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Challenging Traditional Approaches to Computation A Biomolecular Transducer Employing Ternary Language and Rendering a Biological Output Mark Chaskes and Paul Lazarescu Mentor: Tamar Ratner The Schulich Faculty of Chemistry Technion, Haifa, Israel, 32000

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Objective Design a theoretical biomolecular transducer to solve consecutive mathematical equations in ternary. -First divide an input by three and then divide the yeild of that by two

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What is biomolecular computing? A biomolecular computer is a group of molecules that read dsDNA and can print an output.

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What is a DNA based transducer? A transducer is not a PC; it has unique capabilities that an ordinary computer does not. Advantages include: Direct interface with a biological system Can release a biological output Able to compute in parallel Store large amounts of data in a small volume

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Schematic Diagram S0S0 S1S1 Begins in S 0 and changes states Begins in S 0 and changes states depending on read symbols Key S 0 and S 1 are states a and b are symbols

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Design on the Molecular Level Symbols are dsDNA strands Restriction enzymes cleave the sequence at recognition sites States are determined by the location of cleavage within the symbol

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Process Reading 2 from S 0 prints 0 and goes to S 2 Divide by three transducer reading the input State 0200

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Process Divide by three transducer reading the input Reading 0 from S 2 prints 2 and goes to S 0 0State 200

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Process Divide by three transducer reading the input Reading 0 from S 0 prints 0 and encodes the output State 00

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Process S1S1 Reading 0 from S 0 prints 0 and goes to S 0 Divide by two transducer reading the input State 0020

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Process S1S1 Reading 2 from S 0 prints 1 and goes to S 0 Divide by two transducer reading the input State 020

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Process S1S1 Reading 0 from S 0 prints 0 and goes to S 0 Divide by two transducer reading the input State 00

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AATTCGGCCGTT..8 base..CTCCTCGCAGC..8 base..CTCGTTAGTCTTAGTCTTTGCTGAAATT TTAAGCCGGCAA..pairs..GAGGAGCGTCG..pairs..GAGCAATCAGAATCAGAAACGACTTTAA BseRI Recognition Site EagI Recognition Site Spacers BbvI Recognition Site Plasmid Terminator Molecular Design of the Input Encoding in Ternary (18 in base ten)

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AATTCGGCCGTT..8 base..CTCCTCGCAGC..8 base..CTCGTTAGTCTTAGTCTTTGCTGAAATT TTAAGCCGGCAA..pairs..GAGGAGCGTCG..pairs..GAGCAATCAGAATCAGAAACGACTTTAA AGTCTT...8 base...CTCCTCGCAGC...2 base AATCAGAA...pairs...GAGGAGCGTCG...pairs...TCAG S 0 to S 0, read 0, print 0 AGTCTT...8 base...CTCCTCGCAGC...1 base AATCAGAA...pairs...GAGGAGCGTCG...pairs...CCAT S 0 to S 1, read 1, print 0 AGTCTT...8 base...CTCCTCGCAGC AATCAGAA...pairs...GAGGAGCGTCGGAGC S 0 to S 2, read 2, print 0 GGTATT...8 base...CTCCTCGCAGC...3 base AACCATAA...pairs...GAGGAGCGTCG...pairs...CAGA S 1 to S 0, read 0, print 1 GGTATT...8 base...CTCCTCGCAGC...2 base AACCATAA...pairs...GAGGAGCGTCG...pairs...CATA S 1 to S 1, read 1, print 1 GGTATT...8 base...CTCCTCGCAGC...1 base AACCATAA...pairs...GAGGAGCGTCG...pairs...AGCA S 1 to S 2, read 2, print 1 CTCGTT...8 base...CTCCTCGCAGC...4 base AAGAGCAA...pairs...GAGGAGCGTCG...pairs...AGAA S 2 to S 0, read 0, print 2 CTCGTT...8 base...CTCCTCGCAGC...3 base AAGAGCAA...pairs...GAGGAGCGTCG...pairs...ATAA S 2 to S 1, read 1, print 2 CTCGTT...8 base...CTCCTCGCAGC...2 base AAGAGCAA...pairs...GAGGAGCGTCG...pairs...GCAA S 2 to S 2, read 2, print 2 Divide-by-three Computation First Restriction

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AATTCGGCCGTT CTCGTTAGTCTTAGTCTTTGCTGAAATT TTAAGCCGGC AATCAGAATCAGAAACGACTTTAA AGTCTT...8 base...CTCCTCGCAGC...2 base AATCAGAA...pairs...GAGGAGCGTCG...pairs...TCAG S 0 to S 0, read 0, print 0 AGTCTT...8 base...CTCCTCGCAGC...1 base AATCAGAA...pairs...GAGGAGCGTCG...pairs...CCAT S 0 to S 1, read 1, print 0 AGTCTT...8 base...CTCCTCGCAGC AATCAGAA...pairs...GAGGAGCGTCGGAGC S 0 to S 2, read 2, print 0 GGTATT...8 base...CTCCTCGCAGC...3 base AACCATAA...pairs...GAGGAGCGTCG...pairs...CAGA S 1 to S 0, read 0, print 1 GGTATT...8 base...CTCCTCGCAGC...2 base AACCATAA...pairs...GAGGAGCGTCG...pairs...CATA S 1 to S 1, read 1, print 1 GGTATT...8 base...CTCCTCGCAGC...1 base AACCATAA...pairs...GAGGAGCGTCG...pairs...AGCA S 1 to S 2, read 2, print 1 CTCGTT...8 base...CTCCTCGCAGC...4 base AAGAGCAA...pairs...GAGGAGCGTCG...pairs...AGAA S 2 to S 0, read 0, print 2 CTCGTT...8 base...CTCCTCGCAGC...3 base AAGAGCAA...pairs...GAGGAGCGTCG...pairs...ATAA S 2 to S 1, read 1, print 2 CTCGTT...8 base...CTCCTCGCAGC...2 base AAGAGCAA...pairs...GAGGAGCGTCG...pairs...GCAA S 2 to S 2, read 2, print 2 Divide-by-three Computation First Restriction

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AATTCGGCCGTT CTCGTTAGTCTTAGTCTTTGCTGAAATT TTAAGCCGGC AATCAGAATCAGAAACGACTTTAA AGTCTT...8 base...CTCCTCGCAGC AATCAGAA...pairs...GAGGAGCGTCGGAGC Transition Molecule S 0 to S 2, reading 2, printing 0 DNA Ligase AGTCTT...8 base...CTCCTCGCAGC...2 base AATCAGAA...pairs...GAGGAGCGTCG...pairs...TCAG S 0 to S 0, read 0, print 0 AGTCTT...8 base...CTCCTCGCAGC...1 base AATCAGAA...pairs...GAGGAGCGTCG...pairs...CCAT S 0 to S 1, read 1, print 0 AGTCTT...8 base...CTCCTCGCAGC AATCAGAA...pairs...GAGGAGCGTCGGAGC S 0 to S 2, read 2, print 0 GGTATT...8 base...CTCCTCGCAGC...3 base AACCATAA...pairs...GAGGAGCGTCG...pairs...CAGA S 1 to S 0, read 0, print 1 GGTATT...8 base...CTCCTCGCAGC...2 base AACCATAA...pairs...GAGGAGCGTCG...pairs...CATA S 1 to S 1, read 1, print 1 GGTATT...8 base...CTCCTCGCAGC...1 base AACCATAA...pairs...GAGGAGCGTCG...pairs...AGCA S 1 to S 2, read 2, print 1 CTCGTT...8 base...CTCCTCGCAGC...4 base AAGAGCAA...pairs...GAGGAGCGTCG...pairs...AGAA S 2 to S 0, read 0, print 2 CTCGTT...8 base...CTCCTCGCAGC...3 base AAGAGCAA...pairs...GAGGAGCGTCG...pairs...ATAA S 2 to S 1, read 1, print 2 CTCGTT...8 base...CTCCTCGCAGC...2 base AAGAGCAA...pairs...GAGGAGCGTCG...pairs...GCAA S 2 to S 2, read 2, print 2 Divide-by-three Computation First Ligation

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AATTCGGCCGTTAGTCTT...8 base...CTCCTCGCAGCCTCGTTAGTCTTAGTCTTTGCTGAAATT TTAAGCCGGCAATCAGAA...pairs...GAGGAGCGTCGGAGCAATCAGAATCAGAAACGACTTTAA AGTCTT...8 base...CTCCTCGCAGC...2 base AATCAGAA...pairs...GAGGAGCGTCG...pairs...TCAG S 0 to S 0, read 0, print 0 AGTCTT...8 base...CTCCTCGCAGC...1 base AATCAGAA...pairs...GAGGAGCGTCG...pairs...CCAT S 0 to S 1, read 1, print 0 AGTCTT...8 base...CTCCTCGCAGC AATCAGAA...pairs...GAGGAGCGTCGGAGC S 0 to S 2, read 2, print 0 GGTATT...8 base...CTCCTCGCAGC...3 base AACCATAA...pairs...GAGGAGCGTCG...pairs...CAGA S 1 to S 0, read 0, print 1 GGTATT...8 base...CTCCTCGCAGC...2 base AACCATAA...pairs...GAGGAGCGTCG...pairs...CATA S 1 to S 1, read 1, print 1 GGTATT...8 base...CTCCTCGCAGC...1 base AACCATAA...pairs...GAGGAGCGTCG...pairs...AGCA S 1 to S 2, read 2, print 1 CTCGTT...8 base...CTCCTCGCAGC...4 base AAGAGCAA...pairs...GAGGAGCGTCG...pairs...AGAA S 2 to S 0, read 0, print 2 CTCGTT...8 base...CTCCTCGCAGC...3 base AAGAGCAA...pairs...GAGGAGCGTCG...pairs...ATAA S 2 to S 1, read 1, print 2 CTCGTT...8 base...CTCCTCGCAGC...2 base AAGAGCAA...pairs...GAGGAGCGTCG...pairs...GCAA S 2 to S 2, read 2, print 2 Divide-by-three Computation First Ligation AATTCGGCCGTTAGTCTT...8 base...CTCCTCGCAGCCTCGTTAGTCTTAGTCTTTGCTGAAATT TTAAGCCGGCAATCAGAA...pairs...GAGGAGCGTCGGAGCAATCAGAATCAGAAACGACTTTAA

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AATTCGGCCGTTAGTCTT...8 base...CTCCTCGCAGCCTCGTTAGTCTTAGTCTTTGCTGAAATT TTAAGCCGGCAATCAGAA...pairs...GAGGAGCGTCGGAGCAATCAGAATCAGAAACGACTTTAA Continue cycle of restriction, hybridization, and ligation until terminator is cleaved AATTCGGCCGTTAGTCTT...8 base...CTCCTCGCAGCCTCGTTAGTCTTAGTCTTTGCTGAAATT TTAAGCCGGCAATCAGAA...pairs...GAGGAGCGTCGGAGCAATCAGAATCAGAAACGACTTTAA

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AATTCGGCCGTTAGTCTTCTCGTTAGTCTT TGCTGAAATT TTAAGCCGGCAATCAGAAGAGCAATCAG CTTTAA Divide-by-three Computation Final Restriction

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AATTCGGCCGTTAGTCTTCTCGTTAGTCTT TGCTGAAATT TTAAGCCGGCAATCAGAAGAGCAATCAG CTTTAA TGCTGA...Reporter... AAACGACT....Gene 0....ACGA Detection Molecule Divide-by-three Computation Final Ligation

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AATTCGGCCGTTAGTCTTCTCGTTAGTCTTTGCTGA...Reporter...TGCTGAAATT TTAAGCCGGCAATCAGAAGAGCAATCAGAAACGACT....Gene 0....ACGACTTTAA This transducer has printed 020, which is 6 in base ten (6 10 ). Check: 18/3 = 6? Yes. Divide-by-three Computation Final Ligation

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AATTCGGCCGTTAGTCTTCTCGTTAGTCTTTGCTGA...Reporter...TGCTGAAATT TTAAGCCGGCAATCAGAAGAGCAATCAGAAACGACT....Gene 0....ACGACTTTAA Biological Function 0 Biological Function 0 could be releasing a drug, changing the bacteria phenotype, etc.

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AATTCGGCCGTTAGTCTTCTCGTTAGTCTTTGCTGA...Reporter...TGCTGAAATT TTAAGCCGGCAATCAGAAGAGCAATCAGAAACGACT....Gene 0....ACGACTTTAA Divide-by-two Computation Transition Stage A third restriction enzyme that cleaves within its recognition site is necessary only when consecutive computation (using two separate transducers) occurs.

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AATTC GGCCGTTAGTCTTCTCGTTAGTCTTTGCTGA...Reporter...TGCTGAAATT TTAAGCCGG CAATCAGAAGAGCAATCAGAAACGACT....Gene 0....ACGACTTTAA GGCCTTTCTCCTCGCAGCT AAAGAGGAGCGTCGACCGG Reinsertion Molecule Divide-by-two Computation Transition Stage Reinsertion of the recognition sites is also required for consecutive computation.

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AATTCGGCCTTTCTCCTCGCAGCTGGCCGTTAGTCTTCTCGTTAGTCTTTGCTGA...Reporter...TGCTGAAATT TTAAGCCGGAAAGAGGAGCGTCGACCGGCAATCAGAAGAGCAATCAGAAACGACT....Gene 0....ACGACTTTAA Divide-by-two Computation Transition Stage

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AATTCGGCCTTTCTCCTCGCAGCTGGCCGTTAGTCTTCTCGTTAGTCTTTGCTGA...Reporter...TGCTGAAATT TTAAGCCGGAAAGAGGAGCGTCGACCGGCAATCAGAAGAGCAATCAGAAACGACT....Gene 0....ACGACTTTAA Entire cycle repeats again until terminator is cleaved once more Divide-by-two Computation

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AATTAGTCTTGGTATTAGTCTT TGCTGA...Reporter...TGCTGAAATT TTAATCAGAACCATAATCAG CT....Gene 0....ACGACTTTAA Divide-by-two Computation Final Restriction

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AATTAGTCTTGGTATTAGTCTT TGCTGA...Reporter...TGCTGAAATT TTAATCAGAACCATAATCAG CT....Gene 0....ACGACTTTAA TGCTGA...Reporter... AAACGACT....Gene 0....ACGA Detection Molecule Divide-by-two Computation Final Ligation

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AATTAGTCTTGGTATTAGTCTTTGCTGA...Reporter...TGCTGA...Reporter...TGCTGAAATT TTAATCAGAACCATAATCAGAAACGACT....Gene 0....ACGACT....Gene 0....ACGACTTTAA Divide-by-two Computation Final Ligation (3 10 ) This transducer has printed 010, which is 3 in base ten. Check: (18/3)/2 = 3? Yes.

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AATTAGTCTTGGTATTAGTCTTTGCTGA...Reporter...TGCTGA...Reporter...TGCTGAAATT TTAATCAGAACCATAATCAGAAACGACT....Gene 0....ACGACT....Gene 0....ACGACTTTAA Biological Function 0

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Discussion & Conclusions This project worked as expected. 18 ÷ 3= 6 ; 6 ÷ 2= 3 No molecule encoded the recognition site of an enzyme Proof of concept worked however not done in practicality. Transducers engineered functioned as coded

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Acknowledgements We would like to sincerely thank Mr. Russell N. Stern for his generosity and donation. We would like to sincerely thank Mr. Russell N. Stern for his generosity and donation. Thank you to the Louis Herman Israel Experience Fund for their contribution. Thank you to the Louis Herman Israel Experience Fund for their contribution. We would also like to thank our mentor Tamar Ratner, for her continued dedication and help. We would also like to thank our mentor Tamar Ratner, for her continued dedication and help. Finally, we would like to thank Professor Ehud Keinan for allowing us to use his laboratory and his student. Finally, we would like to thank Professor Ehud Keinan for allowing us to use his laboratory and his student.

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