CH391L/S13/CleanGenomes

A clean or minimal genome refers to the minimum set of genes that an organism needs to survive and reproduce in a defined environment. This implies that there are genes that are “nonessential” to the organism’s survival and can be removed without destroying the cell or disrupting its growth cycle. Examples of nonessential DNA would include duplicate genes, transposable elements, regions coding for phenotypic plasticity and catabolic pathways used for the intake and breakdown of complex biomolecules.[1][2][3]

Advantages of a Minimal Genome

As the complexity of synthetic biology projects increases, extraneous parts inside each cell will complicate future endeavors. A minimal genome would give synthetic biologists a reliable and predictable “chassis” that could provide an ideal platform for perusing new research. Minimizing a genome would eliminate the unknown by only including parts with a known function; symplifying synthetic engineering.

Current model organisms used in modern research contain deleterious DNA and gene products (like insertion sequences and unforeseen molecular interactions) which could disable or prohibit future endeavors by interacting unfavorably with whatever the “subject of interest” is.[1][4] "Depending on the genetic and physiological context, [Insertion Sequence] contribution to gene inactivation ranges from 3.9% to 98%."[5]

Smaller amounts of extraneous gene products, like secondary metabolites, would simplify and reduce the cost of extraction and purification of cell parts, biomolecules and pharmaceuticals, many of which would be more expensive and time consuming to separate from a population of cells by conventional means.[6]

Gene stability in reduced genomes has been improved by removing transposable elements (TEs), error prone DNA polymerases and the enzymes responsible for the SOS response. Spontaneous, random genetic changes would be extremely harmful toward a minimal cell lacking a number of redundant systems. A stable genome would be a very desirable trait for research and experiment replication.[6][7]

Future advances in genome replication, unnatural amino acids, drug development, fuel production, biofilm formation[8] biomaterial synthesis and other cellular applications and processes[7] could be accelerated by simplifying and streamlining the genome used to code for a cell.[1][4]

Minimal/Clean Genomes vs. Wild Type Genomes

Cells with reduced genomes have less relative fitness than similar wild type cells and cannot easily cope with changes in its environment. For example, reduced cells are more sensitive to reactive oxygen species, even when the cells still retain the genes needed to respond towards oxidative stress. [7]

The mutation rate in cells with minimal genomes is significantly reduced compared to wild type genomes.[7] This would decrease the genetic diversity of the reduced genome cell population. It is conceivable that if a bacteriophage ever infected a reduced or minimal genome line, then it would quickly become a phage genome line.

Estimating the Number of Essential Genes

There are several different methods used for estimating the minimum number of essential genes an organism needs to survive in a controlled environment. Each method has its own shortcomings which limits their applications.

Comparative Genomics

Comparative genomics looks for genomic homology between different organisms and strains. Genetic homology over a wide number of similar organisms could be an indicator of essential genes since they were conserved throughout those strains or species.[1][6]

A comparative approach could underestimate the number of essential genes since in only accounts for true genetic orthologs. For example, this gene estimate would not account for genes with different morphologies that code for functionally similar gene products.[9][10] In some instances, it could also overestimate the number of essential genes since homologous genes do not have to be useful or essential. For example, virulence factors that are homologous in many pathogenic microbes are not essential genes. [6]

Gene Disruption using Transposable Mutagenesis

Targeted gene disruption using transposable mutagenesis involves attempting to inactivate genes using a large number of transposable elements, then sequencing the resulting genome. If a transposable element is unable to insert itself into a gene, then that gene could be more essential to the cell than other genes that are susceptible to disruption.[11] S

Some of the genes screened may read a false positive for essentiality since there is the chance that some transducable elements may not have been transduced into that gene, i.e. a nonessential gene may not have had a transposable element inserted into it. Also, one transposon may disable multiple gene products of varying essentiality (like in alternatively spliced genes). An essential gene could also function normally with a transposable element inside it, which could result in a false negative error.[1][9][12]

mRNA Disruption using Antisense RNA

Antisense RNA (asRNA) is a single strand of RNA that complementary to an mRNA inside a cell. When antisense RNA base pairs with mRNA, the mRNA is unable to be translated. Cells that can't survive in the presence of certain asRNAs indicate that the untranslated gene products bound by asRNA are essential to cellular survival.

Antisense RNA disruption can only work is there is an adequate amount of mRNA to disrupt. For example, intracellular signaling polypeptides may not be targeted by asRNA disruption since they don't need to be highly expressed.[9][13]

Genome Reduction

One of the most straight forward approaches to determining a minimal gene set is to reduce the number of genes in a cells genome until it can no longer survive. This method would be able to definitively determine the essentiality of trans and cis regions of DNA inside a chromosome.

Genome reduction would take a longer time to accomplish since determining a minimal gene set trough genome reduction involves, relatively, more trial and error than the other aforementioned mehtods.

A 2012 study by Iwadate et. al. have shown that cells E. coli can live with 38.9% of its original genome removed.[7]

Genome Synthesis

Whole genome assembly is the creation of an artificial genome, with the intent to install that genome into a pre-existing cell. The new genome is assembled in steps, starting with relatively short (~1000bp) sequences that are then assembled into larger and larger pieces. Synthesis of an artificial bacterial genome has been achieved twice: the first was the complete synthesis of the Mycoplasma genitalium genome[14], followed by the complete synthesis (and transplantation into another cell) of M. mycoides[15]. The next synethic genome will be M. laboratorium, which will feature a reduced genome, due to the targeted removal of non-essential genes[16].

Genome synthesis is prohibitive because of its high cost due the price of DNA synthesis. The cost of DNA synthesis is expected to decrease in the future.

Genome Reduction

One approach to creating more reliable, efficient host organisms for synthetic constructs is the reduction of the genome to eliminate extraneous genes, mutagenic mobile elements, and other unnecessary or destabilizing factors. This can be viewed as a form of reverse engineering of extant strains.

Systematic Genome Reduction

One natural approach to engineering strains with a reduced genome is to systematically identify and delete regions of the genome not necessary for host cell survival. Posfai et al. created the MDS strains (multiple deletion strains) by aligning the genomes of multiple genomes of E. coli, identifying regions which were absent in multiple strains, and deleting them via Lambda Red recombination. All IS elements were removed as well, lowering the mutation rate and increasing the stability of genetic constructs introduced into the cell. The strain had comparable growth rate compared to wild type.[6][2]

Ara et al. constructed a minimal version of the B. subtilis genome in 2007. [17] This strain had slightly decreased growth rate compared to wild-type, but displayed normal morphology and similar protein production capabilities.

Iwadate et al. in 2011 improved on the "CRS cassette method"[12] to remove up to 38.9% of E. coli's chromosome.[7]

Explanation of Methods used in Genome Reduction

One method used in 2002 by Kolisnychenko et al. involves the use of site-specific recombination to remove selected DNA from a genome. This method was used to delete DNA fragments anywhere from 7 to 82 kb in length from E. coli MG1655.[6]

Method used by Kolisnychenko in 2002. Image by: Kolisnychenko et al., edited by: Evan J. Weaver for clarity. More information regarding this picture can be found clicking on the picture.

The method used by Ara et al. in 2007 to create a reduced B. subtilis genome was similar to the method used by Kolisnychenko.

Selection for Reduced Genome

Long-term evolution of strains under the correct conditions could select for a genome of minimal size. Such conditions may include growth in rich media lacking sugars to favor the loss of biosynthetic pathways or sugar metabolism operons, growth in structured environments which favor a smaller cell, or growth under other conditions which favor the loss of unnecessary genes.

Mycoplasma mycoides genome synthesis strategy

A study by Lee et al. removed about 10% of M. extorquens AM1's genome through evolutionary selection by growing it in a controlled environment. The cells were grown on plates containing mixtures of methanol and succinate for 1500 generations. Most of the DNA lost contained accessory genes.[18]

Minimal Genome Synthesis

For more details, see: DNA Assembly

Another approach is the synthesis of a minimal, designed genome from scratch using DNA synthesis technology and the transformation of this genome into cells to create a viable, novel, synthetic organism. This approach can be viewed as forward engineering of a novel organism, but would likely be informed by studies which determine the minimal set of genes necessary for a living organism.

Mycoplasma mycoides Synthesis

Gibson et al synthesized the first artificial cell by generating the Mycoplasma mycoides genome from digitized genome information and transforming it into Mycoplasma capricolum cells devoid of genomic information. These cells were capable of continuous self-replication and were identified by "watermarks" inserted in the genome. This technology could be utilized in the future to create cells with novel and useful properties from scratch.[14][15]

Image of M. genitalium that contain synthetic genomes.

iGEM Connection

An iGEM team from Korea created a program to analyze genomes to help find a minimal genome. Their definition of a minimal genome states that a minimal genome is a set of genes that every living organism contains; a comparative genomics approach. They plan on using this software to design their own minimalistic genome.

References

1. Forster AC and Church GM. Towards synthesis of a minimal cell. Mol Syst Biol. 2006;2:45. DOI:10.1038/msb4100090 |
2. Pósfai G, Plunkett G 3rd, Fehér T, Frisch D, Keil GM, Umenhoffer K, Kolisnychenko V, Stahl B, Sharma SS, de Arruda M, Burland V, Harcum SW, and Blattner FR. Emergent properties of reduced-genome Escherichia coli. Science. 2006 May 19;312(5776):1044-6. DOI:10.1126/science.1126439 |
3. Hutchison CA, Peterson SN, Gill SR, Cline RT, White O, Fraser CM, Smith HO, and Venter JC. Global transposon mutagenesis and a minimal Mycoplasma genome. Science. 1999 Dec 10;286(5447):2165-9. DOI:10.1126/science.286.5447.2165 |
4. Jewett MC and Forster AC. Update on designing and building minimal cells. Curr Opin Biotechnol. 2010 Oct;21(5):697-703. DOI:10.1016/j.copbio.2010.06.008 |

5. K. Umenhoffer, T. Fehér, G. Balikó, F. Ayaydin, J. Pósfai, F. R Blattner, and G. Pósfai. Reduced evolvability of Escherichia coli MDS42, an IS-less cellular chassis for molecular and synthetic biology applications. Microb Cell Fact. 2010; 9: 38

6. Kolisnychenko V, Plunkett G 3rd, Herring CD, Fehér T, Pósfai J, Blattner FR, and Pósfai G. Engineering a reduced Escherichia coli genome. Genome Res. 2002 Apr;12(4):640-7. DOI:10.1101/gr.217202 |
7. Iwadate Y, Honda H, Sato H, Hashimoto M, and Kato J. Oxidative stress sensitivity of engineered Escherichia coli cells with a reduced genome. FEMS Microbiol Lett. 2011 Sep;322(1):25-33. DOI:10.1111/j.1574-6968.2011.02331.x |
8. May T and Okabe S. Enterobactin is required for biofilm development in reduced-genome Escherichia coli. Environ Microbiol. 2011 Dec;13(12):3149-62. DOI:10.1111/j.1462-2920.2011.02607.x |
9. Kobayashi K, Ehrlich SD, Albertini A, Amati G, Andersen KK, Arnaud M, Asai K, Ashikaga S, Aymerich S, Bessieres P, Boland F, Brignell SC, Bron S, Bunai K, Chapuis J, Christiansen LC, Danchin A, Débarbouille M, Dervyn E, Deuerling E, Devine K, Devine SK, Dreesen O, Errington J, Fillinger S, Foster SJ, Fujita Y, Galizzi A, Gardan R, Eschevins C, Fukushima T, Haga K, Harwood CR, Hecker M, Hosoya D, Hullo MF, Kakeshita H, Karamata D, Kasahara Y, Kawamura F, Koga K, Koski P, Kuwana R, Imamura D, Ishimaru M, Ishikawa S, Ishio I, Le Coq D, Masson A, Mauël C, Meima R, Mellado RP, Moir A, Moriya S, Nagakawa E, Nanamiya H, Nakai S, Nygaard P, Ogura M, Ohanan T, O'Reilly M, O'Rourke M, Pragai Z, Pooley HM, Rapoport G, Rawlins JP, Rivas LA, Rivolta C, Sadaie A, Sadaie Y, Sarvas M, Sato T, Saxild HH, Scanlan E, Schumann W, Seegers JF, Sekiguchi J, Sekowska A, Séror SJ, Simon M, Stragier P, Studer R, Takamatsu H, Tanaka T, Takeuchi M, Thomaides HB, Vagner V, van Dijl JM, Watabe K, Wipat A, Yamamoto H, Yamamoto M, Yamamoto Y, Yamane K, Yata K, Yoshida K, Yoshikawa H, Zuber U, and Ogasawara N. Essential Bacillus subtilis genes. Proc Natl Acad Sci U S A. 2003 Apr 15;100(8):4678-83. DOI:10.1073/pnas.0730515100 |
10. Lagesen K, Ussery DW, and Wassenaar TM. Genome update: the 1000th genome--a cautionary tale. Microbiology (Reading). 2010 Mar;156(Pt 3):603-608. DOI:10.1099/mic.0.038257-0 |

11. C. M. Trepod,J. E. Mott. Elucidation of Essential and Nonessential Genes in the Haemophilus influenzae Rd Cell Wall Biosynthetic Pathway by Targeted Gene Disruption Antimicrob Agents Chemother. 2005 February; 49(2): 824–826

12. J. Kato, M. Hashimoto. Construction of consecutive deletions of the Escherichia coli chromosome Molecular Systems Biology 3; Article number 132

13. Ji Y, Zhang B, Van SF, Horn, Warren P, Woodnutt G, Burnham MK, and Rosenberg M. Identification of critical staphylococcal genes using conditional phenotypes generated by antisense RNA. Science. 2001 Sep 21;293(5538):2266-9. DOI:10.1126/science.1063566 |
14. Gibson DG, Benders GA, Andrews-Pfannkoch C, Denisova EA, Baden-Tillson H, Zaveri J, Stockwell TB, Brownley A, Thomas DW, Algire MA, Merryman C, Young L, Noskov VN, Glass JI, Venter JC, Hutchison CA 3rd, and Smith HO. Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science. 2008 Feb 29;319(5867):1215-20. DOI:10.1126/science.1151721 |
15. Gibson DG, Glass JI, Lartigue C, Noskov VN, Chuang RY, Algire MA, Benders GA, Montague MG, Ma L, Moodie MM, Merryman C, Vashee S, Krishnakumar R, Assad-Garcia N, Andrews-Pfannkoch C, Denisova EA, Young L, Qi ZQ, Segall-Shapiro TH, Calvey CH, Parmar PP, Hutchison CA 3rd, Smith HO, and Venter JC. Creation of a bacterial cell controlled by a chemically synthesized genome. Science. 2010 Jul 2;329(5987):52-6. DOI:10.1126/science.1190719 |
16. Glass JI, Assad-Garcia N, Alperovich N, Yooseph S, Lewis MR, Maruf M, Hutchison CA 3rd, Smith HO, and Venter JC. Essential genes of a minimal bacterium. Proc Natl Acad Sci U S A. 2006 Jan 10;103(2):425-30. DOI:10.1073/pnas.0510013103 |
17. Ara K, Ozaki K, Nakamura K, Yamane K, Sekiguchi J, and Ogasawara N. Bacillus minimum genome factory: effective utilization of microbial genome information. Biotechnol Appl Biochem. 2007 Mar;46(Pt 3):169-78. DOI:10.1042/BA20060111 |
18. Lee MC and Marx CJ. Repeated, selection-driven genome reduction of accessory genes in experimental populations. PLoS Genet. 2012;8(5):e1002651. DOI:10.1371/journal.pgen.1002651 |