ࡱ > / 1 . 7 bjbjUU "* 7| 7| l h h h h t > ~ $ = ] X R R R R ~ R ~ R R r r @) h R r r 0 r R r R Project Summary for Fiscal Year (FY) 2002 project 28-Sep-2001 to 12-Jul-2002 Technical Report Input Fields Principal Investigator: Firstname: George Lastname: Church Address 1: 200 Longwood Avenue Address 2: Harvard Medical School City: Boston State: MA Zip: 02115 Phone:617-432-7562 Fax: 617-432-7663 Email: church@arep.med.harvard.edu Level Of Participation - Billed: 10% Unbilled: 20% Project URL: http://arep.med.harvard.edu/darpabiocomp/ Objective: "DNA computing" so far has focused on computing (which is slow relative to silicon computing). This project, in contrast, explores one of the few working sub-nanometer input/output, memory & manufacturing processes. The major challenge is integration with silicon computing while maintaining the nano-size advantages. The motivations are unattended bio-monitoring, 3D memory arrays cubic-nanometer per bit (currently 100 trillion), input from light, chemicals, and toxins; output as nm-scale positioning. Approach: The project focuses on real-world Input/Output issues including analog-to-digital (A/D) and digital-to-analog (D/A). For system input, we harvest a diverse set of biochemical and biophysical (photon) sensors. We also propose a novel fusion of DNA and RNA polymerases to decouple positioning from synthesis. For output we use polymerases for positioning mechanical effectors and hence rapidly synthesize three-dimensionally complex patterns of DNA and/or electro-optical (EO) computer circuits. This should be compatible with the DNA-bit programming done in the system input
In order to improve the performance of the fabrication and memory tools, we will develop in vitro replication/translation arrays for experimental feedback. We will design a 90kbp minigenome capable of replication and protein-synthesis. This minigenome will be 6 times smaller than the smallest living cellular genomes, and display up to 800-fold faster replication, with 1000-fold fewer molecular components. These in vitro systems are ideal for integrating with detailed computational models, due to simplicity, knowledge of the 3D structure of nearly all components and extreme experimental accessibility. Also coupling the extremes of modeling (from single base changes to 3D structures to molecular networks to population doubling selection) is likely to be dramatically more transparent and tractable.
Novel, Useful Applications & technology transfer We will focus from the start on practical applications that take advantage of the unique features of DNA rather than competing head-on with EO. Examples are: (a) proven information archiving and retrieval (up to a billion years as mineralized fossils or living DNA records); (b) interfacing with biochemical, photon, or thermal sensors. (c) A DNA recorder analogous to black-box flight recorder would take early advantage of our ability to record on DNA more easily than reading it. Only rarely would the archived materials be accessed. (d) Polymerases take 0.34 nm steps under control of available dNTPs. Novel methods for separating the positioning from the incorporation of reactive bases will allow nanofabrication. Recent Accomplishments: We demonstrated a in vitro coupled replication/transcription/translation of a We have now demonstrated this for a poly-GFP construct reporter using E.coli translation extracts. We are working with expression clones for all 21 ribosomal 30S proteins and DnaK chaperone(in collaboration with Gloria Culver). We have constructed one drug-resistant rRNA and are constructing an affinity-tagged protein.
We have tested pure error-prone polymerases E. coli Pol IV mutant and the DinB homologue (Dbh) from Sulfolobus solfataricus (in collaboration with Tom Ellenberger & Laura Silvian). In assessing them for incorporation of mismatched dNTPs, we observed high activity on templateless primers and are exploiting this activity. We have demonstrated use of photocleavable (nitrophenylethyl) linkages between nucleotides and fluorescent groups. We will next combine these two strategies.
We have developed a Minimum Perturbation Analysis software for optimization of metabolic network utilization in mutant genotypes. We have tested in extensively using metabolic fluxes (from Uwe Sauers group) and a new high-throughput method for measuring growth rates of hundreds of mutants in parallel. Current Plan: In FY2003, we will synthesize arrays of DNAs & polymerases for use with in situ sequencing in gels & chemically & photochemically cleavable links between fluorescent markers and dNTPs. This is critical to querying DNA memory systems in general as well as a variety of commercially established genomics applications.
We will extend our work on error-prone polymerases to synthesize stretches longer than 10 bp. Polony DNA fluorescent-base extension will be used for output. This would constitute proof of the key input and output methods.
We will assess ways to use synthetic genomes to program in vitro synthesis and assembly of small ribosomal subunits. This will allow development and optimization of commercially useful protein expression and display systems. Technology Transition: How the impact of this work is measured: Citations and milestones of licensees set by HMS OTL.
Prototype available for dissemination: In situ fluorescent base extension. Purpose: Identity and quantitation based on single DNA molecules. Environment requirements: Research laboratory. Point of contact / email address: Rob Mitra System available for dissemination: Minimum Perturbation Analysis. Purpose: Optimization of metabolic network utilization in engineered (or mutant) genomes. Environment requirements: Research computers supporting Perl & C.
Point of contact / email address: Daniel Segre