Timing molecular motion and production with a synthetic transcriptional clock

The realization of artificial biochemical reaction networks with unique functionality is one of the main challenges for the development of synthetic biology. Due to the reduced number of components, biochemical circuits constructed in vitro promise to be more amenable to systematic design and quantitative assessment than circuits embedded within living organisms. To make good on that promise, effective methods for composing subsystems into larger systems are needed. Here we used an artificial biochemical oscillator based on in vitro transcription and RNA degradation reactions to drive a variety of “load” processes such as the operation of a DNA-based nanomechanical device (“DNA tweezers”) or the production of a functional RNA molecule (an aptamer for malachite green). We implemented several mechanisms for coupling the load processes to the oscillator circuit and compared them based on how much the load affected the frequency and amplitude of the core oscillator, and how much of the load was effectively driven. Based on heuristic insights and computational modeling, an “insulator circuit” was developed, which strongly reduced the detrimental influence of the load on the oscillator circuit. Understanding how to design effective insulation between biochemical subsystems will be critical for the synthesis of larger and more complex systems

Science To Support DOE Site Cleanup: The Pacific Northwest National Laboratory Environmental Management Science Program Awards

Pacific Northwest National Laboratory (PNNL) was awarded ten Environmental Management Science Program (EMSP) research grants in fiscal year 1996, six in fiscal year 1997, nine in fiscal year 1998, seven in fiscal year 1999, and five in fiscal year 2000. All of the fiscal year 1996 award projects have published final reports. The 1997 and 1998 award projects have been completed or are nearing completion. Final reports for these awards will be published, so their annual updates will not be included in this document. This section summarizes how each of the 1999 and 2000 grants address significant U.S. Department of Energy (DOE) cleanup issues, including those at the Hanford Site. The technical progress made to date in each of these research projects is addressed in more detail in the individual progress reports contained in this document. The 1999 and 2000 EMSP awards at PNNL are focused primarily in two areas: Tank Waste Remediation, and Soil and Groundwater Cleanup

Mechanistic insights into an engineered riboswitch: a switching element which confers riboswitch activity

While many different RNA aptamers have been identified that bind to a plethora of small molecules only very few are capable of acting as engineered riboswitches. Even for aptamers binding the same ligand large differences in their regulatory potential were observed. We address here the molecular basis for these differences by using a set of unrelated neomycin-binding aptamers. UV melting analyses showed that regulating aptamers are thermally stabilized to a significantly higher degree upon ligand binding than inactive ones. Regulating aptamers show high ligand-binding affinity in the low nanomolar range which is necessary but not sufficient for regulation. NMR data showed that a destabilized, open ground state accompanied by extensive structural changes upon ligand binding is important for regulation. In contrast, inactive aptamers are already pre-formed in the absence of the ligand. By a combination of genetic, biochemical and structural analyses, we identified a switching element responsible for destabilizing the ligand free state without compromising the bound form. Our results explain for the first time the molecular mechanism of an engineered riboswitch

Analysis and Computational Dissection of Molecular Signature Multiplicity

Molecular signatures are computational or mathematical models created to diagnose disease and other phenotypes and to predict clinical outcomes and response to treatment. It is widely recognized that molecular signatures constitute one of the most important translational and basic science developments enabled by recent high-throughput molecular assays. A perplexing phenomenon that characterizes high-throughput data analysis is the ubiquitous multiplicity of molecular signatures. Multiplicity is a special form of data analysis instability in which different analysis methods used on the same data, or different samples from the same population lead to different but apparently maximally predictive signatures. This phenomenon has far-reaching implications for biological discovery and development of next generation patient diagnostics and personalized treatments. Currently the causes and interpretation of signature multiplicity are unknown, and several, often contradictory, conjectures have been made to explain it. We present a formal characterization of signature multiplicity and a new efficient algorithm that offers theoretical guarantees for extracting the set of maximally predictive and non-redundant signatures independent of distribution. The new algorithm identifies exactly the set of optimal signatures in controlled experiments and yields signatures with significantly better predictivity and reproducibility than previous algorithms in human microarray gene expression datasets. Our results shed light on the causes of signature multiplicity, provide computational tools for studying it empirically and introduce a framework for in silico bioequivalence of this important new class of diagnostic and personalized medicine modalities

Post traumatic brain perfusion SPECT analysis using reconstructed ROI maps of radioactive microsphere derived cerebral blood flow and statistical parametric mapping

<p>Abstract</p> <p>Background</p> <p>Assessment of cerebral blood flow (CBF) by SPECT could be important in the management of patients with severe traumatic brain injury (TBI) because changes in regional CBF can affect outcome by promoting edema formation and intracranial pressure elevation (with cerebral hyperemia), or by causing secondary ischemic injury including post-traumatic stroke. The purpose of this study was to establish an improved method for evaluating regional CBF changes after TBI in piglets.</p> <p>Methods</p> <p>The focal effects of moderate traumatic brain injury (TBI) on cerebral blood flow (CBF) by SPECT cerebral blood perfusion (CBP) imaging in an animal model were investigated by parallelized statistical techniques. Regional CBF was measured by radioactive microspheres and by SPECT 2 hours after injury in sham-operated piglets versus those receiving severe TBI by fluid-percussion injury to the left parietal lobe. Qualitative SPECT CBP accuracy was assessed against reference radioactive microsphere regional CBF measurements by map reconstruction, registration and smoothing. Cerebral hypoperfusion in the test group was identified at the voxel level using statistical parametric mapping (SPM).</p> <p>Results</p> <p>A significant area of hypoperfusion (P < 0.01) was found as a response to the TBI. Statistical mapping of the reference microsphere CBF data confirms a focal decrease found with SPECT and SPM.</p> <p>Conclusion</p> <p>The suitability of SPM for application to the experimental model and ability to provide insight into CBF changes in response to traumatic injury was validated by the SPECT SPM result of a decrease in CBP at the left parietal region injury area of the test group. Further study and correlation of this characteristic lesion with long-term outcomes and auxiliary diagnostic modalities is critical to developing more effective critical care treatment guidelines and automated medical imaging processing techniques.</p

DNA Aptamers against the Lup an 1 Food Allergen

Using in vitro selection, high affinity DNA aptamers to the food allergen Lup an 1, ß-conglutin, were selected from a pool of DNA, 93 bases in length, containing a randomised sequence of 49 bases. ß-conglutin was purified from lupin flour and chemically crosslinked to carboxylated magnetic beads. Peptide mass fingerprinting was used to confirm the presence of the ß-conglutin. Single stranded DNA was generated from the randomised pool using T7 Gene 6 Exonuclease and was subsequently incubated with the magnetic beads and the captured DNA was released and amplified prior to a further round of Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Evolution was monitored using enzyme linked oligonucleotide assay and surface plasmon resonance. Once a plateau in evolution was reached, the isolated DNA sequences were cloned and sequenced. The consensus motif was identified via alignment of the sequences and the affinities of these sequences for immobilised ß-conglutin were determined using surface plasmon resonance. The selected aptamer was demonstrated to be highly specific, showing no cross-reactivity with other flour ingredients or with other conglutin fractions of lupin. The secondary structures of the selected aptamers were predicted using m-fold. Finally, the functionality of the selected aptamers was demonstrated using a competitive assay for the quantitative detection of ß-conglutin. . Future work will focus on structure elucidation and truncation of the selected sequences to generate a smaller aptamer for application to the analysis of the Lup an 1 allergen in foodstuffs

Synthetic biology: Understanding biological design from synthetic circuits

An important aim of synthetic biology is to uncover the design principles of natural biological systems through the rational design of gene and protein circuits. Here, we highlight how the process of engineering biological systems — from synthetic promoters to the control of cell–cell interactions — has contributed to our understanding of how endogenous systems are put together and function. Synthetic biological devices allow us to grasp intuitively the ranges of behaviour generated by simple biological circuits, such as linear cascades and interlocking feedback loops, as well as to exert control over natural processes, such as gene expression and population dynamics

A human breast cell model of pre-invasive to invasive transition

A crucial step in human breast cancer progression is the acquisition of invasiveness. There is a distinct lack of human cell culture models to study the transition from pre-invasive to invasive phenotype as it may occur 'spontaneously' in vivo. To delineate molecular alterations important for this transition, we isolated human breast epithelial cell lines that showed partial loss of tissue polarity in three-dimensional reconstituted-basement membrane cultures. These cells remained non-invasive; however, unlike their non-malignant counterparts, they exhibited a high propensity to acquire invasiveness through basement membrane in culture. The genomic aberrations and gene expression profiles of the cells in this model showed a high degree of similarity to primary breast tumor profiles. The xenograft tumors formed by the cell lines in three different microenvironments in nude mice displayed metaplastic phenotypes, including squamous and basal characteristics, with invasive cells exhibiting features of higher grade tumors. To find functionally significant changes in transition from pre-invasive to invasive phenotype, we performed attribute profile clustering analysis on the list of genes differentially expressed between pre-invasive and invasive cells. We found integral membrane proteins, transcription factors, kinases, transport molecules, and chemokines to be highly represented. In addition, expression of matrix metalloproteinases MMP-9,-13,-15,-17 was up regulated in the invasive cells. Using siRNA based approaches, we found these MMPs to be required for the invasive phenotype. This model provides a new tool for dissection of mechanisms by which pre-invasive breast cells could acquire invasiveness in a metaplastic context