аЯрЁБс > ўџ D F ўџџџ C џџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџџьЅС q П л. bjbjt+t+ -@ A A Ш* џџ џџ џџ ] В В В В 8 ъ і , В Ў V 6 6 L L L ' ' ' а в в в ; Д С Д u $ є ј! | ' ' ' ' ' O L L л 6 O O O ' ( L L а В В ' а O в O ! X h а L " @/]qУВ В O Р Project Summary for Fiscal Year (FY) 2003 project 12-Jul-2002 to 11-Jul-2003 Thomas.Renz@rl.af.mil, Robert.Kaminski@rl.af.mil, skumar@darpa.mil, jmartin@snap.org 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/ Quad Chart: http://arep.med.harvard.edu/darpabiocomp/ChurchQ01.ppt Objective: "DNA computing" so far has focused on computing calculations with DNA (which is slow relative to silicon computing). This project, in contrast, explores the other aspects of computing (i.e. input/output, memory & manufacturing processes). We do this using the only class of programmable nanometer scale replicators (i.e. polymerase-ribosome-based). 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, protein, 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 have an in vitro coupled replicating and translating system based on bacterial E.coli translation extracts. For this we have developed (1) a linear expression clone system compatible with the most powerful in vitro replication system (PCR). (2) a modular method for computer gene design and automated gene synthesis including affinity-tagging for all ribosomal proteins.
We have all 22 of the 30S subunit RNAs synthesized and most of the proteins. The remainder have revealed surprising properties of these most abundant cell proteins and suggest a strategy for overcoming the initial recalcitrance.
In collaboration with Dr. Olejnik at Ambergen we have tested photocleavable fluorophore-base connection and are trying nitrobenzyl-3' blocking groups. In addition Dr. Pirrung from Duke University has sent NPPOC-3'blocked-dTTP which we (Jay Shendure and Greg Porreca) are testing. We have developed a design for modifying the DNA polymerase which we use to accomodate these bulky reversible 3' blocking group. It is becoming evident that steric hindrance of these 3' blockers has thwarted other research groups in the past from making progress and engineering the polymerase is an important and novel approach.
We have developed a Minimization of Metabolic Adjustment (MOMA) 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.
We have automated and made SBML & BioSpice compatible versions of MOMA, plus related web resources.
We have developed methods for 3D & 4D modeling of bacterial cells and replication translation of their circular chromosomes. In addition we have 1D to 4D models of expansion of an in vitro DNA colony.
We have completed genome sequencing for Mycoplasma mobile and a "complete" proteome comparison of M. mobile and M. pneumoniae. These are proving crucial for integration and 4D-modeling efforts. Current Plan:
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: Jay Shendure 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