121 Artificial Genomes

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Artificial Genomes and Synthetic OrganismsABRINA, Joanna; ABUEL, Rachelle; BEA, Junelyn; CAFINO, Ernest; DALISAY, Roi; PABILANE, AlmaBiochem 121 Special Report Dr. Carillo

REPORT OUTLINE(Guide to Reporter – NOT to be put in PowerPoint)

I. Genetic EngineeringA. Definition and Scope

Field of science that caters to the alteration of genetic code by artificial means, and is, therefore,different from traditional selective breeding (Dixon, February 2011)

Uses recombinant DNA or RNA techniques to introduce foreign, heritable material prepared in vitro or directly to the organism, and then fused or hybridized with the host

Human cloning (duplication of gene) and stem cell research are under genetic engineering Forming new combinations of heritable genetic material followed by the incorporation of that

material either indirectly through a vector system or directly through micro-injection, macro-injection and micro-encapsulation techniques.

TRANSGENIC - If genetic material from another species is added to the host CISGENIC - if genetic material from the same species or a species that can naturally breed with

the host is used can also be used to remove genetic material from the target organism, creating a gene

knockout organism genetic engineering = gene manipulation + cell synthesis

B. Applications and Examples Insertion of human genes into sheep so that they secrete alpha-1 antitrypsin in their milk, a useful

substance in treating some cases of lung disease Combining the gene that programs poison in the tail of a scorpion with a cabbage forming

genetically modified cabbages that kill caterpillars (poison as an insecticide) Creating a chicken with four legs and no wings Creating a goat with spider genes that creates "silk" in its milk (Dixon, February 2011) One of the most popular and first breakthroughs of genetic engineering is the insulin-producing

bacteria commercialized in 1982.II. Artificial/Synthetic Genomes

A. Definition

B. Process of SynthesisC. Artificial vs Synthetic

III. Synthetic Organisms – products of genetic engineering and artificial genomes A. Examples

a. Spider goats

Freckles, result of genetic engineering by RandyLewis, a professor of Genetics at Utah StateUniversity

Freckles‘ milk is full of spider-silk protein. ―In the medical field, we already know that we can

produce spider silk that's good enough to be usedin ligament repair. . . .we already know we canmake it strong enough as an elastic. We've donesome studies that show that you can put it in thebody and you don't get inflammation and get ill.‖ – Randy Lewis\

b. Synthetic bacteria (unicellular organism) by J. Craig Venter Synthia, aka Mycoplasma mycoides JCVI-syn 1.0, a cell

which genetic code, copied and modified from an existingbacterium, had been assembled by a computer.

The genome of Mycoplasma mycoides was constructedfrom more than 1,000 sections of preassembled units ofDNA. Researchers transplanted the artificially assembledgenome into a M. capricolum cell which had been



emptied of its own genome. Once the DNA "booted up", the bacteria began to function andreproduce in the same manner of naturally occurring M. mycoides

Not given new species name, but its synthetic genome was given an official version number,Mycoplasma mycoides JCVI-syn1.0.

B. Benefits and Uses The U.S. government together with the leading oil, agribusiness and chemical companies are

already big funders of synthetic biology research, providing capitals to start-up syntheticbiology companies.

Proponents hope this emerging field will create a new "bioeconomy " in which any and alltypes of plant matter can feed synthetic organisms that will be " living factories " that can beused for production fuels, industrial chemicals, bio-plastics, medicines and even food.

Environmental applications include functions such as detecting environmental contaminantsusing biosensors and removing such contaminants using specifically tailored plants ormicroorganisms.

Health-related applications include diagnosing, monitoring, and responding to diseaseconditions in humans and animals and developing andmanufacturing new drugs and vaccines.

Industrial applications comprise employing plants,microorganisms, and specifically tailored enzymes fordeveloping biofuels, as well as devising more efficientbiomanufacturing and synthesis processes using chemicaltechnology.

Green energy companies are using the emerging field ofsynthetic biology to modify bacteria into creatinghydrocarbons for gasoline, diesel, and jet fuel.

C. Social Issues - Many concerns have arisen about the dangers of synthetic biology. Emerging risks areclassified into 4 headings: biosafety, biosecurity, intellectual property and trade, and ethical concerns.Some of these issues include:

The release into the environment of novel, genetically modified organisms – either accidentallyor deliberately – potentially resulting in harmful consequences for ecological systems and/orhuman health;

The possible exploitation of synthetic biology for bioterrorism – including the construction ofmodified or novel microorganisms with lethal or incapacitating effects: synthesis of severalpathogenic viruses from scratch, such as poliovirus and severe acute respiratory syndrome(SARS);

Increasingly routine nature of many synthetic biology procedures, making them moreaccessible to those with no specialized training;

Possibility of recreation existing, extinct, or eradicated pathogens of humans, animals, orplants;

Patenting strategies, potentially creating monopolies inhibiting basic research and restriction ofproduct development to only large companies;

Trade and global justice issues, such as preventing exploitation of indigenous resources byenabling chemical synthesis of valuable products in industrial countries (e.g. production of theantimalarial drug artemisinin in genetically engineered bacteria rather than extracting it from aplant source);

Claims that synthetic biology is involved in creating artificial life, raising philosophical andreligious concerns.

D. Current Development Synthetic Genomics Inc., a company founded by Dr. Venter, provided $30 million to fund

experiments in synthetic biology. It also owns the intellectual-property rights to cell-creationtechniques. It has a $600 million contract with Exxon Mobil Corp. to schemean algae that cancapture carbon dioxide and make fuel.

At least three other companies—Amyris Biotechnologies in Emeryville, Calif.; LS9 Inc. in SanFrancisco; and Joule Unlimited in Cambridge, Mass.—are working on synthetic cells toproduce renewable fuels.

References: Capecchi, M. R. (2001). Generating mice with targeted mutations . Nature medicine 7 (10): 1086 –1090



Dixon, P. (12 February 2011). Genetic engineering: what is genetic engineering? Retrieved fromwww.globalchange.com

Jacobsen, E.; Schouten, H. J. (2008). Cisgenesis, a New Tool for Traditional Plant Breeding, Should be Exempted from the Regulation on Genetically Modified Organisms in a Step by Step Approach . PotatoResearch 51: 75 –88

The European Parliament and the council of the European Union (12 March 2001). Directive on the release of genetically modified organisms (GMOs) Directive 2001/18/EC ANNEX. Official Journal of theEuropean Communities. p. page 17

http://www.guardian.co.uk/science/2012/jan/14/synthetic-biology-spider-goat-genetics http://www.livescience.com/6486-live-organism-synthetic-genome-created.html http://www.foe.org/projects/food-and-technology/synthetic-biology http://www.actionbioscience.org/newfrontiers/lowrie.html http://www.synbioproject.org/topics/synbio101/links/

―The first self -replicating species we‘ve had on the planet whose parent is a computer.‖ –Dr. Venter

John Craig Venter and his team at the J. Craig Venter Institute (JCVI) in Rockville, Maryland, and San Diego,successfully created the first living organism with a completely synthetic genome. The project was achieved at greatexpense, an estimated $40 million (most of it paid for by Synthetic Genomics, a company Dr. Venter founded) and effortof 20 people working for almost 15 years.

Upsides

hitherto, the largest piece of DNA synthesized (~a million units in length)able to make it accurate enough to substitute for the cell‘s own DNA

Dissuasiondesigning new organisms will take yearsprogress toward making biofuels is already being achieved with conventional genetic engineering approaches in whichexisting organisms are modified a few genes at a timefurther research should be halted until sufficient regulations are in place, as such may pose ―dangerous newtechnology‖

Further Goals:Creating heavily customized genomes (i.e. ones that make fuels or pharmaceuticals, vaccines)getting them to ―boot‖ up the same way in a cell

Timeline of the Artificial Genome Project

1995John Craig Venter and JCVI colleagues Clyde Hutchison III and Hamilton Smith sequenced the 600,000-basechromosome of a bacterium called Mycoplasma genitalium , the smallest genome of a free-living organism. The microbehas about 500 genes, and researchers found they could delete 100 individual genes without ill effect

However, synthesizing a full bacterial chromosome and making it to work in a recipient cell are needed in order to confirm the minimal genome suggested by those experiments. But these steps have taken years because the technology to make and manipulate whole chromosomes did not exist.

2007Venter, Smith, Hutchison, and colleagues finally demonstrated that they could transplant natural chromosomes fromone microbial species to another

2008They showed that they could make an artificial chromosome that matched M. genitalium ‘s but also contained―watermark‖ DNA sequences that would enable them to tell the synthetic genome from the natural one

However, these steps became bogged down, partly due to slow growth of M. genitalium that one experiment can take weeks to complete. The team decided to change microbes in midstream, sequencing the 1-million-base genome of the faster growing M. mycoidesand beginning to build a synthetic copy of its chromosome.

2009They showed they could extract the M. mycoides natural chromosome, place it into yeast, modify the bacterial genome,and then transfer it to a close microbial relative, M. capricolum

http://www.livescience.com/6486-live-organism-synthetic-genome-created.html


http://www.foe.org/projects/food-and-technology/synthetic-biology


http://www.actionbioscience.org/newfrontiers/lowrie.html







2010

After numerous attempts, they successfully transplanted the recombinant genome in M capricolum . They sequencedthe DNA in the blue bacterial colonies they had retrieved and later on confirmed that the bacteria had the syntheticgenome, and the microbes were indeed making proteins characteristic of M. mycoides rather than M capricolum . Thecolony grew like a typical M. mycoides as well.The scientists start off with a genome sequence from a bacterium that already has a very small genome, alter it on thecomputer to add or delete genes, then synthesize it from chemicals, transplant it into a cell, and see how the changesaffect the cell's function.

The scientists constructed the genome of the bacterium Mycoplasma mycoides from more than 1,000 sections ofpreassembled units of DNA, ordered from Blue Heron, a company that specializes in synthesizing DNA. Theytransplanted the artificially assembled genome—named Mycoplasma mycoides JCVI-syn1.0—into a Mycoplasma capricolum cell that had been emptied of its own genome. Once the DNA "booted up," the bacteria began to functionand reproduce in the same manner as naturally occurring M. mycoides . Booting up results once the recombinant DNAutilizes elements of the M. capricolum host cells, which still contain certain machinery that let them carry out the processof expressing a gene, or taking the genetic code and using it to build proteins (transcription). When the artificial genomeentered the cell, the cellular machines that run DNA transcription recognized the DNA, and began doing their job.

The researchers used yeast to glue together thousands of DNA snippets, each containing 1,080 base pairs. To assist inassembly, each section of DNA contained 80 base pairs at every end that instructed the yeast where to join the twostrands. Slowly, the DNA strands came together in runs of tens of thousands of base pairs, and then hundreds ofthousands, until the yeast produced a complete 1,080,000-base-pair synthetic genome.

However, the synthesis process still introduced some mutations into the M. mycoides genome. For the assembledgenome to be recognizable as synthetic, the researchers deliberately inserted four sequences of DNA that serve aswatermarks. The watermarks contain a code that translates DNA into English letters with punctuation, allowing thescientists to literally write messages with the genes. When translated, the watermarks spell out the names of the 46researchers who helped with the project, quotations from James Joyce, physicist Richard Feynman and J. RobertOppenheimer, and a URL that anyone who deciphers the code can e-mail.

In the course of the project, they found out that even a tiny inaccuracy could prevent the inert DNA from activating into alive bacterium. At one point, a single base pair mistake set the entire program back three months. But DNA sequencingaccuracy has become so advanced that at least finding the mistakes took only days, not the months needed a decadeago during the infancy of genetic engineering.

Sources:(2010). Synthetic genome brings new life to bacterium. Science Magazine , 328, 958-959Bourzac, K. (2010). How to make an artificial cell: Researchers at the Venter Institute explain their ground-

breaking techniques. Technology Review, MIT, Retrieved from http://www.How to Make an Artificial Cell -Technology Review.htmFox, S. (2010). First live organism with synthetic genome created. Retrieved from 6486-live-organism- synthetic-genome-created.com on March 15, 2012Wade, N. (2010). Researchers say they created ‗Synthetic cell‘ . The New York Times . Retrieved from

http://www.21cell/NYTimes.com

Figure 1.The bacterial colonies growing on these petri dishes contain a genome modified on a computer and then pieced together in the

lab



Figure 2.Blue colonies of Mycoplasma mycoidesindicate a successfully transplanted genome

Figure 3.Self-replicatingM.mycoides bacteria revealed in an electron micrograph

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