【正文】 0 – Ambitious prediction of nottoodistant future (Church et al, 2023): $$29 Synthesis lengths are increasing Commercial DNA Synthesis Companies Data Source: Rob Carlson, U of W, Seattle Bioneer South Korea Cinnagen Tehran, Iran Takara Biosciences Dalian, China Inqaba Biotec Pretoria, South Africa Fermentas Vilnius, Lithuania Bio ST, Alpha DNA, Biocorp Montreal, Canada GENEART Regensberg, Germany MWG Bangalore, India Zelinsky Institute Moscow, Russia ScinoPharm Shanhua, Taiwan Genosphere Paris, France Biolegio Malden, Netherlands Ambion Austin, Texas Biosearch Novato, California BioSynthesis Lewisville, Texas Chemgenes Wilmington, Mass. BioSpring Frankfurt am Main, Germany Biosource Camarillo, CA Dharmacon Lafaette, Co. CyberGene AB Novum, Sweden Cortec DNA Kingston, Ontario, CA Eurogentec Belgium, . DNA Technology Aarhus, Denmark Genemed Synthesis S. San Francisco, CA DNA Menlo Park, CA Metabion Munich, Germany Microsynth Balgach, Switzerland Japan Bio Services Japan Blue Heron Biotechnology Bothell, WA Geneworks Adelaide, Australia Imperial BioMedic Chandigarh, India Bioserve Biotechnologies Hyderabad, India Genelink Hawthorne, NY. DNA Synthesis (Caruthers method) Error Rate: 1% = 300 seconds per step Microarray oligonucleotide synthesis The power of parallelism Chipbased versus linear synthesis Oligonucleotides synthesized Singlestranded fragments of 5090 nucleotides 3’overlapping next fragment by 17 nucleotides (Tm calculated 5256176。, cooling to 56176。). Number of rounds depends on number of fragments. Carried out by PCR machine. Final step of amplification of plete gene driven by use of excess of terminal singlestranded fragments PCRbased oligo ligation In theory, the scale of synthesis is unlimited Biotechnology Research Workflow 1. Concept 2. Design / debug/ test 4. Design oligos 6. Transform 7. Test 5. Synthesize DNA 3. Run code What are the implications of DNA synthesis capacity + freedom of information? The problem: ―Dual Use‖ Research ? Dual use research includes life sciences research: ? With legitimate scientific purpose ? That may be misused to pose a biologic threat to public health and/or national security. How easy is it to get this technology? What can we do? Number of Individuals Individual’s Intent honorable dishonorable Bin Laden Geics, Inc. Disgruntled Researcher Garage BioHacker Basic Researcher Risk spectrum Basic logic circuits Borrowing from electrical engineering Protein Expression Basics ? RNA polymerase binds to promoter ? RNAP transcribes gene into messenger RNA ? Ribosome translates messenger RNA into protein Z Z Promoter Z Gene Protein Transcription RNA Polymerase DNA Translation Messenger RNA Regulation Through Repression and Induction ? Repressor proteins can bind to the promoter and block the RNA polymerase from performing transcription ? The DNA site near the promoter recognized by the repressor is called an operator ? The target gene can code for another repression protein enabling regulatory cascades Z Promoter Operator Z Gene R Gene R R R Promoter Transcription Translation DNA Binding RNA Polymerase Logic Circuits ? Proteins are the wires/signals ? Promoters + decay implement the gates ? Any finitestate digital circuit can be built ? For example, X or Y ? Z X Y R1 Z R1 R1 X Y Z = gene gene gene TranscriptionBased Inverter ? Protein concentrations are analogous to electrical current ? BUT… proteins do not function in an isolated system and need to be unique 0 1 1 0 R R Z Simple Inverter Model 00 . 20 . 40 . 60 . 810 0 . 2 0 . 4 0 . 6 0 . 8 1I n p u t P r o t e i n C o n c e n t r a t i o nOutput Protein ConcentrationR Operator Z Gene Z R Cooperativity ? Cooperative DNA binding is where the binding of one protein increases the likelihood of a second protein binding ? Cooperativity adds more nonlinearity to the system – Increases switching sensitivity – Improves robustness to noise Z Promoter Operator Z Gene R Gene R R R Promoter Transcription Translation Cooperative DNA Binding RNA Polymerase R 00 . 20 . 40 . 60 . 810 0 . 2 0 . 4 0 . 6 0 . 8 1I n p u t P r o t e i n C o n c e n t r a t i o nOutput Protein ConcetrationN o C o o pC o o pCooperative Inverter Model R R Operator Z Gene Z R BioCircuit ComputerAided Design SPICE BioSPICE steady state dynamics intercellular ? BioSPICE: a prototype biocircuit CAD tool – simulates protein and chemical concentrations – intracellular circuits, intercellular munication – single cells, small cell aggregates Geic Circuit Elements input mRNA ribosome promoter output mRNA ribosome operator translation transcription RNAp RBS RBS A BioSPICE Inverter Simulation input output repressor promoter ? They work in vivo – Flipflop (Gardner Collins, 2023) – Ring oscillator (Elowitz Leibler, 2023) ? However, cells are very plex environments – Current modeling techniques poorly predict behavior “ Proof of Concept‖ Circuits time (x100 sec) [A] [C] [B] B _ S _ R A _ [R] [B] _ [S] [A] time (x100 sec) time (x100 sec) RSLatch (“flipflop”) Ring oscillator Cellular Logic Summary ? Current systems are limited to less than a dozen gates – Three inverter ring oscillator (Elowitz, 2023) – RS latch (Gardner, 2023) – Intercell munication (Weiss, 2023) ? A natural repressorbased logic technology presents ser