A Suicide Plasmid System for Targeted Gene Deletion in Shewanella oneidensis MR-1 utilizing a cre-lox Based Recombination Method. ANGELA REEVELY (Tuskegee University Tuskegee, AL 36088) SOUMITRA BARUA (Oak Ridge National Laboratory, Oak Ridge, TN, 37831)

The versatile respiratory capabilities of the Gram-negative facultative anaerobic bacterium Shewanella oneidensis MR-1 cause it to be at the height of environmental interest for purposes of developing new bioremediation strategies for removing toxic chemicals and pollutants from groundwater. S. oneidensis is a metal-ion reducing bacteria that can convert nitrate to ammonium and soluble forms of Uranium and Chromium to a reduced and insoluble form. The Shewanella Federation, which includes the DOE's Oak Ridge National Laboratory, is presently conducting a comprehensive study of the sequenced genome S. oneidensis. The protocol for research included setting up a suicide plasmid system for targeted gene deletion inS. oneidensis MR-1 utilizing a cre-lox based recombination method. A pJK102 suicide plasmid was prepared involving PCR amplification and cloning of the N' and C'- terminal flanking regions of the targeted deleted gene in separate reactions. PCR purified restriction digests of both the N'and C'-terminal inserts were ligated in the respective restriction enzyme digested pJK100 in separated reactions. Screenings of Km^R/Tet^R colonies of Escherichia coli containing pJK102 suicide plasmids with the correct size insert were tested and reviewed by PCR. This suicide plasmid was then moved from the DAP (diaminopimelic acid) auxotroph E. coli to S. oneidensis MR-1 through conjugation. The resultant MR-1 strain is Km^R/Tet^S which contains the loxP-Km^R-loxP cassette. Then another cre-recombinase enzyme producing plasmid, pCM157/Tet^R was moved into the MR-1 strain to resolve the loxP-Km^R-loxP cassette through conjugation. Finally, the Tet^R plasmid pCM157 was cured from the S. oneidensis MR-1 by conducting a three to four day continuous culture in non-selective rich Luria-Bertani medium. The c-type cytochrome genes of MR-1 were targeted and obtained mutant strains of SO0479 and SO4484. These constructs will be used to explore the versatile metabolic capability of this organism. The goal of this research is to understand the function of these multiple c-type cytochrome genes of S. oneidensis. Future efforts will continue to correlate the function of these c-type cytochrome genes on bioremediation strategies for removing toxic chemicals and pollutants from groundwater.

Anaerobic Co-Cultures of Purple Non-Sulfur Photosynthetic Bacterium Rhodopseudomonas palustris and Metal-Reducing Bacterium Shewanella oneidensis. WILLIAM DABBS (University of Tennessee Knoxville, TN 37996) ABHIJEET P. BOROLE (Oak Ridge National Laboratory, Oak Ridge, TN, 37831)

Rhodopseudomonas palustris is a metabolically versatile organism whose genome was recently sequenced. R. palustris is capable of fixing nitrogen, sequestering carbon dioxide (greenhouse gas reduction) and producing hydrogen gas (alternate energy solution). It is also capable of growing under different metabolic states, namely photoheterotrophic, photoautotrophic, chemoheterotrophic and chemoautotrophic. Under these different metabolic states, the organism produces different proteins to carry out various functions. These proteins act as markers for identifying the metabolic state of the bacterium using Proteomics. Therefore, by knowing the metabolic status of R. palustris, in theory, one could infer the status of an entire community. Due to R. palustris' versatility and familiarity, it has been selected by a group at ORNL to be used as a sentinel organism in a study of microbial communities. The goal of this specific study is to examine the possibility of designing a minimal medium to grow an anaerobic co-culture of R. palustris and Shewanella oneidensis. S. oneidensis is of interest to DOE due to its metal reducing ability which could aid in the cleanup of heavy metals. One major concern, while designing this medium, is ensuring each organism has an optimal carbon source for growth. On its own, R. palustris has had positive results by using both succinate and acetate as a carbon source. In studies with lactate as a carbon source and fumarate as an electron acceptor, S. oneidensis has grown well reducing fumarate to succinate and partially oxidizing lactate to acetate. Therefore, there is a possibility that R. palustris can utilize the succinate or acetate produced by S. oneidensis for its own benefit. This may mean that S. oneidensis could supply the carbon source for R. palustris for this experiment without one being provided in the medium. R. palustris and S. oneidensis have been grown in separate anaerobic cultures in a variety of different media. The results thus far, for R. palustris, have been concordant with the predicted results, and it has grown optimally in the medium with succinate. Testing the growth of R. palustris and S. oneidensis will be the final step in creating the anaerobic medium which will support the co-culture of R. palustris and S. oneidensis.

Detection of Hydrogen Peroxide (H2O2) in a Sol-Gel Embedded Amplex Red/ Horse Radish Peroxidase (HRP) Solution Using a Miniature Fluorescence Spectrometer. MARK FISHER (Yale University New Haven, CT 06520) GUY GRIFFIN (Oak Ridge National Laboratory, Oak Ridge, TN, 37831)

Under appropriate conditions, the non-fluorescent compound amplex red is selectively oxidized by H2O2, the posited precursor of other reactive oxygen species believed to be involved in cellular inflammation and damage, in the presence of HRP catalyst to form the fluorescent dye resorufin. Entrapment of the dye /enzyme (Amplex Red/ HRP) molecules in a matrix enables their better-controlled localization and increased robustness in non-ideal conditions. Based on a fluorescence intensity measurement and data from a calibrated concentration curve, concentrations of H2O2 in an unknown solution can be determined. A sol stock was prepared by stirring a mixture of tetramethylorthosilicate (TMOS), water, and 0.1M HCl for three hours. The dye solution was prepared by mixing 50 mM phosphate buffer with HRP (10 U/mL) and 10 mM amplex red, after which 0.125 mL was vortexed with 0.125 mL of the sol stock. The mixture was then pipetted into a test tube, allowed to solidify on the side of a cuvette, or coated on a polished 800 nm fiber. After being rinsed or dipped in water for one minute, the gel was dried for 24 or 48 hours in darkness at ambient temperature. Concentrations of H2O2 ranging from 0.5 uM to 10 uM were introduced to the sol-gel tubes in 0.25 mL aliquots and were allowed to diffuse for an hour in darkness at room temperature. The intensity of fluorescence for each tube was then measured. A similar process was repeated using 1 mL of various concentrations of H2O2 for ten minutes on the cuvette slides. A few droplets of 0.5 uM H2O2 were added to the tips of the fibers, and the fluorescence intensity was again measured. The results so far suggest that sol-gel encapsulation of the dye does not inhibit the linear relationship between H2O2 concentration and the fluorescence intensity of resorufin in the tubes or cuvettes. The intensities are overall lower than those measured in liquid solution, but this is most likely caused by the kinetics of H2O2 diffusion through the sol-gel and the decrease in volume of Amplex Red inherent in the sol-gel encapsulation process. In the cases of the fibers, detection of resorufin has been achieved, but no linear relationship has yet been established. Resorufin detection has also proved to be extremely sensitive to drying time of the sol-gel. The long-term goal of the project is to coat a pulled nano-scale fiber with the sol-gel dye matrix and use it as a reagentless optic sensor for intracellular detection of hydrogen peroxide.

Development of Corrugated Patterning of CVD Diamond Foils. VASHTI DAVIS (Grinnell College Grinnell, IA 50112) ROBERT SHAW (Oak Ridge National Laboratory, Oak Ridge, TN, 37831)

Stripper foils are used in the technology of linear accelerators to change a negative ion beam to an ion beam that is positively charged. Most commonly, carbon stripper foils are employed. However the Spallation Neutron Source (SNS) at Oak Ridge National Laboratory requires a foil that can withstand a 1GeV beam at 10's of mA, which is beyond the capability of carbon foils. Research investigating the use of diamond stripper foils found that the material could meet the 1GeV requirement. In order to use diamond foils in the SNS the diamond stripper foil is suspended into the beam by only a one edge handle and must be 12mm x 20 mm. Unfortunately the stress created by the film growth process causes the diamond foil to curl. Research was conducted to find a method to keep the foil lying flat. A silicon etching process was used to create corrugations in the diamond foils, making the film lie flat. Variations of several patterns in the corrugations were investigated to determine the best design for keeping the foil lying flat and also maintaining the integrity of the diamond film. Patterns resembling a horseshoe shape with various radii of curvature and frequency of corrugations were investigated. When comparing patterns, preliminary results indicated that 100 corrugations per inch produce the best results. Furthermore, the pattern that maintained the desired results on a regular basis was designed with a radius of curvature of 80 mils and only a 40% corrugation coverage of the film. Ideally the corrugations will cover only a small amount of the diamond foil, so the results that have been initially observed are well accepted. As part of investigation into the use of diamond stripper foils, research will continue to explore the possibilities of nanocrystalline technology to further the lifetime of stripper foils used at linear accelerators.

Parallel Computation of Blood Flow in Arteries with Hyperelastic Walls. HANNAH FLATH (Columbia University New York, NY 10027) RICHARD WARD (Oak Ridge National Laboratory, Oak Ridge, TN, 37831)

A computational model of realistic blood flow in a section of artery could improve diagnosis and treatment of health problems such as aneurysms and wounds to artery vessel walls. Systems of equations describing fluid and wall behavior are numerically simulated in PICMSS (Parallel Interoperable Computational Mechanics System Simulator), a parallel computational software environment for solving equations using finite element analysis. Models currently under investigation are linearized versions of systems published by A. Quarteroni et al. in 2001 and in the 1998 Thesis of M. S. Olufsen. These systems include Navier-Stokes and incompressible fluid equations coupled through boundary conditions to wall motion. A linear response of the wall to pulsatile blood flow is currently being incorporated, but actual vessel walls have a nonlinear viscoelastic response which will be approximated as hyperelastic later in the project. During the summer SULI term, verification and development of the model will continue, including 1) comparison to the results of Quarteroni and Olufsen, 2) hyperelastic wall motion, and 3) application to arteries with vessel wall damage.

Sampling Strategies of Populus tremuloides in Rocky Mountain National Park for Assessment of Somatic Mutations and Diversity Within Clonal Populations. TODD FLEMING (Ball State University Muncie, IN 49307) LEE GUNTER (Oak Ridge National Laboratory, Oak Ridge, TN, 37831)

Quaking aspen (Populus tremuloides Michx.), has the widest distribution of any tree species in North America, and may also have one of the longest lifespans due to its clonal propagation. Large aspen stands in the west primarily consist of clones made up of individual ramets connected by extensive root systems. Estimates of the age of some clones extend upwards of 10,000 years. There is no current way of measuring the age of these clones due the fact that individual ramets live only 100 - 150 years. Accumulated somatic mutation rates may be used to age aspen clones if a method can be developed for determining the basal genotype and thus allowing for the assessment of total mutations generated over time. Comparing Single Nucleotide Polymorphisms (SNPs) from several meristems sampled across the crown of a single ramet to isolate the most frequent meristematic genotype should allow the identification of the basal genotype, and combining genotypes for an entire stand would allow the average number of mutations within a given ramet cohort to be calculated. Investigators will travel to Rocky Mountain National Park in early August to sample leaf tissue from aspen stands. Park officials have also requested a diversity study be done on sampled aspen. Suitable stands will be selected and sampling points and distances measured prior to arrival using geographical information system software Arcmap. Meristems of ramets will be sampled individually, but the possible pooling of these samples for DNA extraction prior to SNP analysis could save thousands of hours of lab work. The process of pooling leaf sample tissue prior to extraction is being tested using individual genotypes of the hybrid cottonwood family 52124 (TD '52-225' by 'D124' ) developed by the University of Minnesota - Duluth (UMD) Natural Resources Research Institute (NRRI). In addition, the use of commercial cat litter in place of silica gel as an affordable, convenient desiccant for preserving tissue samples in the field is being tested.

Single-Cell Noise Spectrum Analysis for Gene Circuit Characterization. DAR ROY (The Hebrew University of Jerusalem Jerusalem, ISRAEL 91904) MICHAEL L. SIMPSON (Oak Ridge National Laboratory, Oak Ridge, TN, 37831)

The capability to characterize natural and engineered gene circuits could be beneficial on both academic and industrial fronts by gaining more understanding of intra- and inter-cellular biological processes and structure-function relationships. To explore the frequency distribution of stochastic processes in gene circuits, Green Fluorescent Protein (GFP) expressing gene circuits were constructed on high copy number plasmids in Escherichia Coli bacterial cells. Experiments concentrated on two different types of circuits: (1) Non-regulated gene constructs differing in protein degradation rates, and (2) Autoregulated negative-feedback gene constructs where gene expression is repressed by the binding of a repressor protein at an operator site in the promoter. Laser confocal microscopy (Leica SP2) with a 488 nm Ar source was used to acquire 4-8 hour time-series fluorescence data (500-550 nm) from excited bacterium populations. Cell doubling time was controlled by varying growth temperatures. Single-cell fluorescent output was extracted from microscopy images by tracking individual cells using computer-imaging software to calculate mean pixel intensity. GFP noise was defined as this output's deviation from the population's mean fluorescence. A common reference point from calculated biased Autocorrelation Functions (ACFs) for this time-series noise data (time lag at which the ACF decayed by ½) was used as a measure of noise bandwidth (Fnbw) for gene circuits. An increase in Fnbw with decreasing cell doubling time was observed for all the gene constructs. In addition, an increased bandwidth of about two-fold was seen for the autoregulated gene circuit as opposed to the unregulated circuit. This increase in bandwidth was predicted by a previous theoretical study for these gene circuits and is consistent with negative feedback theory. Additional spectral measurements and model interpretation may reveal significant information on enzyme kinetics, feedback mechanisms, and kinetic parameter fitting of these gene circuits. Ultimately the physical sciences approach to biological systems may be at the forefront of molecular-scale engineering and help enhance the biologist's tools for discovering underlying structure of gene circuits and networks.

Skin & Hair Development: Examining the Role of Tg737, Kif3a, & the Primary Cilium. MICHAEL MILLER (Albion College Albion, MI 49224) EDWARD J. MICHAUD (Oak Ridge National Laboratory, Oak Ridge, TN, 37831)

The primary cilium is an organelle that is found singly on the surface of most vertebrate cell types. The primary cilium functions as a cellular antenna, receiving both mechanical and chemical signals from neighboring cells. This organelle plays a critical role in the development and function of many mammalian organ systems, as exemplified by mutations in Tg737 and Kif3a, two genes needed to build the primary cilium. Previous work has also shown that mutations in the Tg737 and Kif3a genes alter the Sonic hedgehog (Shh) and Wnt signaling pathways, which are vital to the development of many organ systems, including the skin and hair follicles. Additionally, uncontrolled activation of the Shh and Wnt pathways causes a variety of cancers, including basal cell carcinoma (BCC) of the skin, which is the most common form of human cancer. Here mice with a hypomorphic mutation in Tg737 or a conditional mutation in Kif3a were utilized to examine the roles of these two genes in skin and hair follicle development. Specifically, quantitative real-time PCR (QRT-PCR) was used to examine the expression profiles of Shh and Wnt pathway genes in the skin of the mutant mice. By using a statistical analysis program and comparing each mutant mouse to its wild-type littermate control, relative gene expression levels were determined. The results demonstrate that both mutations altered Shh signaling; however, they affected the pathway at two different levels. In the Tg737 mutant, the Ptch1, Gli1, and Gli3 genes were down-regulated, but the Shh and Gli2 genes were unaffected. In the Kif3a mutant, the Shh, Gli1, and Gli3 genes were down-regulated, but the Ptch1 and Gli2 genes were unaltered. In the Wnt pathway, four different genes were studied. Catnb, an activator of the pathway, was not significantly affected in the Tg737 mutant but was down-regulated in the Kif3a mutant. Lef1, a repressor of the pathway, was down-regulated in both mutants. However, Ccnd1 and Myc, two genes activated by the Wnt pathway, were normal in both mutants. Taken together, these results demonstrate that the Shh and Wnt pathways are altered in the skin of Tg737 and Kif3a mutant mice. As the next step, in situ hybridization, immunohistochemistry, and immunofluorescence microscopy will be performed to complement the present results. Ultimately, the goal of this study is to determine the function of primary cilia in skin and hair follicle development, as well as the mechanisms involved in BCC.