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Challenging Traditional Approaches to Computation: A Biomolecular Transducer Employing Ternary Language and Rendering a Biological Output Paul Lazarescu and Mark Chaskes Mentor: Tamar Ratner The Schulich Faculty of Chemistry, Technion-Israel Institute of Technology Design and Development Abstract Biomolecular computing is a new field of research, merging several sciences. Previous works were based on finite state automata and have had limited computing capabilities. In this project, a transducer model was used in order to design a biomolecular machine with greater computing potential. In this research, two automata were designed as software for the transducer: one able to divide a ternary input by three and the other by two. By making use of a plasmid this transducer could perform multiple computations consecutively. Figure r1p0 S0 S0 r2p1 S0 S0 r1p0 S1 S1 r1p2 S0 S1 r0p1 S1 S1 r2p2 S1 S1 S0 r1p2 r2p2 r0p0 Figure 3 r2p0 A. r0p0 B. S1 S0 S2 r0p1 r2p2 S1 r0p1 S0 S2 r0p2 S0 S1 r1p1 S1 S2 r1p2 S1 S1 r2p1 S2 S2 r2p2 S2 r1p2 S0 to S0, read 0, print 0 AGTCTT...8 base...CTCCTCGCAGC...2 base AATCAGAA...pairs ...GAGGAGCGTCG...pairs...TCAG 0 BseRI BbvI S0 to S1, read 1, print 0 AGTCTT...8 base...CTCCTCGCAGC...1 base AATCAGAA...pairs ...GAGGAGCGTCG...pairs...CCAT AGTCTT...8 base...CTCCTCGCAGC AATCAGAA...pairs ...GAGGAGCGTCGGAGC S1 to S0, read 0, print 1 GGTATT...8 base...CTCCTCGCAGC...3 base AACCATAA...pairs ...GAGGAGCGTCG...pairs...CAGA S1 to S1, read 1, print 1 Figure 4 r0p2 Figure 4: The divide-by-three transducer used in this project. A. A schematic diagram of the transducer. ‘r’ represents the ‘read’ symbol and ‘p’ represents the ‘printed’ symbol. B. Transition rules of this transducer. 0 B. Sequences decided upon AGTCTT for each symbol TCAGAA Sticky ends left by AGTCTT sequences cleaved in S0 AA 1 2 GGTATT CCATAA CTCGTT GAGCAA GGTATT AA Figure 5: A. The state of the transducer depends Sticky ends left by GTCTT GTATT on the way the symbol sequences cleaved in S1 A A was cleaved. B. The symbols cleaved in Sticky ends left by TCTT TATT different states leaving sequences cleaved in S2 unique sticky ends Figure 5 Terminator TGCTGA ACGACT CTCGTT AA TGCTGA CT TCGTT A GCTGA T CGTT CTGA Results 1[1] GGTATT...8 base...CTCCTCGCAGC...2 base AACCATAA...pairs ...GAGGAGCGTCG...pairs...CATA S1 to S2, read 2, print 1 GGTATT...8 base...CTCCTCGCAGC...1 base AACCATAA...pairs ...GAGGAGCGTCG...pairs...AGCA S2 to S0, read 0, print 2 CTCGTT...8 base...CTCCTCGCAGC...4 base AAGAGCAA...pairs ...GAGGAGCGTCG...pairs...AGAA Terminator AATTCGGCCGTT..8 base..CTCCTCGCAGC..8 base..CTCGTTAGTCTTAGTCTTTGCTGAAATT TTAAGCCGGCAA..pairs ..GAGGAGCGTCG..pairs ..GAGCAATCAGAATCAGAAACGACTTTAA BseRI Recognition Site + A (18 in base ten) 2 0 0 BbvI Recognition Site Spacers BbvI BseRI Plasmid First cut by restriction enzymes AATTCGGCCGTT TTAAGCCGGC B CTCGTTAGTCTTAGTCTTTGCTGAAATT AATCAGAATCAGAAACGACTTTAA (S0,2) + All TM First Ligation addition of transition molecule S0 to S2 (read 2, print 0) C AATTCGGCCGTTAGTCTT..8 base..CTCCTCGCAGCCTCGTTAGTCTTAGTCTTTGCTGAAATT TTAAGCCGGCAATCAGAA..pairs ..GAGGAGCGTCGGAGCAATCAGAATCAGAAACGACTTTAA Figure 2[2] DNA Based Transducer: a more complex version of an automaton, that can both ‘read’ and ‘print’ information using double-stranded DNA (dsDNA). + BbvI BseRI Restriction Enzyme Type II (Fig. 2A): cleaves dsDNA at a certain distance from a recognition site. Final Cut AATTCGGCCGTTAGTCTTCTCGTTAGTCTT TTAAGCCGGCAATCAGAAGAGCAATCAG (S0,T) + All DM Soreni, S. Yogev, E. Kossoy, Y. Shoham, E. Keinan, Parallel Biomolecular Computation on Surfaces with Advanced Finite Automata. J. AM. CHEM. SOC. 127, 3935-3943 (2005). TGCTGAAATT CTTTAA Final Ligation bonding of detection molecule to sticky ends of terminator AATTCGGCCGTTAGTCTTCTCGTTAGTCTTTGCTGA...Reporter...TGCTGAAATT TTAAGCCGGCAATCAGAAGAGCAATCAGAAACGACT....Gene 0....ACGACTTTAA 0 Plasmid: a circular vector found in bacteria, in which a foreign DNA sequence is easily inserted. from the National Human Genome Project TCTTAGTCTTTGCTGAAATT TCAGAAACGACTTTAA Repeat cycle of restriction, hybridization, and ligation until the terminator is cleaved DNA Ligase (Fig. 2A): covalently bonds different fragments of dsDNA to each other. Sticky End (Fig. 2C): unpaired single strand DNA (ssDNA) overhangs. These sequences bond to other DNA sticky ends with complementary base pair sequences. Second Cut AATTCGGCCGTTAGTCTT TTAAGCCGGCAATCAG 2 0 Terminator CTCGTT...8 base...CTCCTCGCAGC...3 base AAGAGCAA...pairs ...GAGGAGCGTCG...pairs...ATAA S2 to S2, read 2, print 2 CTCGTT...8 base...CTCCTCGCAGC...2 base AAGAGCAA...pairs ...GAGGAGCGTCG...pairs...GCAA Figure 8: The TM for the divide-by-three transducer. For every transition rule of the transducer, one transition molecule had to be designed. Another six transition molecules were created for the divide-by-two transducer. Detection Molecule (DM) Input (divide-by-three transducer) EagI Recognition Site D [2] M. S0 r0p0 S0 S0 r1p0 S1 S0 r2p0 S2 r1p1 A. Transition Molecules (TM) S0 to S2, read 2, print 0 r2p1 r1p0 DNA Based Automaton (Fig. 2): a model that can ‘read’ DNA symbols and change it’s state according to the read data. [1] Adapted Figure 3: The divide-by-two transducer used in this project. The transduer begins in state 0 (S0). A. A schematic diagram of the transducer. 'r' represents the 'read' symbol and 'p' represents the 'printed‘ symbol. B. Transition rules of this transducer. For each transition rule there is a transition molecule. r 0 p 1 B. S0 r0p0 S2 to S1, read 1, print 2 Introduction Terms DNA (Fig. 1): double strand molecule. Made of base pairs: cytosine (C) only bonds to guanine (G), and adenine (A) only bonds to thymine (T). A. r 2 p 1 Molecules Design Terminator Reporter Gene 0 inserted and can be expressed by bacteria, or, a second computation can occur. Figure 6 Figure 6: An example of a computation process on an input string of ‘200’ (equivalent to 1810), using the DNA based transducer that divides by three. Figure 9: The DM TGCTGA...Reporter... AAACGACT....Gene 0....ACGA bonds with the cleaved terminator. TGCTGA...Reporter... These molecules AAACGACT....Gene 1....CGAC instigate a biological function (releasing a TGCTGA...Reporter... drug, giving a bacterial AAACGACT....Gene 2....GACT Figure 9 phenotype output, etc.) and print the terminator symbol to continue the computation. Discussion Two software for a DNA based transducer were created. A variety of molecules were also designed as the components of the transducers. The models and the molecules were simulated and checked using a computer program. The molecules were processed and functioned as expected, and the design worked properly. Creating a biomolecular transducer has not yet been accomplished experimentally. However, this science can have many applications. Conclusions In the future biomolecular computers will hopefully integrate into biological systems. Because these machines are capable of a biological output, this project is literally cutting-edge science. These devices are unlikely to replace the common computer. Instead, due to their capability for direct interface, the importance of biomolecular computing lies within the integration of biological systems, in fields ranging from medicine to agriculture. Acknowledgements: We would like to sincerely thank our mentor Tamar Ratner for her dedication, as well as Professor Ehud Keinan for allowing us to use his www.PosterPresenta tions.com laboratory. We also would like to thank Mr. Russell N. Stern and the Louis Herman Israel Experience Fund for their generosity and donation. POSTER TEMPLATE BY: