Bioluminescent imaging is an emerging biomedical surveillance strategy that uses external cameras to detect in vivo light generated in small animal models of human physiology or in vitro light generated in tissue culture or tissue scaffold mimics of human anatomy. The most widely utilized of reporters is the firefly luciferase (luc) gene; however, it generates light only upon addition of a chemical substrate, thus only generating intermittent single time point data snapshots. To overcome this disadvantage, we have demonstrated substrate-independent bioluminescent imaging using an optimized bacterial bioluminescence (lux) system. The lux reporter produces bioluminescence autonomously using components found naturally within the cell, thereby allowing imaging to occur continuously and in real-time over the lifetime of the host. We have validated this technology in human cells with demonstrated chemical toxicological profiling against exotoxin exposures at signal strengths comparable to existing luc systems (~1.33 × 107 photons/second). As a proof-in-principle demonstration, we have engineered breast carcinoma cells to express bioluminescence for real-time screening of endocrine disrupting chemicals and validated detection of 17β-estradiol (EC50 = ~ 10 pM). These and other applications of this new reporter technology will be discussed as potential new pathways towards improved models of target chemical bioavailability, toxicology, efficacy, and human safety.
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.
Bioluminescent production represents a facile method for bioreporter detection in mammalian tissues. The lack of
endogenous bioluminescent reactions in these tissues allows for high signal to noise ratios even at low signal strength
compared to fluorescent signal detection. While the luciferase enzymes commonly employed for bioluminescent
detection are those from class Insecta (firefly and click beetle luciferases), these are handicapped in that they require
concurrent administration of a luciferin compound to elicit a bioluminescent signal. The bacterial luciferase (lux) gene
cassette offers the advantages common to other bioluminescent proteins, but is simultaneously capable of synthesizing
its own luciferin substrates using endogenously available cellular compounds. The longstanding shortcoming of the lux
cassette has been its recalcitrance to function in the mammalian cellular environment. This paper will present an
overview of the work completed to date to overcome this limitation and provide examples of mammalian lux-based
bioreporter technologies that could provide the framework for advanced, biomedically relevant real-time sensor
development.
KEYWORDS: Imaging systems, Green fluorescent protein, Signal detection, Tissues, In vivo imaging, In vitro testing, Preclinical imaging, Animal model studies, Bioluminescence, Magnesium
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 × 104 cells but at the cost of increasing overall image integration time.
Biophotonic imaging is a versatile and powerful tool, that when combined with living microbial bioreporters, can be
applied in diagnostic technologies for sensitive, nondestructive, real-time monitoring of chemical and biological targets.
Bioreporters, consisting of bacteria as well as the viruses (bacteriophage) that infect them, can be genetically engineered
to emit visible light upon interaction with a specific chemical or biological entity. By interfacing these bioreporters with
imaging cameras or miniaturized integrated circuit microluminometers, fully standalone detection units are formed that
can be deployed for intelligent distributed multi-target chem/bio monitoring.
Bioluminescent bioreporters carrying the bacterial lux gene cassette have been well established for the sensing and
monitoring of select chemical agents. Their ability to generate target specific visible light signals with no requirement
for extraneous additions of substrate or other hands-on manipulations affords a real-time, repetitive assaying technique
that is remarkable in its simplicity and accuracy. Although the predominant application of lux-based bioluminescent
bioreporters has been towards chemical compound detection, novel genetic engineering schemes are yielding a variety of
new bioreporter systems that extend the lux sensing mechanism beyond mere analyte discrimination. For example, the
unique specificity of bacteriophage (bacterial viruses) has been exploited in lux bioluminescent assays for specific
identification of foodborne bacterial pathogens such as Escherichia coli O157:H7. With the concurrent ability to
interface bioluminescent bioreporter assays onto integrated circuit microluminometers (BBICs; bioluminescent
bioreporter integrated circuits), the potential exists for the development of sentinel microchips that can function as
environmental monitors for multiplexed recognition of chemical and biological agents in air, food, and water. The size
and portability of BBIC biosensors may ultimately provide a deployable, interactive network sensing technology
adaptable towards chem/bio defense.
The objective of this investigation is to develop a bioluminescent bioreporter system for the detection and monitoring of pathogenic microbial species. Current detection methodologies typically rely on time-consuming sample pre-enrichment steps to elevate pathogen concentrations to detectable levels or DNA based polymerase chain reaction (PCR) techniques that require extensive user training and expensive instrumentation. Detection utilizing bioluminescent bioreporter organisms, however, can provide a simple and rapid means of monitoring foodborne pathogens. Bioluminescent bioreporters are engineered to produce light in response to specific environmental inducers. The light signal is then measured with photodetector devices to generate a quantitative assessment of inducer concentration. The immediate goal of this research effort is to integrate key quorum sensing signal transduction elements into pathogen specific bacteriophages. Upon infection of a unique pathogenic species by the bacteriophages, quorum sensing signals will be generated that will subsequently stimulate bioluminescence in neighboring bioluminescent bioreporter cells. Utilizing both bacteriophages and bioluminescent bioreporters, we realize exceptional pathogen specificity while attaining enhanced bioluminescence production. This integrative approach will lead to rapid pathogen identification without requisite sample pre-enrichment. Additionally, since the bioluminescent response is completely intrinsic to the bioreporter organism, no user interventions are required for generating light signals; the protocol requires only addition of the food sample with the bacteriophage/bioluminescent bioreporter system. Measurement of light responses can be achieved using high-throughput microtiter plate readers, hand-held photomultiplier units, or microchip luminometers.
Histamine is the primary etiological agent in the foodborne disease scombrotoxicosis, one of the most common food toxicities related to fish consumption. Procedures for detecting histamine in fish products are available, but are often too expensive or too complex for routine use. As an alternative, a bacterial bioluminescent bioreporter has been constructed to develop a biosensor system that autonomously responds to low levels of histamine. The bioreporter contains a promoterless Photorhabdus luminescens lux operon (luxCDABE) fused with the Vibrio anguillarum angR regulatory gene promoter of the anguibactin biosynthetic operon. The bioreporter emitted 1.46 times more bioluminescence than background, 30 minutes after the addition of 100mM histamine. However, specificity was not optimal, as this biosensor generated significant bioluminescence in the presence of L-proline and L-histidine. As a means towards improving histamine specificity, the promoter region of a histamine oxidase gene from Arthrobacter globiformis was cloned upstream of the promotorless lux operon from Photorhabdus luminescens. This recently constructed whole-cell, lux-based bioluminescent bioreporter is currently being tested for optimal performance in the presence of histamine in order to provide a rapid, simple, and inexpensive model sensor for the detection of foodborne toxins.
As a means towards advanced, early-warning detection of microbial growth in enclosed structures, we have constructed a bioluminescent bioreporter for the detection of the microbial volatile organic compound (MVOC) p-cymene. MVOCs are produced as metabolic by-products of bacteria and fungi and are detectable before any visible signs of microbial growth appear, thereby serving as very early indicators of potential biocontamination problems. The bioreporter, designated Pseudomonas putida UT93, contains a Vibrio fischeri luxCDABE gene fusion to a p-cymene/p-cumate inducible promoter. Exposure of strain UT93 to p-cymene from approximately 0.02 to 850 ppm produced self-generated bioluminescence in less than 1.5 hours. The bioreporter was also interfaced with an integrated circuit microluminometer to create a miniaturized hybrid sensor for remote monitoring of p-cymene signatures. This bioluminescent bioreporter integrated circuit (BBIC) device was capable of detecting fungal presence within approximately 3.5 hours of initial exposure to Penicillium roqueforti.
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.
The presence of biologically derived toxins in foods is of utmost significance to food safety and human health concerns. Biologically active amines, referred to as biogenic amines, serve as a noteworthy example, having been implicated as the causative agent in numerous food poisoning episodes. Of the various biogenic amines encountered, histamine, putrescine, cadaverine, tyramine, tryptamine, beta-phenylethylamine, spermine, and spermidine are considered to be the most significant, and can be used as hygienic-quality indicators of food. Biogenic amines can be monitored using whole-cell bioluminescent bioreporters, which represent a family of genetically engineered microorganisms that generate visible light in response to specific chemical or physical agents in their environment. The light response occurs due to transcriptional activation of a genetically incorporated lux cassette, and can be measured using standard photomultiplier devices. We have successfully engineered a lux-based bioreporter capable of detecting and monitoring the biogenic amine beta-phenylethylamine. This research represents a biologically-based sensor technology that can be readily integrated into Hazard Analysis Critical Control Point programs to provide a rugged monitoring regime that can be uniformly applied for field-based and in-house laboratory quality control analyses. Since the bioreporter and biosensing elements are completely self-contained within the sensor design, this system provides ease of use, with operational capabilities realized by simply combining the food sample with the bioreporter and allowing the sensor to process the ensuing bioluminescent signal and communicate the results. The application of this technology to the critically important issue of food safety and hygienic quality represents a novel method for detecting, monitoring, and preventing biologically active toxins in food commodities.
As the workhorse of the integrated circuit (IC) industry, the capabilities of CMOS have been expanded well beyond the original applications. The full spectrum of analog circuits from switched-capacitor filters to microwave circuit blocks, and from general-purpose operational amplifiers to sub- nanosecond analog timing circuits for nuclear physics experiments have been implemented in CMOS. This technology has also made in-roads into the growing area of monolithic sensors with devices such as active-pixel sensors and other electro-optical detection devices. While many of the processes used for MEMS fabrication are not compatible with the CMOS IC process, depositing a sensor material onto a previously fabricated CMOS circuit can create a very useful category of sensors. In this work we report a chemical sensor composed of bioluminescent bioreporters (genetically engineered bacteria) deposited onto a micro-luminometer fabricated in a standard CMOS IC process. The bioreporter used for this work emitted 490-nm light when exposed to toluene. This luminescence was detected by the micro- luminometer giving an indication of the concentration of toluene. Other bioluminescent bioreporters sensitive to explosives, mercury, and other organic chemicals and heavy metals have been reported. These could be incorporated (individually or in combination) with the micro-luminometer reported here to form a variety of chemical sensors.
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