Network-Scale Engineering: Systems Approaches to Synthetic Biology

The field of Synthetic Biology seeks to develop engineering principles for biological systems. Modular biological parts are repurposed and recombined to develop new synthetic biological devices with novel functions. The proper functioning of these devices is dependent on the cellular context provided by the host organism, and the interaction of these devices with host systems. The field of Systems Biology seeks to measure and model the properties of biological phenomena at the network scale. We present the application of systems biology approaches to synthetic biology, with particular emphasis on understanding and remodeling metabolic networks. Chapter 2 demonstrates the use of a Flux Balance Analysis model of the Saccharomyces cerevisiae metabolic network to identify and construct strains of S. cerevisiae that produced increased amounts of formic acid. Chapter 3 describes the development of synthetic metabolic pathways in Escherichia coli for the production of hydrogen, and a directed evolution strategy for hydrogenase enzyme improvement. Chapter 4 introduces the use of metabolomic profiling to investigate the role of circadian regulation in the metabolic network of the photoautotrophic cyanobacterium Synechococcus elongatus PCC 7942. Together, this work demonstrates the utility of network-scale approaches to understanding biological systems, and presents novel strategies for engineering metabolism

A Search for Nitrogen Enriched Quasars in the Sloan Digital Sky Survey Early Data Release

A search for nitrogen-rich quasars in the Sloan Digital Sky Survey Early Data Release (SDSS EDR) catalog has yielded 16 candidates, including five with very prominent emission, but no cases with nitrogen emission as strong as in Q0353-383. The quasar Q0353-383 has long been known to have extremely strong nitrogen intercombination lines at lambda 1486 and lambda 1750 Angstroms, implying an anomalously high nitrogen abundance of about 15 times solar. It is still the only one of its kind known. A preliminary search through the EDR using the observed property of the weak C IV emission seen in Q0353-383 resulted in a sample of 23 objects with unusual emission or absorption-line properties, including one very luminous redshift 2.5 star-forming galaxy. We present descriptions, preliminary emission-line measurements, and spectra for all the objects discussed here.Comment: 20 pages, 5 figures, submitted to AJ; final refereed versio

The BTC40 Survey for Quasars at 4.8 < z < 6

The BTC40 Survey for high-redshift quasars is a multicolor search using images obtained with the Big Throughput Camera (BTC) on the CTIO 4-m telescope in V, I, and z filters to search for quasars at redshifts of 4.8 < z < 6. The survey covers 40 sq. deg. in B, V, & I and 36 sq. deg. in z. Limiting magnitudes (3 sigma) reach to V = 24.6, I = 22.9 and z = 22.9. We used the (V-I) vs. (I-z) two-color diagram to select high-redshift quasar candidates from the objects classified as point sources in the imaging data. Follow-up spectroscopy with the AAT and CTIO 4-m telescopes of candidates having I < 21.5 has yielded two quasars with redshifts of z = 4.6 and z = 4.8 as well as four emission line galaxies with z = 0.6. Fainter candidates have been identified down to I = 22 for future spectroscopy on 8-m class telescopes.Comment: 27 pages, 8 figures; Accepted for publication in the Astronomical Journa

A dominant mutation in a neuronal acetylcholine receptor subunit leads to motor neuron degeneration in Caenorhabditis elegans

Inappropriate or excessive activation of ionotropic receptors can have dramatic consequences for neuronal function and, in many instances, leads to cell death. In Caenorhabditis elegans, nicotinic acetylcholine receptor (nAChR) subunits are highly expressed in a neural circuit that controls movement. Here, we show that heteromeric nAChRs containing the acr-2 subunit are diffusely localized in the processes of excitatory motor neurons and act to modulate motor neuron activity. Excessive signaling through these receptors leads to cell-autonomous degeneration of cholinergic motor neurons and paralysis. C. elegans double mutants lacking calreticulin and calnexin-two genes previously implicated in the cellular events leading to necrotic-like cell death (Xu et al. 2001)-are resistant to nAChR-mediated toxicity and possess normal numbers of motor neuron cell bodies. Nonetheless, excess nAChR activation leads to progressive destabilization of the motor neuron processes and, ultimately, paralysis in these animals. Our results provide new evidence that chronic activation of ionotropic receptors can have devastating degenerative effects in neurons and reveal that ion channel-mediated toxicity may have distinct consequences in neuronal cell bodies and processes

A synthetic system links FeFe-hydrogenases to essential E. coli sulfur metabolism

<p>Abstract</p> <p>Background</p> <p>FeFe-hydrogenases are the most active class of H₂-producing enzymes known in nature and may have important applications in clean H₂energy production. Many potential uses are currently complicated by a crucial weakness: the active sites of all known FeFe-hydrogenases are irreversibly inactivated by O₂.</p> <p>Results</p> <p>We have developed a synthetic metabolic pathway in <it>E. coli </it>that links FeFe-hydrogenase activity to the production of the essential amino acid cysteine. Our design includes a complementary host strain whose endogenous redox pool is insulated from the synthetic metabolic pathway. Host viability on a selective medium requires hydrogenase expression, and moderate O₂levels eliminate growth. This pathway forms the basis for a genetic selection for O₂tolerance. Genetically selected hydrogenases did not show improved stability in O₂and in many cases had lost H₂production activity. The isolated mutations cluster significantly on charged surface residues, suggesting the evolution of binding surfaces that may accelerate hydrogenase electron transfer.</p> <p>Conclusions</p> <p>Rational design can optimize a fully heterologous three-component pathway to provide an essential metabolic flux while remaining insulated from the endogenous redox pool. We have developed a number of convenient <it>in vivo </it>assays to aid in the engineering of synthetic H₂metabolism. Our results also indicate a H₂-independent redox activity in three different FeFe-hydrogenases, with implications for the future directed evolution of H₂-activating catalysts.</p

Insulation of a synthetic hydrogen metabolism circuit in bacteria

<p>Abstract</p> <p>Background</p> <p>The engineering of metabolism holds tremendous promise for the production of desirable metabolites, particularly alternative fuels and other highly reduced molecules. Engineering approaches must redirect the transfer of chemical reducing equivalents, preventing these electrons from being lost to general cellular metabolism. This is especially the case for high energy electrons stored in iron-sulfur clusters within proteins, which are readily transferred when two such clusters are brought in close proximity. Iron sulfur proteins therefore require mechanisms to ensure interaction between proper partners, analogous to many signal transduction proteins. While there has been progress in the isolation of engineered metabolic pathways in recent years, the design of insulated electron metabolism circuits <it>in vivo </it>has not been pursued.</p> <p>Results</p> <p>Here we show that a synthetic hydrogen-producing electron transfer circuit in <it>Escherichia coli </it>can be insulated from existing cellular metabolism via multiple approaches, in many cases improving the function of the pathway. Our circuit is composed of heterologously expressed [Fe-Fe]-hydrogenase, ferredoxin, and pyruvate-ferredoxin oxidoreductase (PFOR), allowing the production of hydrogen gas to be coupled to the breakdown of glucose. We show that this synthetic pathway can be insulated through the deletion of competing reactions, rational engineering of protein interaction surfaces, direct protein fusion of interacting partners, and co-localization of pathway components on heterologous protein scaffolds.</p> <p>Conclusions</p> <p>Through the construction and characterization of a synthetic metabolic circuit <it>in vivo</it>, we demonstrate a novel system that allows for predictable engineering of an insulated electron transfer pathway. The development of this system demonstrates working principles for the optimization of engineered pathways for alternative energy production, as well as for understanding how electron transfer between proteins is controlled.</p

Predicting Cochlear Implant Electrode Placement Using Monopolar, Three-Point and Four-Point Impedance Measurements

Objective: This study aimed to investigate the relationship between cochlear implant (CI) electrode distances to the cochleas inner wall (the modiolus) and electrical impedance measurements made at the CIs electrode contacts. We introduced a protocol for three-point impedances in which we recorded bipolar impedances in response to monopolar stimulation at a neighboring electrode. We aimed to assess the usability of three-point impedances and two existing CI impedance measurement methods (monopolar and four-point impedances) for predicting electrode positioning during CI insertion. Methods: Impedances were recorded during stepwise CI electrode array insertions in cadaveric human temporal bones. The positioning of the electrodes with respect to the modiolus was assessed at each step using cone beam computed tomography. Linear mixed regression analysis was performed to assess the relationship between the impedances and electrode-modiolar distances. The experimental results were compared to clinical impedance data and to an existing lumped-element model of an implanted CI. Results: Three-point and four-point impedances strongly correlated with electrode-modiolar distance. In contrast, monopolar impedances were only minimally affected by changes in electrode positioning with respect to the modiolus. An overall model specificity of 62% was achieved when incorporating all impedance parameters. This specificity could be increased beyond 73% when prior expectations of electrode positioning were incorporated in the model. Conclusion: Three-point and four-point impedances are promising measures to predict electrode-modiolar distance in real-time during CI insertion. Significance: This work shows how electrical impedance measurements can be used to predict the CIs electrode positioning in a biologically realistic model

Quasar Candidates in the Hubble Deep Field

We focus on the search for unresolved faint quasars and AGN in the crude combine images using a multicolor imaging analysis that has proven very successful in recent years. Quasar selection was carried out both in multicolor space and in "profile space," defined as the multi-parameter space formed by the radial profiles of the objects in the different images. By combining the dither frames available for each filter, we were able to obtain well-sampled radial profiles of the objects and measure their deviation from that of a stellar source. We also generated synthetic quasar spectra in the range 1.0 < z < 5.5 and computed expected quasar colors. We determined that the data are 90% complete for point sources at 26.2, 28.0, 27.8, 26.8 in the F300W, F450W, F606W and F814W filters, respectively. We find 41 compact objects in the HDF: 8 pointlike objects with colors consistent with quasars or stars, 18 stars, and 15 slightly resolved objects, 12 of which have colors consistent with quasars or stars. We estimate the upper limit of unresolved and slightly resolved quasars/AGNs with V < 27.0 and z < 3.5 to be 20 objects (16,200 per deg^2). We find good agreement among authors on the number of stars and the lack of quasar candidates with z > 3.5. We find more quasar candidates than previous work because of our more extensive modeling and use of all of the available color information. (abridged)Comment: We have clarified our discussion and conclusions, added some references and removed the appendix, which is now available from the first author. 37 pages including 10 embedded postscript figures and 6 tables. To appear in the Feb. 99 issue of A