Minimal genome

The minimal genome concept assumes that genomes can be reduced to a bare minimum, given that they contain many non-essential genes of limited or situational importance to the organism. Therefore, if a collection of all the essential genes were put together, a minimum genome could be created artificially in a stable environment. By adding more genes, the creation of an organism of desired properties is possible. The concept of minimal genome arose from the observations that many genes do not appear to be necessary for survival.[1][2] In order to create a new organism a scientist must determine the minimal set of genes required for metabolism and replication. This can be achieved by experimental and computational analysis of the biochemical pathways needed to carry out basic metabolism and reproduction.[3] A good model for a minimal genome is Mycoplasma genitalium due to its very small genome size. Most genes that are used by this organism are usually considered essential for survival; based on this concept a minimal set of 256 genes has been proposed.[4]

Genome reduction in nature

Bacteria

Many naturally occurring bacteria have reduced genomes even though they may not be reduced to the bare minimum. Although these genomes are thus not "minimal", they are good models for genome reduction and thus "minimal genomes". Genome reduction occurs most commonly in endosymbiotic, parasitic or pathogenic bacteria that live in their hosts. The host provides most of the nutrients such bacteria require, hence the bacteria do not need the genes for producing such compounds themselves. Examples include species of Buchnera, Chlamydia, Treponema, Mycoplasma, and many others. One of the most reduced genomes in free-living bacteria has been found in Pelagibacter ubique which encodes 1,354 proteins. Mycoplasma genitalium has been used as a prime model for minimal genomes. It is a human urogenital pathogen which has the smallest genome of size 580 kb and it consists of only 482 protein-coding genes.[5]

Viruses

Viruses have the smallest genomes in nature. For instance, bacteriophage MS2 consists of only 3569 nucleotides (single-stranded RNA) and encodes just four proteins.[6] Similarly, among eukaryotic viruses, porcine circoviruses are among the smallest.[7] They encode only 2–3 open reading frames.

Rise of the minimal genome and construction of synthetic mycoplasma

This concept arose as a result of a collaborative effort between National Aeronautics and Space Administration (NASA) and two scientists: Harold Morowitz and Mark Tourtellotte. In the 1960s, NASA was searching for extraterrestrial life forms, assuming that if they existed they may be simple creatures. While Morowitz, to attract people's attention published about mycoplasmas as being the smallest and simplest self-replicating creatures. The two grouped together and came up with an idea to assemble a living cell from the components of mycoplasmas. Since, mycoplasmas are built with a minimum set of organelles such as: a plasma membrane, ribosomes and a circular double stranded DNA; it was selected as the best candidate for cell reassembly. Morowitz' major idea was to define the entire machinery of mycoplasmas cell in molecular level. He announced that an international effort would help him accomplish this main objective.

The main plan consisted of:
  1. physical and functional mapping with complete sequencing of the mycoplasma
  2. Determine the open reading frames (ORFs)
  3. Determining the encoded amino acids
  4. Understanding the functions of genes
  5. Final step: reassemble mycoplasma's cellular machinery.

This entire process was hard work, meanwhile even when papers were being published on the construction of minimal genome; by the 1980s Richard Herrmann's laboratory had fully sequenced and genetically characterized the 800kb genome of M. pneumoniae. That small of a genome itself took close to three years of hard work. Later in the 1995s another laboratory located in Maryland the Institute for Genomic Research (TIGR) collaborated with the teams of Johns Hopkins and University of North Carolina. Their organism for genome sequencing was Mycoplasma genitalium consisting of only 580 kb genome, the sequencing of which was done in 6 months.

The sequencing data revealed many interesting facts about M. genitalium such as discovery of some conserved genes, which ultimately helped in defining essentiality to life, of a minimal self- replicating cell. By far M. genitalium has become the prime candidate for minimal genome project. In fact these organisms are closest to minimal genome capable of self-replicating.

Minimal set of essential genes are typically found by selective inactivation or deletions of genes and then testing the effect of each under a given set conditions. The discovery of essential genes have been done by the J. Craig Venter institute, they claim M. genitalium consists of 382 essential genes.

The next venture that the J.Craig Venter institute landed upon was creating a synthetic organism named Mycoplasma laboratorium, through minimal set genes of M. genitalium. This project opens new doors for synthetic biology because this impressive creation is being built upon by bringing together chemical synthesis and recombination cloning methodology.[5]

How to begin reconstructing

Reconstruction of a minimal genome is possible by using the knowledge of existing genomes via which the sets of genes, essential for living can also be determined. Once the set of essential genetic elements are known, one can proceed to define the key pathways and core-players by modeling simulations and wet lab genome engineering. The two organisms upon which the ‘minimal gene set for cellular life' was applied were: Haemophilus influenzae, and M. genitalium. A list of orthologous proteins were compiled in hope that it would contain protein necessary for cell survival, as orthologous analysis determines how two organisms evolved and shed away any non-essential genes. Since, H. influenza and M. genitalium are Gram negative and Gram positive bacteria and due to their vast evolution it was expected that these organisms would be enriched genes that were of universal importance. However, 244 detected orthologs discovered contained no parasitism-specific proteins. The conclusion of this analysis was that similar biochemical functions might be performed by non-orthologous proteins. Even when biochemical pathways of these two organisms were mapped, several pathways were present but many were incomplete. Proteins determined to be common between the two organisms were non-orthologous to each other. Much of the research mainly focuses on the ancestral genome and less on the minimal genome. Studies of these existing genomes have helped determine that orthologous genes found in these two species are not necessarily essential for survival, in fact non-orthologous genes were found to be more important. Also, it was determined that in order for proteins to share same functions they do not need to have same sequence or common three dimensional folds. Distinguishing between orthologs and paralogs and detecting displacements of orthologs have been quiet beneficial in reconstructing evolution and determining the minimal gene set required for a cellular life. Instead, of conducting a strict orthology study, comparing groups of orthologs and occurrence in most clades instead of every species helped encounter genes lost or displaced. Only genomes that have been completely sequenced have enabled in studying orthologs among group of organisms. Without a fully sequenced genome it would not be possible to determine the essential minimal gene set required for survival.[2]

Essential genes of M. genitalium

J. Craig Venter Institute (JCVI) conducted a study to find all the essential genes of M. genitalium through global transposon mutagenesis. As a result, they found that 382 out of 482 protein coding genes were essential. Genes encoding proteins of unknown function constitute 28% of the essential protein coding genes set. Before conducting this study the JCVI had performed another study on the non-essential genes, genes not required for growth, of M.genitalium, where they reported the use of transposon mutagenesis. Despite figuring out the non-essential genes, it is not confirmed that the products that these genes make have any important biological functions. It was only through gene essentiality studies of bacteria that JCVI have been able to compose a hypothetical minimal gene sets.

The study published in 1999 and 2005

In JCVI's 1999 study among the two organisms, M. genitalium and Mycoplasma pneumoniae they mapped around 2,200 transposon insertion sites and identified 130 putative non-essentials genes in M. genitalium protein coding genes or M. pneumoniae orthologs of M. genitalium genes. In their experiment they grew a set of Tn4001 transformed cells for many weeks and isolated the genomic DNA from these mixture of mutants. Amplicons were sequenced to detect the transposon insertion sites in mycoplasma genomes. Genes that contained the transposon insertions were hypothetical proteins or proteins considered non-essential.

Meanwhile, during this process some of the disruptive genes once considered non-essential, after more analyses turned out essential. The reason for this error could have been due to genes being tolerant to the transposon insertions and thus not being disrupted; cells may have contained two copies of the same gene; or gene product was supplied by more than one cell in those mixed pools of mutants. Insertion of transposon in a gene meant it was disturbed, hence non-essential, but because they did not confirm the absence of gene products they mistook all disruptive genes as non-essential genes.

The same study of 1999 was later expanded and the updated results were then published in 2005.

Some of the disruptive genes thought to be essential were isoleucyl and tyrosyl-tRNA synthetases (MG345 and MG455), DNA replication gene dnaA (MG469), and DNA polymerase III subunit a (MG261). The way they improved this study was by isolating and characterizing M. genitalium Tn4001 insertions in each colony one by one. The individual analyses of each colony showed more results and estimates of essential genes necessary for life. The key improvement they made in this study was isolating and characterizing individual transposon mutants. Previously, they isolated many colonies containing a mixture of mutants. The filter cloning approach helped in separating the mixtures of mutants.

Now, they claim completely different sets of non-essential genes. The 130 non-essential genes claimed at first have now reduced to 67. Of the remaining 63 genes 26 genes were only disrupted in M. pneumoniae which means that some M. genitalium orthologs of non-essential M. pneumoniae genes were actually essential.

They have now fully identified almost all of the non-essential genes in M. genitalium, the number of gene disruptions based on colonies analyzed reached a plateau as function and they claim a total of 100 non-essential genes out of the 482 protein coding genes in M. genitalium

The ultimate result of this project has now come down to constructing a synthetic organism, Mycoplasma laboratorium based on the 387 protein coding region and 43 structural RNA genes found in M. genitalium.[8]

Mycoplasma laboratorium
Main article: Mycoplasma laboratorium

This project is currently still going on and it might possibly become the very first life form created by humans. It is quite likely that this line of research may lead to creating a bacterium that could further be engineered to produce fuels, make medicines, take some action on global warming, and make antibiotics.

In May 2010 the JCVI successfully created a "synthetic life form", which will enable them to dissect a genetic instruction set of a bacterial cell and see how it really works.[9] The synthetic life form was constructed by replacing the DNA of an existing bacterium and replacing it with DNA that was artificially designed and constructed.

Minimal genome projects

A number of projects have attempted to identify the essential genes of a species. This number should approximate the "minimal genome". For instance, the genome of E. coli has been reduced by about 30%, demonstrating that this species can live with much fewer genes than the wild-type genome contains.[10]

The following table contains a list of such minimal genome projects (including the various techniques used).[11]

Year Organism Method
1996 H. influenzae, E. coli In silico comparison of genomes[12]
1998 H. influenzae, S. pneumoniae Tn mutagenesis and DNA fingerprinting[13]
1999 M. genitalium Saturating Tn mutagenesis[14]
2000 V. cholerae Tn mutagenesis and arabinose promoter[15]
2001 S. aureus Antisense RNA[16]
2001 M. bovis Tn mutagenesis and microarray[4]
2002 H. influenzae Tn mutagenesis and DNA fingerprinting[17]
2002 Buchnera spp Sequence comparison[18]
2002 S. cerevisiae Systematic gene deletion[19]
2002 S. aureus Antisense RNA[20]
2002 E. coli Red recombinase excision[21]
2002 E. coli Cre/loxP excision[22]

For more information please refer also to section 'Minimal genome project' at 'Mycoplasma laboratorium'.

Number of essential genes
Main article: Essential gene

The number of essential genes is different for each organism. In fact, each organism has a different number of essential genes, depending on which strain (or individual) is tested. In addition, the number depends on the conditions under which an organism is tested. In several bacteria (or other microbes such as yeast) all or most genes have been deleted individually to determine which genes are "essential" for survival. Such tests are usually carried out on rich media which contain all nutrients. However, if all nutrients are provided, genes required for the synthesis of nutrients are not "essential". When cells are grown on minimal media, many more genes are essential as they may be needed to synthesize such nutrients (e.g. vitamins). The numbers provided in the following table typically have been collected using rich media (but consult original references for details).

Organism Essential Genes
Escherichia coli 1617
Bacillus subtiis 271
Haemophilus influenzae 642
Streptococcus pneumoniae 244
Mycoplasma genitalium 381
Vibrio cholerae 779
Staphylococcus aureus 653
Saccharomyces cerevisiae 1110

The number of essential genes were collected from the Database of Essential Genes (DEG),[23] except for B. subtilis, where the data comes from Genome News Network[24][25] The organisms listed in this table have been systematically tested for essential genes. For more information about minimal genome Please refer also to section 'Other Genera' at 'Mycoplasma laboratorium'.

First self replicating synthetic cell

May 20, 2010 – Researchers at the JCVI have successfully created a synthetic bacterial cell that is capable of replicating itself. The team has synthesized a 1.08 million base pair chromosome of a modified Mycoplasma mycoides. The synthetic cell is called: Mycoplasma mycoides JCVI-syn1.0. One of the remarkable thing about this cell is that its DNA was built in the computer and transplanted into cell from which its own (origin) genome was removed. The original molecules and on-going reaction networks of the recipient cell then used the artificial DNA to generate daughter cells. These daughter cells are of synthetic origin and capable of further replication. This proves that genomes can be designed on computers. The steps they applied to build this was first they simulated a model of this genome computationally, they identified DNA via watermarks; next, they chemically produced this genome in the laboratory and finally, transplanted this genome into a recipient cell to produce a synthetic cell solely controlled by this synthetic genome.

The first half of the project has taken 15 years to complete. The team designed an accurate, digitized genome of M. mycoides. A total of 1,078 cassettes all 1,080 base pair long were built. These cassettes were designed in a way that the end of each DNA cassette overlapped by 80 base pairs. The whole assembled genome was transplanted in yeast cells and grown as yeast artificial chromosome. This synthetic cell will be now able to show scientists how truly a cell works.

Now that they have synthetic cells growing in their laboratory, the JCVI group can focus on their ultimate goal of synthesizing a minimal cell containing just the essential genes necessary for life.[26]

Future direction and uses

Future direction: Based on JCVI's progress in the field of synthetic biology, it is possible that in near future scientists will be able to propagate M. genitalium's genome in the form of naked DNA, into recipient mycoplasmas cells and replace their original genome with a synthetic genome. Since, mycoplasmas have no cell wall, the transfer of a naked DNA into their cell is possible. The only requirement now is the technique to include the synthetic genome of M. genitalium into mycoplasma cells. To some extent this has become possible, the first replicating synthetic cell has already been developed by the JCVI and they are now on to creating their first synthetic life, consisting of minimal number of essential genes. This new breakthrough in synthetic biology will certainly bring in a new approach to understand biology; and this redesigning and prototyping genomes will later become beneficial to biotechnology companies, enabling them to produce synthetic microbes that produce new, cheaper and better bio-products.[5]

Uses of minimal genome:

  1. Identification of essential genes
  2. Reduced genetic complexity that allows greater predictability of engineered strains.
  3. Engineer plants to resist herbicides or harsh environmental conditions.
  4. Synthetically produce pharmaceuticals
  5. Large scale benefits: clean energy
  6. Renewable chemicals
  7. Sequestering carbon from the atmosphere.
  8. Create beneficial microbes to make them produce bio-products.[27]

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