Biomod/2011/SRISHTI/ArtScienceBangalore/Project

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Project

RASA-01: Open-Souce DNA Computing RASA-01 is a simple Do-it-yourself DNA computing kit for the general hobbyist interested in DNA computing. By collaborating with artists, designers, scientists and chefs, ArtScienceBangalore presents alternate scenarios of Human Computer Interaction with bio-molecular computers and DNA computing.

The Altair 8800 was a Do It Yourself microcomputer kit introduced in 1975. Sold through mail order advertisements, the kit, based on an Intel 8080 CPU was instrumental in sparking the micro-computer and personal computer revolution. The Altair was not an easy computer to program, the user had to toggle switches to positions corresponding to an 8080 microprocessor instruction or opcode in binary. With RASA-01 ArtScienceBangalore proposes an updated, simpler DNA version of the Altair. RASA-01 uses the Shapiro-Rothemund Unit which is a programmable two state DNA automaton. It uses a double stranded DNA as input, endonuclease and DNA ligase as main hardware and transition molecules as software.


PROJECT

This project, carried out by artists, designers and scientific collaborators, is a speculative investigation into the possibilities of using Biolomolecular design to make a simple 2-state finite machine. We prototyped an educational open-source biocomputer that takes the form of a kit which includes:

  • a DIY PCR machine
  • a DIY Gel electrophoresis box
  • input strands of DNA

We hope this project makes the theories and methods behind bimolecular design and biomolecular computing accessible to designers, artists, enthusiasts and amateurs. We believe that burgeoning technologies have more interesting outcomes when a plurality of voices are included in their formative years.


SWEET & SOUR NANOBOTS: EDIBLE INTERFACES?

During the development of our project we imagined with a variety of interfaces. Researching the history of early kit-computing and Human-Computer-Interaction, we realized how essential rich media interfaces such as screens, keyboards, speakers and mouse were to making digital computing accessible to a wider audience. We came to the conclusion that there are many advantages and interesting design constraints by focusing on taste as the sensory interface for the nano computer. Imagining and prototyping taste as a means of human-nanocomputing-interface has forced us to ask many questions about the possibilities and limitations of design for BioMod. The focus on medicinal uses of nanotechnologies has also made us to revisit the historical relationship between medicine and food, and ask if there are any synergies in these domains for nanotechnology. From our research into the history of kit computing it appears that early design decisions in the human-computer-interfaces and peripherals can lead to lock-in effects in both thinking and actual production. By imagining the possibilities of tastable computing we hope to open unexplored areas of the solution space for computing interfaces. Our first version of an Open Source DNA Computer is called RASA - "taste" in Sanskrit.


PROJECT PROCESS

The Project involved 2 phases:

Building the Hardware to carry out the computation- This involved building a PCR machine and a GEL BOX

The wetware that carries out the computation

The process and the protocols are given below:


PROTOCOL FOR BUILDING A DNA COMPUTER

Methods Synthetic DNA Double-stranded synthetic DNA molecules were prepared by annealing 2,000 pmol of commercially obtained deoxyoligonucleotides (Sigma-Genosys) in a final volume of 10ml of 10mM Tris-HCl buffer, pH8.0, containing 1mM EDTA and 50mM NaCl. The annealing was performed by heating the solution to 94 VC followed by slow cooling. The formation of a duplex was confirmed by native PAGE (20%). The oligomers were 5W-phosphorylated and PAGE-purified by the supplier and used without further purification. Input molecules These were constructed stepwise by ligating one or more synthetic DNA segments of thedesired sequence to a 1,457-bp fragment obtained by digestion of the pBluescript II SK+ plasmid (Stratagene) with FokI, followed by polymerase chain reaction (PCR) amplification of the coding segment and a 300- or 325-bp tail region. The sequences of the resulting input molecules were confirmed by sequencing.


Output-detecting molecules The output-detecting molecule for the S0 output (S0-D) was formed by ligating a synthetic adapter of 30 bp containing a FokI recognition site to a 181-bp fragment obtained by digesting the pBluescript II SK+ plasmid with FokI, PCR amplification and additional FokI digestion to form the 160-bp fragment bearing the desired sticky end. The output-detecting molecule for the S1 output (S1-D) was obtained by PCR amplification of a 285-bp fragment corresponding to positions 1,762±2,047 of the pBluescript II SK+ plasmid followed by FokI digestion of the PCR product to form a 250-bp fragment.


Computation reactions Reactions were set by mixing 2.5 pmol of the input molecule, 1.5 pmol of each output- detection molecule and 15 pmol of each transition molecule with 12 units of FokI and 120 units of T4 DNA Ligase (both from New England Biolabs) in 120 ml of NEB4 buffer supplemented with 1 mM ATP and incubating at 18 VC for 70 min. In case of multiple inputs in the same reaction, equal amounts were used, summing up to 2.5 pmol. The mixtures were purified by the Qiagen PCR purification kit and eluted using 30 ml EB buffer (Qiagen). Aliquots (10 ml) were assayed by gel electrophoresis using 3% MetaPhor agarose (FMC Bioproducts) unless indicated otherwise. The lengths of the DNA species were verified using a commercial 50-bp DNA step ladder (Promega). To further confirm that output reporting molecules were formed as expected, we amplified by PCR and sequenced the output-detection molecule/output molecule junction region in both output-reporting molecules S0-R and S1-R and found the expected sequences . PROTOCOL FOR BUILDING A DNA COMPUTER

Methods Synthetic DNA Double-stranded synthetic DNA molecules were prepared by annealing 2,000 pmol of commercially obtained deoxyoligonucleotides (Sigma-Genosys) in a final volume of 10ml of 10mM Tris-HCl buffer, pH8.0, containing 1mM EDTA and 50mM NaCl. The annealing was performed by heating the solution to 94 VC followed by slow cooling. The formation of a duplex was confirmed by native PAGE (20%). The oligomers were 5W-phosphorylated and PAGE-purified by the supplier and used without further purification. Input molecules These were constructed stepwise by ligating one or more synthetic DNA segments of thedesired sequence to a 1,457-bp fragment obtained by digestion of the pBluescript II SK+ plasmid (Stratagene) with FokI, followed by polymerase chain reaction (PCR) amplification of the coding segment and a 300- or 325-bp tail region. The sequences of the resulting input molecules were confirmed by sequencing.


Output-detecting molecules The output-detecting molecule for the S0 output (S0-D) was formed by ligating a synthetic adapter of 30 bp containing a FokI recognition site to a 181-bp fragment obtained by digesting the pBluescript II SK+ plasmid with FokI, PCR amplification and additional FokI digestion to form the 160-bp fragment bearing the desired sticky end. The output-detecting molecule for the S1 output (S1-D) was obtained by PCR amplification of a 285-bp fragment corresponding to positions 1,762±2,047 of the pBluescript II SK+ plasmid followed by FokI digestion of the PCR product to form a 250-bp fragment.


Computation reactions Reactions were set by mixing 2.5 pmol of the input molecule, 1.5 pmol of each output- detection molecule and 15 pmol of each transition molecule with 12 units of FokI and 120 units of T4 DNA Ligase (both from New England Biolabs) in 120 ml of NEB4 buffer supplemented with 1 mM ATP and incubating at 18 VC for 70 min. In case of multiple inputs in the same reaction, equal amounts were used, summing up to 2.5 pmol. The mixtures were purified by the Qiagen PCR purification kit and eluted using 30 ml EB buffer (Qiagen). Aliquots (10 ml) were assayed by gel electrophoresis using 3% MetaPhor agarose (FMC Bioproducts) unless indicated otherwise. The lengths of the DNA species were verified using a commercial 50-bp DNA step ladder (Promega). To further confirm that output reporting molecules were formed as expected, we amplified by PCR and sequenced the output-detection molecule/output molecule junction region in both output-reporting molecules S0-R and S1-R and found the expected sequences .






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