GMU:DIY Biolab “Driver’s License”/DNA extraction

Related Scientific and Artistic Projects

Dolly

Fig. 2. Dolly. Photo courtesy of The Roslin Institute, The University of Edinburgh. Source: http://dolly.roslin.ed.ac.uk/

Dolly is a sheep that was cloned in Roslin Institute in Scotland in 1996 (Fig. 2.). The cell taken from a mammary gland of the six year old sheep was altered and implanted into an egg of a surrogate Scottish Blackface sheep, who, through the normal pregnancy gave a birth to a healthy offspring. Interestingly, Dolly was born from a somatic and not a reproductive cell, meaning, that at any time any taken cell of an adult could be reprogrammed into a reproductive stem cell.

The technology of cloning Dolly was used to clone also other mammals, including dogs and cats. The owner of the patent, a Korean company BioArts International stoped providing commercial service in 2008 because of lack of interest and a competition in a black market. The companies cloning vendor, the Sooam Biotech Research Foundation of Seoul still offers the service. On the other hand, it’s announced last competition to clone a beloved dog was announced for the 25th of November 2013. According to the foundation, the biggest hurdle to clone mammals from an adult cell is still a low rate of the pregnancy reaching as low as 2%, and the price, reaching 100.000 US dollars per clone.

The idea of being able to clone a mammal from an adult cell gives an idea of cloning a human, which by now is banned by civil law in all the world. Nevertheless the research is being still done at an embryo level for gaining further scientific knowledge. The used technique – the somatic cell nuclear transfer – combined with CRISPR/Cas9 gene editing technique might change expectations in cloning mammals, including humans.

• http://www.nature.com/articles/ncomms12359
• http://dolly.roslin.ed.ac.uk/facts/the-life-of-dolly/index.html
• http://www.bioartsinternational.com/press_release/ba09_09_09.htm
• http://en.sooam.com/dogcn/sub03.html
• http://dolly.roslin.ed.ac.uk/facts/cloning-faqs/index.html
• http://en.sooam.com/dogcn/sub06.html

GloFish

Fig. 3. Glofish, orange and green tetra. Source: http://glofish.com

At least 74 attempts at animal modification where counted in 2015 by A. Lievens et al (2015). While genetically modified animals are not yet produced for human consumption on a large scale, news associated with them come up. In 2015 genetically modified salmon was approved in US by the Food and Drug administration and showed up on a market in 2017*. GloFish (Fig. 3.), the first genetically modified pet was around pet shops in US since 2004 (Nash, 2004).

GloFish is interesting because of its aesthetic value. Being a pet, its function is to entertain their hosts. Secondly, GloFish includes green fluorescent protein (GFP), which is widely used in scientific research. GFP modification is also used for educational purposes in high schools and colleges to teach students recombinant DNA or protein purification techniques. Finally, even if genetic modification is restricted for the domestic experiments, the GFP modification was legalized by Berlin authorities in 2016 for educational purposes for the use in home environment.*

Originally isolated from the jellyfish Aequorea victoria, scientists use the protein as markers within DNA sequence or as a concept of proof for genetic modification. Although GFP modification is considered being harmless in organisms, some studies suggest its influence on animal aging, their locomotor ability or eye morphology. Therefore it is suggested to use GFP cautiously for genetical modifications (Mawhinney & Staveley 2011).

• https://www.cnbc.com/2017/08/09/salmon-becomes-worlds-first-genetically-modified-animal-to-enter-food-supply.html
• In 2016 Rüdiger Trojok was using GFP for modifying E. Coli bacteria in his home laboratory. For more info see https://www.meetup.com/Biotinkering-Berlin/events/235107360/?_cookie-check=zc-l_hyy9qe64U94

Eduardo Kac, GFP Bunny

Fig. 4. GFP bunny. Source: http://oslolux.wordpress.com/eduardo-kac

The transgenic artwork "GFP Bunny" was completed in February 2000 with the birth of rabbit named "Alba" (Fig. 4.). The project is an outcome of Kac’s collaboration with zoosystemician Louis Bec and scientists Louis-Marie Houdebine and Patrick Prunet. The rabbit has an altered genomics carrying a fluorescent GFP, a same protein, which was used to modify GloFish. Genetically modified Alba is an albino rabbit with no skin pigment and pink eyes. Under the day light it would look white and under the blue light with maximum excitation of 488 nm, the rabbit would glow with a bright green light (Kac 2000). In the description of the project Kac writes:

"My transgenic artwork /GFP Bunny/ comprises the creation of a green fluorescent rabbit, the public dialogue generated by the project, and the social integration of the rabbit."*

While the project generated a huge attention in the media and was followed by a number of interpretations,3 the social integration of the rabbit has failed as it was never released from the laboratory.* Despite that Alba, like any other rabbit, was seeking interaction and was, for example, sitting comfortably on Kac’s hands (Kac 2000).

For genetic modification of Alba, scientists integrated GFP into the genome through zygote microinjection, the method most extensively used in the production of transgenic animals including rabbits. The method suggests the in vitro fertilization which is done outside of the organism and therefore could be easily combined with the somatic cell nuclear transfer method used for cloning Dolly.

There is a number of controversial discussions around “GFP Bunny” project. On one hand the photo of a fluorescent rabbit is manipulated digitally. Secondly, there are different versions of how long the rabbit was alive. Finally, according to Wired magazine, it is not clear if the rabbit was genetically modified as an artistic project from the beginning on.* Nevertheless, the project gained enough attention from media to consider the project as one of the most influential in Bioart.

• http://www.ekac.org/gfpbunny.html#gfpbunnyanchor
• http://www.ekac.org/gfpbunny.html
• https://today.duke.edu/2000/11/bunnyn03.html
• https://www.wired.com/2002/08/rip-alba-the-glowing-bunny/

Revital Cohen and Tuur Van Balen, Sterile

Fig. 5. Sterile. Source: http://containerartistresidency01.org

Sterile is a project by Revital Cohen and Tuur Van Balen, which is a genetically modified Albino goldfish (Fig. 5.). At the Schering Foundation exhibition space in Berlin it is shown along other works, including the video Kingyo Kingdom, which follows fish breeders in Japan and at the same time contextualizes the genetically modified fish and Sensei Ichi-gō, a machine capable of reproducing sterile goldfish. Within the project description, Sterile is described as follows:

"Albino goldfish engineered to hatch without reproductive organs. They were not conceived as animals but made as objects, unable to partake in the biological cycle. An edition of 45 goldfish was produced for the artists by Professor Yamaha Etsuro in his laboratory in Hokkaido, Japan, following an intricate collaboration process which began in 2011."

The important part of the project is that the fishes were not sterilized after they were born but genetically modified in order not to have reproductive organs. In such a way, artists show their responsibility in front of all eco sphere as the genetically modified animals are not breeding modified in the next generations, leaving the project limited until the fishes die naturally. Having the fish born sterile, the project questions genetic modifications made by humans in relation to a naturally evolving environment.

Also, an important message in Sterile is a blurring out a border line between objects and subjects: a fish being technically engineered to become an object or machine programmed to become similar to a living organism. The idea of an object becoming a subject and vice versa is conceptualized around a machine-human rhizome within the context of the umbrella project of this paper Introduction to Posthuman Aesthetics, which proposes a subjective perspective on discourses in contemporary aesthetics.

• http://www.cohenvanbalen.com/work/sterile
• http://www.artscatalyst.org/sterile-sensei-ichi-go

Joe Davis, Microvenus

Fig. 6. Microvenus. Source: http://unmondemoderne.wordpress.com

Microvenus is a genetically modified Escherichia coli bacteria strain carrying a piece of synthetically composed sequence of amino acid molecules. It was first cloned into several laboratory strains of E. coli in collaboration with molecular geneticist Dana Boyd at Jon Beckwith’s laboratory at Harvard Medical School in 1988. As bacteria is small and invisible for the human eye, the artwork instead of being “aesthetic” is rather imaginable. Davis introduces the Microvenus as follow:

"Each Microvenus organism contains many copies of a special molecule designed by the artist and his colleagues. The artistic molecule is a short piece of synthetic DNA containing a coded visual icon that has been incorporated into a living strain of bacteria (E. Coli)."*

The coded icon is a symbol of life from a germanic rune. Resembling letter Y it also represents a female earth in different mythologies. The graphical image was converted into the bit-map image and further into DNA base pairs in order to synthetically program and attach to the original DNA code of the plasmids carried by bacteria (Fig. 6.). Davis has imagined that the code might be interpreted by extraterrestrials as the final stage of the project was supposed to include shipping of the modified bacteria into the universe.

Microvenus is interesting from different perspectives. First of all it is the first genetically modified art work. Secondly, Davis’ idea to encode and store data into the DNA in the end of 80s was a couple of decades ahead of a scientific project presented by CUHK team at the iGEM competition in 2010.* Finally, thinking of extraterrestrial intelligence, it probably makes sense to think of communication happening at the very nature of life, within the chemical interactions between different molecules. So encoding and reading genetical information, might give many answers about different life forms.

• Davis, J. (1996). "Microvenus". Art Journal. 55 (1): 70–74. Available at https://www.jstor.org/stable/777811
• http://2010.igem.org/Team:Hong_Kong-CUHK

Terms

Enzymes

“Enzymes are known to catalyze more than 5,000 biochemical reaction types.[4] Most enzymes are proteins, although a few are catalytic RNA molecules. The latter are called ribozymes. Enzymes' specificity comes from their unique three-dimensional structures.”(https://en.wikipedia.org/wiki/Enzyme)

Proteins

“Proteins are large biomolecules, or macromolecules, consisting of one or more long chains of amino acid residues.” (https://en.wikipedia.org/wiki/Protein)

Amino acids

“Amino acids are organic compounds containing amine (-NH2) and carboxyl (-COOH) functional groups, along with a side chain (R group) specific to each amino acid.[1][2][3] The key elements of an amino acid are carbon (C), hydrogen (H), oxygen (O), and nitrogen (N), although other elements are found in the side chains of certain amino acids. About 500 amino acids are known (though only 20 appear in the genetic code) and can be classified in many ways.”(https://en.wikipedia.org/wiki/Amino_acid)

genetic code

“The genetic code is the set of rules by which information encoded within genetic material (DNA or mRNA sequences) is translated into proteins by living cells.”(https://en.wikipedia.org/wiki/Genetic_code)

DNA

“Deoxyribonucleic acid is a molecule that carries the genetic instructions used in the growth, development, functioning and reproduction of all known living organisms and many viruses. DNA and ribonucleic acid (RNA) are nucleic acids; alongside proteins, lipids and complex carbohydrates (polysaccharides), they are one of the four major types of macromolecules that are essential for all known forms of life. Most DNA molecules consist of two biopolymer strands coiled around each other to form a double helix.”(https://en.wikipedia.org/wiki/DNA)

“In molecular biology, the term double helix[1] refers to the structure formed by double-stranded molecules of nucleic acids such as DNA. The double helical structure of a nucleic acid complex arises as a consequence of its secondary structure, and is a fundamental component in determining its tertiary structure. The term entered popular culture with the publication in 1968 of The Double Helix: A Personal Account of the Discovery of the Structure of DNA, by James Watson.”(https://en.wikipedia.org/wiki/Nucleic_acid_double_helix)

“The double-helix model of DNA structure was first published in the journal Nature by James Watson and Francis Crick in 1953”(https://en.wikipedia.org/wiki/Nucleic_acid_double_helix)

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  Onion (Allium) cells in different phases of the cell cycle, drawn by E. B. Wilson, 1900 (wikipedia)

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  A eukaryotic cell (left) and a prokaryotic cell (right) (wikipedia)

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  The structure of the DNA double helix. The atoms in the structure are colour-coded by element and the detailed structures of two base pairs are shown in the bottom right.(wikipedia)

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PCR

"Polymerase chain reaction (PCR) is a technique used in molecular biology to amplify a single copy or a few copies of a segment of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. It is an easy, cheap, and reliable way to repeatedly replicate a focused segment of DNA, a concept which is applicable to numerous fields in modern biology and related sciences." (https://en.wikipedia.org/wiki/Polymerase_chain_reaction)

Primers (molecules) forward strand and reverse strand

How does it work:

• We start at 95C two strands divides, hydrogen bonds will break at this temperature; DNA is accessible
• Annealing: primers bind to the targets Roughly at the 65C
• 72C polymerase start elongation of dna. Copies into one direction. It is called TAQ polymerase; comes from specific species called /thermus aquaticus/; lives in gazers and very stable; For every 1000 base pairs you need to copy we need around 60s.

95C the started molecules bind from the other side

Exponential reaction, at some point it cannot cope and more, because the volume of the epi is limited

DNA extraction

https://www.youtube.com/watch?v=7wU97pTs-xE

Meat tissue cells

• mince thoroughly a small piece of meat tissue
• Go to DNA-Extraktions-Kit Protocol

Blood cells

• Take your blood
• Go to DNA-Extraktions-Kit Protocol

Mouth cells

• 50ml Falkon mit 50ml isotonischer Kochsalzlösung
• damit den Mund ordentlich ausspülen (1-2min)
• zurück in den Falkon spucken
• zentrifugieren 10min volle Geschwindigkeit
• überstand verwerfen (vorsichtig, nicht schütten) die Zellen sammeln sich unten
• zellen in 1ml Kochsalzlösung aufnehmenund in Eppi transferieren
• nochmal Zentrifugieren
• überstand verwerfen *dann hat man menschliche Zellen im Eppi
• DNA-Extraktions-Kit anwenden

DNA-Extraktions-Kit Protocol