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Michael L Simpson

Publications and source records attributed to Michael L Simpson.

17 recordsLinked to original sources
PubMed2007

Quantitative analysis of EDC-condensed DNA on vertically aligned carbon nanofiber gene delivery arrays.

Vertically aligned carbon nanofibers (VACNFs) with immobilized DNA have been developed as a novel tool for direct physical introduction and expression of exogenous genes in mammalian cells. Immobilization of DNA base amines to the carboxylic acids on nanofibers can influence the accessibility and transcriptional activity of the DNA template, making it necessary to determine the number of accessible gene copies on nanofiber arrays. Polymerase chain reaction (PCR) and in vitro transcription (IVT) were used to investigate the transcriptional accessibility of DNA tethered to VACNFs by correlating the yields of both IVT and PCR to that of non-tethered, free DNA. Yields of the promoter region and promoter/gene region of bound DNA plasmid were high. Amplification using primers designed to cover 80% of the plasmid failed to yield any product. These results are consistent with tethered, longer DNA sequences having a higher probability of interfering with the activity of DNA and RNA polymerases. Quantitative PCR (qPCR) was used to quantify the number of accessible gene copies tethered to nanofiber arrays. Copy numbers of promoters and reporter genes were quantified and compared to non-tethered DNA controls. In subsequent reactions of the same nanofiber arrays, DNA yields decreased dramatically in the non-tethered control, while the majority of tethered DNA was retained on the arrays. This decrease could be explained by the presence of DNA which is non-tethered to all samples and released during the assay. This investigation shows the applicability of these methods for monitoring DNA immobilization techniques.

Carbon↗
PubMed2006

Reversible electrowetting of vertically aligned superhydrophobic carbon nanofibers.

Reversible electrostatically induced wetting (electrowetting) of vertically aligned superhydrophobic carbon nanofibers has been investigated. Carbon nanofibers on a 5 x 5 microm pitch were grown on Si substrates, electrically insulated with a conformal dielectric, and hydrophobized with fluoropolymer. This nanostructured scaffold exhibited superhydrophobic behavior for saline (theta approximately 160 degrees). Electrowetting induced a contact angle reduction to theta approximately 100 degrees. Competitive two-liquid (dodecane/saline) electrowetting exhibited reversibility on the same nanostructured scaffold. Without applied bias, ultra-fine-point tip (approximately 25 nm radius) nanofibers result in effectively zero capacitance with the overlying saline layer. Complete electrowetting of the substrate is confirmed as capacitance values increase by several orders of magnitude with increased wetting. These results demonstrate the applicability of reversible electrowetting on nanostructured scaffolds and use of nanofabricated structures that can be integrated with various micro- and nanoelectronic technologies.

Electrons↗
PubMed2006

Direct quantification of N-(3-oxo-hexanoyl)-L-homoserine lactone in culture supernatant using a whole-cell bioreporter.

The autoinducer N-(3-oxo-hexanoyl)-L-homoserine lactone (3-oxo-C6-HSL) plays a significant role in the quorum-sensing system of the marine bacterium Vibrio fischeri. Upon forming a transcriptional activation complex with LuxR, 3-oxo-C6-HSL induces transcription of the luxICDABEG operon, leading to the increased production of both the 3-oxo-C6-HSL synthase (LuxI) and the bioluminescent proteins. In order to quantitatively analyze this regulatory mechanism, a novel approach was developed to measure 3-oxo-C6-HSL concentrations in V. fischeri cell culture supernatant. A bioluminescent strain of Escherichia coli that responds to 3-oxo-C6-HSL was used as a bioreporter. Although a linear response of the bioreporter to exogenously added synthetic 3-oxo-C6-HSL was found over several orders of magnitude, we show that bioreporter performance was dramatically impacted by variations in the supernatants using samples from a V. fischeri LuxI- strain. However, when maintained in the same supernatant background, the normalized peak bioluminescence maintained a linear response to 3-oxo-C6-HSL concentrations. Therefore, a standard additions technique was developed in which a known concentration of 3-oxo-C6-HSL was added to supernatant samples from wild-type V. fischeri cultures, and the incremental increase of the normalized peak bioluminescence relative to the untreated sample was determined. The concentration of 3-oxo-C6-HSL in the supernatant of the unknown sample was then quantified from the slope of the response between the normalized bioluminescent peaks with and without the addition of 3-oxo-C6-HSL. Advantages of this method are that it is rapid, does not require concentration or extraction, uses a small sample volume (ca. 2 ml), and accounts for effects caused by the composition of the supernatant. Furthermore, the findings can be broadly applicable to other bioreporter systems involving variable background conditions.

4-Butyrolactone↗
PubMed2006

Resident neuroelectrochemical interfacing using carbon nanofiber arrays.

Carbon nanofiber electrode architectures are used to provide for long-term, neuroelectroanalytical measurements of the dynamic processes of intercellular communication between excitable cells. Individually addressed, vertically aligned carbon nanofibers are incorporated into multielement electrode arrays upon which excitable cell matrixes of both neuronal-like derived cell lines (rat pheochromocytoma, PC-12) and primary cells (dissociated cells from embryonic rat hippocampus) are cultured over extended periods (days to weeks). Electrode arrays are characterized with respect to their response to easily oxidized neurotransmitters, including dopamine, norepinephrine, and 5-hydroxytyramide. Electroanalysis at discrete electrodes following long-term cell culture demonstrates that this platform remains responsive for the detection of easily oxidized species generated by the cultured cells. Preliminary data also suggests that quantal release of easily oxidized transmitters can be observed at nanofiber electrodes following direct culture and differentiation on the arrays for periods of at least 16 days.

Animals↗
PubMed2006

The sorting direct method for stochastic simulation of biochemical systems with varying reaction execution behavior.

A key to advancing the understanding of molecular biology in the post-genomic age is the development of accurate predictive models for genetic regulation, protein interaction, metabolism, and other biochemical processes. To facilitate model development, simulation algorithms must provide an accurate representation of the system, while performing the simulation in a reasonable amount of time. Gillespie's stochastic simulation algorithm (SSA) accurately depicts spatially homogeneous models with small populations of chemical species and properly represents noise, but it is often abandoned when modeling larger systems because of its computational complexity. In this work, we examine the performance of different versions of the SSA when applied to several biochemical models. Through our analysis, we discover that transient changes in reaction execution frequencies, which are typical of biochemical models with gene induction and repression, can dramatically affect simulator performance. To account for these shifts, we propose a new algorithm called the sorting direct method that maintains a loosely sorted order of the reactions as the simulation executes. Our measurements show that the sorting direct method performs favorably when compared to other well-known exact stochastic simulation algorithms.

Algorithms↗
PubMed2006

Frequency domain analysis of noise in simple gene circuits.

Recent advances in single cell methods have spurred progress in quantifying and analyzing stochastic fluctuations, or noise, in genetic networks. Many of these studies have focused on identifying the sources of noise and quantifying its magnitude, and at the same time, paying less attention to the frequency content of the noise. We have developed a frequency domain approach to extract the information contained in the frequency content of the noise. In this article we review our work in this area and extend it to explicitly consider sources of extrinsic and intrinsic noise. First we review applications of the frequency domain approach to several simple circuits, including a constitutively expressed gene, a gene regulated by transitions in its operator state, and a negatively autoregulated gene. We then review our recent experimental study, in which time-lapse microscopy was used to measure noise in the expression of green fluorescent protein in individual cells. The results demonstrate how changes in rate constants within the gene circuit are reflected in the spectral content of the noise in a manner consistent with the predictions derived through frequency domain analysis. The experimental results confirm our earlier theoretical prediction that negative autoregulation not only reduces the magnitude of the noise but shifts its content out to higher frequency. Finally, we develop a frequency domain model of gene expression that explicitly accounts for extrinsic noise at the transcriptional and translational levels. We apply the model to interpret a shift in the autocorrelation function of green fluorescent protein induced by perturbations of the translational process as a shift in the frequency spectrum of extrinsic noise and a decrease in its weighting relative to intrinsic noise.

Animals↗
PubMed2005

Use of Saccharomyces cerevisiae BLYES expressing bacterial bioluminescence for rapid, sensitive detection of estrogenic compounds.

An estrogen-inducible bacterial lux-based bioluminescent reporter was developed in Saccharomyces cerevisiae for applications in chemical sensing and environmental assessment of estrogen disruptor activity. The strain, designated S. cerevisiae BLYES, was constructed by inserting tandem estrogen response elements between divergent yeast promoters GPD and ADH1 on pUTK401 (formerly pUA12B7) that constitutively express luxA and luxB to create pUTK407. Cotransformation of this plasmid with a second plasmid (pUTK404) containing the genes required for aldehyde synthesis (luxCDE) and FMN reduction (frp) yielded a bioluminescent bioreporter responsive to estrogen-disrupting compounds. For validation purposes, results with strain BLYES were compared to the colorimetric-based estrogenic assay that uses the yeast lacZ reporter strain (YES). Strains BLYES and YES were exposed to 17beta-estradiol over the concentration range of 1.2 x 10(-8) through 5.6 x 10(-12) M. Calculated 50% effective concentration values from the colorimetric and bioluminescence assays (n = 7) were similar at (4.4 +/- 1.1) x 10(-10) and (2.4 +/- 1.0) x 10(-10) M, respectively. The lower and upper limits of detection for each assay were also similar and were approximately 4.5 x 10(-11) to 2.8 x 10(-9) M. Bioluminescence was observed in as little as 1 h and reached its maximum in 6 h. In comparison, the YES assay required a minimum of 3 days for results. Strain BLYES fills the niche for rapid, high-throughput screening of estrogenic compounds and has the ability to be used for remote, near-real-time monitoring of estrogen-disrupting chemicals in the environment.

Biological Assay↗
PubMed2005

Synthetic nanoscale elements for delivery of materials into viable cells.

Arrays of vertically aligned carbon nanofibers (VACNFs) provide structures that are well suited for the direct integration and manipulation of molecular-scale phenomena within intact, live cells. VACNFs are fabricated via a combination of microfabrication techniques and catalytic plasma-enhanced chemical vapor deposition. In this chapter, we discuss the synthesis of VACNFs and detail the methods for introducing these arrays into the intracellular domain of mammalian cells for the purpose of delivering large macromolecules, specifically plasmid DNA, on a massively parallel basis.

Animals↗
PubMed2004

Surface patterning of silica nanostructures using bio-inspired templates and directed synthesis.

Natural systems excel in directing the synthesis of inorganic materials for various functional purposes. One of the best-studied systems is silica synthesis, as occurs in diatoms and marine sponges. Various biological and synthetic polymers have been shown to template and catalyze silica formation from silicic acid precursors. Here, we describe the use of poly-L-lysine to promote the synthesis of silica in neutral, aqueous solution and when immobilized onto a silicon support structure under similar conditions. Either reagent jetting or conventional photolithography techniques can be used to pattern the templating polymer. Spots created by reagent jetting led to the creation of silica structures in the shape of a ring that may be a result of the spotting process. Photolithographically defined poly-L-lysine spots led to thin laminate structures after exposure to a dilute aqueous silicic acid solution. The laminate structures were nanostructured and highly interconnected. Photolithographic patterning of (3-aminopropyl)trimethoxysilane, a reagent that mimics the lysine functional group, led to similar silica coatings even though low-molecular-weight materials do not rapidly promote silica synthesis in solution. This result highlights the importance of functional-group arrangement for templating and promoting the synthesis of inorganic materials. The described surface-patterning techniques offer a route to integrate conventional silicon-patterning technologies with biologically based material synthesis. Such combined fabrication techniques enable controlled assembly over multiple length scales and an approach to understanding interfacial silica synthesis, as occurs in natural systems.

Biomimetics↗
PubMed2004

Frequency domain chemical Langevin analysis of stochasticity in gene transcriptional regulation.

We present a frequency domain Langevin approach for stochastic analysis that remains valid for many important gene circuit elements even as molecular populations approach zero. We begin by considering the case of low-rate transcription and show that the previously reported shot noise representation is exact at all mRNA population levels for a constant transcription rate. Next, we consider transcriptional control through protein-DNA interactions at an operator site within the gene promoter region. This analysis results in expressions for the dynamics and noise behavior of this important gene sub-circuit, including the spectral density of the intrinsic operator noise and the processing of extrinsic noise by this transcriptional regulation system. This analysis shows that mRNA synthesis noise is composed of wideband shot noise and band-limited operator binding generated noise components. We find that the bandwidth of operator noise and its ultimate effect on total mRNA and protein noise is controlled by operator binding and unbinding dynamics. The most substantial impact of the operator noise is seen at transcription rates just above basal expression. This analysis captures the full behavior of this transcriptional regulation system, and points to potentially serious flaws in simplified mathematical relationships often used to model transcriptional regulation.

Gene Expression Regulation↗
PubMed2004

Rewiring the cell: synthetic biology moves towards higher functional complexity.

A steady stream of research has fueled excitement in the field of synthetic biology. Logic gates, oscillators, and memory elements constructed using genetic and biochemical components have all been demonstrated. However, the nagging question remains as to how higher levels of complexity can be designed into these synthetic systems. A recent paper from Collins' group provides some answers to this question.

Biomimetics↗
PubMed2003

Frequency domain analysis of noise in autoregulated gene circuits.

We describe a frequency domain technique for the analysis of intrinsic noise within negatively autoregulated gene circuits. This approach is based on the transfer function around the feedback loop (loop transmission) and the equivalent noise bandwidth of the system. The loop transmission, T, is shown to be a determining factor of the dynamics and the noise behavior of autoregulated gene circuits, and this T-based technique provides a simple and flexible method for the analysis of noise arising from any source within the gene circuit. We show that negative feedback not only reduces the variance of the noise in the protein concentration, but also shifts this noise to higher frequencies where it may have a negligible effect on the noise behavior of following gene circuits within a cascade. This predicted effect is demonstrated through the exact stochastic simulation of a two-gene cascade. The analysis elucidates important aspects of gene circuit structure that control functionality, and may provide some insights into selective pressures leading to this structure. The resulting analytical relationships have a simple form, making them especially useful as synthetic gene circuit design equations. With the exception of the linearization of Hill kinetics, this technique is general and may be applied to the analysis or design of networks of higher complexity. This utility is demonstrated through the exact stochastic simulation of an autoregulated two-gene cascade operating near instability.

Biophysical Phenomena↗
PubMed2003

Expression of the Photorhabdus luminescens lux genes (luxA, B, C, D, and E) in Saccharomyces cerevisiae.

The luxA, B, C, D, and E genes from Photorhabdus luminescens were cloned and functionally expressed in Saccharomyces cerevisiae to construct a bacterial lux-based yeast bioreporter capable of autonomous bioluminescence emission. The bioreporter was engineered using a series of pBEVY yeast expression vectors that allowed for bi-directional constitutive or inducible expression of the individual luxA, B, C, and E genes. The luxD gene, encoding the acyl-ACP transferase that ultimately supplies the requisite aldehyde substrate for the bioluminescent reaction, was fused to a yeast internal ribosomal entry site (IRES) sequence to ensure high bi-cistronic expression. Although self-generation of bioluminescence was achieved by the bioreporter, the signal was relatively weak and decayed rapidly. To overcome this instability, a flavin oxidoreductase gene (frp) from Vibrio harveyi was co-expressed to provide sufficient concentrations of the FMNH(2) co-factor required for the bioluminescent reaction. Expression of frp with the lux genes not only stabilized but also enhanced bioluminescence to levels approaching 9.0x10(5) times above background.

Acyltransferases↗
PubMed2003

Analysis of noise in quorum sensing.

Noise may play a pivotal role in gene circuit functionality, as demonstrated for the genetic switch in the bacterial phage lambda. Like the lambda switch, bacterial quorum sensing (QS) systems operate within a population and contain a bistable switching element, making it likely that noise plays a functional role in QS circuit operation. Therefore, a detailed analysis of the noise behavior of QS systems is needed. We have developed a set of tools generally applicable to the analysis of gene circuits, with an emphasis on investigations in the frequency domain (FD), that we apply here to the QS system in the marine bacterium Vibrio fischeri. We demonstrate that a tight coupling between exact stochastic simulation and FD analysis provides insights into the structure/function relationships in the QS circuit. Furthermore, we argue that a noise analysis is incomplete without consideration of the power spectral densities (PSDs) of the important molecular output signals. As an example we consider reversible reactions in the QS circuit, and show through analysis and exact stochastic simulation that these circuits make significant and dynamic modifications to the noise spectra. In particular, we demonstrate a "whitening" effect, which occurs as the noise is processed through these reversible reactions.

Calibration↗
PubMed2002

Integrated CMOS photodetectors and signal processing for very low-level chemical sensing with the bioluminescent bioreporter integrated circuit.

We report an integrated CMOS microluminometer optimized for the detection of low-level bioluminescence as part of the bioluminescent bioreporter integrated circuit (BBIC). This microluminometer improves on previous devices through careful management of the sub-femtoampere currents, both signal and leakage, that flow in the front-end processing circuitry. In particular, the photodiode is operated with a reverse bias of only a few mV, requiring special attention to the reset circuitry of the current-to-frequency converter (CFC) that forms the front-end circuit. We report a sub-femtoampere leakage current and a minimum detectable signal (MDS) of 0.15 fA (1510 s integration time) using a room temperature 1.47 mm2 CMOS photodiode. This microluminometer can detect luminescence from as few as 5000 fully induced Pseudomonas fluorescens 5RL bacterial cells.

Biosensing Techniques↗