Stacey S. Patterson

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

ABSTRACT 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 17β-estradiol over the concentration range of 1.2 × 10−8 through 5.6 × 10−12 M. Calculated 50% effective concentration values from the colorimetric and bioluminescence assays (n = 7) were similar at (4.4 ± 1.1) × 10−10 and (2.4 ± 1.0) × 10−10 M, respectively. The lower and upper limits of detection for each assay were also similar and were approximately 4.5 × 10−11 to 2.8 × 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.

Autonomous Bioluminescent Expression of the Bacterial Luciferase Gene Cassette (lux) in a Mammalian Cell Line

Background The bacterial luciferase (lux) gene cassette consists of five genes (luxCDABE) whose protein products synergistically generate bioluminescent light signals exclusive of supplementary substrate additions or exogenous manipulations. Historically expressible only in prokaryotes, the lux operon was re-synthesized through a process of multi-bicistronic, codon-optimization to demonstrate for the first time self-directed bioluminescence emission in a mammalian HEK293 cell line in vitro and in vivo. Methodology/Principal Findings Autonomous in vitro light production was shown to be 12-fold greater than the observable background associated with untransfected control cells. The availability of reduced riboflavin phosphate (FMNH2) was identified as the limiting bioluminescence substrate in the mammalian cell environment even after the addition of a constitutively expressed flavin reductase gene (frp) from Vibrio harveyi. FMNH2 supplementation led to a 151-fold increase in bioluminescence in cells expressing mammalian codon-optimized luxCDE and frp genes. When injected subcutaneously into nude mice, in vivo optical imaging permitted near instantaneous light detection that persisted independently for the 60 min length of the assay with negligible background. Conclusions/Significance The speed, longevity, and self-sufficiency of lux expression in the mammalian cellular environment provides a viable and powerful alternative for real-time target visualization not currently offered by existing bioluminescent and fluorescent imaging technologies.

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 [5×105 vs 2.9×106 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 (2.7×107 RLU/mg total protein). These results show promise toward the potential development of an autonomous light generating lux reporter system in mammalian cells

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.

Phytoplankton-Group Specific Quantitative Polymerase Chain Reaction Assays for RuBisCO mRNA Transcripts in Seawater

The gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) has been shown to be a useful target for molecular assays that quantify form- or clade-specific RNA transcript concentrations as a proxy for the carbon fixation activity of marine phytoplankton. To improve the phylogenetic specificity and sensitivity of RNA probe hybridization methods, a quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay has been reported for diatom and pelagophyte rbcL RNA. Here we detail enhancements made to this PCR method and development of additional assays to specifically quantify rbcL expression from haptophytes, Synechococcus and high-light Prochlorococcus. In vitro RNA transcripts were tested to demonstrate specificity and quantitative accuracy. Application of these methods on seawater samples from two depth profiles in the northern Gulf of Mexico showed a fair degree of agreement between PCR and hybridization results, with results for the chromophytic or form ID rbcL-containing organisms having better agreement between the two methods. Diatoms and other heterokonts were shown to be the primary carbon fixers at these locations by PCR, in agreement with greater form ID rbcL RNA measured by hybridization.

Comparison of human optimized bacterial luciferase, firefly luciferase, and green fluorescent protein for continuous imaging of cell culture and animal models

Bioluminescent and fluorescent reporter systems have enabled the rapid and continued growth of the optical imaging field over the last two decades. Of particular interest has been noninvasive signal detection from mammalian tissues under both cell culture and whole animal settings. Here we report on the advantages and limitations of imaging using a recently introduced bacterial luciferase (lux) reporter system engineered for increased bioluminescent expression in the mammalian cellular environment. Comparison with the bioluminescent firefly luciferase (Luc) system and green fluorescent protein system under cell culture conditions demonstrated a reduced average radiance, but maintained a more constant level of bioluminescent output without the need for substrate addition or exogenous excitation to elicit the production of signal. Comparison with the Luc system following subcutaneous and intraperitoneal injection into nude mice hosts demonstrated the ability to obtain similar detection patterns with in vitro experiments at cell population sizes above 2.5 × 10(4) cells but at the cost of increasing overall image integration time.

Remote detection of human toxicants in real time using a human-optimized, bioluminescent bacterial luciferase gene cassette bioreporter

Traditionally, human toxicant bioavailability screening has been forced to proceed in either a high throughput fashion using prokaryotic or lower eukaryotic targets with minimal applicability to humans, or in a more expensive, lower throughput manner that uses fluorescent or bioluminescent human cells to directly provide human bioavailability data. While these efforts are often sufficient for basic scientific research, they prevent the rapid and remote identification of potentially toxic chemicals required for modern biosecurity applications. To merge the advantages of high throughput, low cost screening regimens with the direct bioavailability assessment of human cell line use, we re-engineered the bioluminescent bacterial luciferase gene cassette to function autonomously (without exogenous stimulation) within human cells. Optimized cassette expression provides for fully endogenous bioluminescent production, allowing continuous, real time monitoring of the bioavailability and toxicology of various compounds in an automated fashion. To access the functionality of this system, two sets of bioluminescent human cells were developed. The first was programed to suspend bioluminescent production upon toxicological challenge to mimic the non-specific detection of a toxicant. The second induced bioluminescence upon detection of a specific compound to demonstrate autonomous remote target identification. These cells were capable of responding to μM concentrations of the toxicant n-decanal, and allowed for continuous monitoring of cellular health throughout the treatment process. Induced bioluminescence was generated through treatment with doxycycline and was detectable upon dosage at a 100 ng/ml concentration. These results demonstrate that leveraging autonomous bioluminescence allows for low-cost, high throughput direct assessment of toxicant bioavailability.

Development of bacteriophage-based bioluminescent bioreporters for monitoring of microbial pathogens

Microorganisms pose numerous problems when present in human occupied enclosed environments. Primary among these are health related hazards, manifested as infectious diseases related to contaminated drinking water, food, or air circulation systems or non-infectious allergy related complications associated with microbial metabolites (sick building syndrome). As a means towards rapid detection of microbial pathogens, we are attempting to harness the specificity of bacterial phage for their host with a modified quorum sensing amplification signal to produce quantifiable bioluminescent (lux) detection on a silicon microluminometer. The bacteriophage itself is metabolically inactive, only achieving replicative capabilities upon infection of its specific host bacterium. Bacteriophage bioluminescent bioreporters contain a genomically inserted luxI component. During an infection event, the phage genes and accompanying luxI construct are taken up by the host bacterium and transcribed, resulting in luxI expression and subsequent activation of a homoserine lactone inducible bioluminescent bioreporter. We constructed a vector carrying the luxI gene under the control of a strong E. coli promoter and cloned it into E. coli. We have shown that it can induce luminescence up to 14,000 counts per second when combined with the bioreporter strain. In their final embodiment, these sensors will be fully independent microelectronic monitors for microbial contamination, requiring only exposure of the biochip to the sample, with on-chip signal processing downloaded directly to the local area network of the environmental control system.

Expression and stabilization of bacterial luciferase in mammalian cells

Current mammalian bioreporters using either firefly luciferase (luc) or GFP constructs require lysis and/or exogenous excitation to evoke a measurable response. Consequently, these cells cannot serve as continuous, on-line monitoring devices for in vivo imaging. Bacterial luciferase, lux, produces a photonic reaction that is cyclic, resulting in autonomous signal generation without the requirement for exogenous substrates or external activation. Therefore, lux-based bioluminescent bioreporters are the only truly autonomous light-generating sensors in existence. Unfortunately, the bacterial lux system has not yet been efficiently expressed in mammalian cells. In this research, three approaches for optimal expression of the a and b subunits of the bacterial luciferase protein were compared and reporter signal stability was evaluated from stably transfected human embryonic kidney cells. Maximum light levels were obtained from cells expressing the luciferase subunits linked with an internal ribosomal entry site (IRES). Cells harboring this construct produced bioluminescence equaling 2.6 X 106 photons/sec compared to 7.2 X 104 photons/sec obtained from cells expressing the luciferase from a dual promoter vector and 3.5 X 104 photons/sec from a Lux fusion protein. Furthermore, the bioluminescence levels remained stable for more than forty cell passages (5 months) in the absence of antibiotic selection. After this time, bioluminescence signals dropped at a rate of approximately 5% per cell passage. These data indicate that mammalian cell lines can be engineered to efficiently express the bacterial lux system, thus lending themselves to possible long-term continuous monitoring or imaging applications in vivo.