming the genome into the acceptor was accomplished using previous methods developed by the JCVI [1].

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 [7]. The amber stop codon was removed at all 321 natural sites in E coli, and the release factor 1 (prf1)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 acid incorporation.
  • Reduced Genome E. coli - In 2003, a reduced E. coli genome was published which had been reduced by 8.1% in total genome size, and 9.3% in total gene number [8]. This heavily modified E. coli is designed for increased genome stability, with 24 of the 44 transposable elements of the E. coli genome removed.

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." This minimal genome organism has been nicknamed Mycoplasma laboritorium. If successful, the Mycoplasma laboritorium minimal genome would be transformed into an acceptor cell (e.g. M. capricolum) in the same manner as Synthia's genome was transformed.

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
A figure showing the 2009 University of Alberta iGEM thoughts on recombination of an E. coli genome, replacing a wild-type region containing both essential and non-essential fragments of DNA, with a minimal genome analog, stringing all green fragments together while the non-essential red fragments have been removed.
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.

References

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Error fetching PMID 12114528:
  1. Error fetching PMID 19696314: [Lartigue2009]
    Methods for preparing the receptor species for genome transformation
  2. Error fetching PMID 12114528: [Cello2002]
    Synthesis of the polio genome, resulting in active virus capable of infection
  3. Error fetching PMID 16236728: [Itaya2005]
    First example of cloning an entire genome, minus some ribosomal RNA
  4. Error fetching PMID 18218864: [Gibson2008a]
    First published chemically synthesized genome
  5. Error fetching PMID 19073939: [Gibson2008b]
    Second published chemically synthesized genome
  6. Error fetching PMID 20488990: [Gibson2010]
    First cell under control of a chemically synthesized genome
  7. Error fetching PMID 24136966: [Lajoie2013]
    E. coli with all amber stop codons changed to ochre stop codons
  8. Error fetching PMID 11932248: [Kolisnychenko2002]
    A reduced genome E. coli
All Medline abstracts: PubMed | HubMed