Biomedical subjects

Stacey S Patterson

Publications and source records attributed to Stacey S Patterson.

5 recordsLinked to original sources

PubMed2005

Codon optimization of bacterial luciferase (lux) for expression in mammalian cells.

Expression of the bacterial luciferase (lux) system in mammalian cells would culminate in a new generation of bioreporters for in vivo monitoring and diagnostics technology. Past efforts to express bacterial luciferase in mammalian cells have resulted in only modest gains due in part to low overall expression of the bacterial genes. To optimize expression, we have designed and synthesized codon-optimized versions of the luxA and luxB genes from Photorhabdus luminsecens. To evaluate these genes in vivo, stable HEK293 cell lines were created harboring wild type luxA and luxB (WTA/WTB), codon-optimized luxA and wild type luxB (COA/WTB), and codon-optimized versions of both luxA and luxB genes (COA/COB). Although mRNA levels within these clones remained approximately equal, LuxA protein levels increased significantly after codon optimization. On average, bioluminescence levels were increased by more than six-fold [5x10(5) vs 2.9x10(6) relative light units (RLU)/mg total protein] with the codon-optimized luxA and wild type luxB. Bioluminescence was further enhanced upon expression of both optimized genes (2.7x10(7) RLU/mg total protein). These results show promise toward the potential development of an autonomous light generating lux reporter system in mammalian cells.

Animals↗

PubMed2005

Increased precision of microbial RNA quantification using NASBA with an internal control.

Detection and quantification of low abundance target RNA has wide utility in the fields of clinical diagnostics, environmental monitoring, gene expression analysis, and biodefense. Nucleic acid based sequence amplification (NASBA) is an isothermal amplification method that provides the sensitivity needed for these applications. However, the requirement for three separate enzymes in NASBA often results in a greater variability between replicate samples than that seen in PCR-based assays. To overcome this problem, we have adapted the bioMérieux Nuclisens Basic Kit and Nuclisens EasyQ Analyzer along with the introduction of a synthetic internal control RNA (IC-RNA) for quantification of potentially any RNA sequence. Using the rbcL gene from the Florida red tide organism Karenia brevis as our target, we describe a simple method to accurately quantify the native target by computing the ratio of the time to positivity (TTP) values for both the wild-type and IC-RNA, and plotting this ratio against the starting number of target molecules or cells. By utilizing this simple method, we have significantly increased our accuracy and precision of prediction over the standard TTP calculations.

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↗

PubMed2004

Development and evaluation of a method to detect and quantify enteroviruses using NASBA and internal control RNA (IC-NASBA).

We have developed a rapid, sensitive, and specific assay for the detection and quantification of enteroviruses using nucleic acid sequence-based amplification (NASBA). The inclusion of an internal control (IC) increased the precision and accuracy of the method over a standard NASBA assay and provided a way to detect assay inhibition. The assay was sensitive to 10 viral particles with amplification and detection occurring in as little as 18 min. The assay detected a variety of different enteroviruses to the exclusion of non-target viruses. The standard NASBA method resulted in predictions of viral load to within an order of magnitude of the expected number, as compared with prediction to within less than a half order of magnitude using the IC-NASBA method. Rapid and sensitive detection of enteroviruses is important in both clinical samples to diagnose illness and in environmental samples to assess risk of wastewater contamination and potential health hazards.

Enterovirus↗

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↗