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The Evolution of the Bacterial Luciferase Gene Cassette (lux) as a Real-Time Bioreporter

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The bacterial luciferase gene cassette (lux) is unique among bioluminescent bioreporter systems due to its ability to synthesize and/or scavenge all of the substrate compounds required for its production of light. As a result, the lux system has the unique ability to autonomously produce a luminescent signal, either continuously or in response to the presence of a specific trigger, across a wide array of organismal hosts. While originally employed extensively as a bacterial bioreporter system for the detection of specific chemical signals in environmental samples, the use of lux as a bioreporter technology has continuously expanded over the last 30 years to include expression in eukaryotic cells such as Saccharomyces cerevisiae and even human cell lines as well. Under these conditions, the lux system has been developed for use as a biomedical detection tool for toxicity screening and visualization of tumors in small animal models. As the technologies for lux signal detection continue to improve, it is poised to become one of the first fully implantable detection systems for intra-organismal optical detection through direct marriage to an implantable photon-detecting digital chip. This review presents the basic biochemical background that allows the lux system to continuously autobioluminesce and highlights the important milestones in the use of lux-based bioreporters as they have evolved from chemical detection platforms in prokaryotic bacteria to rodent-based tumorigenesis study targets. In addition, the future of lux imaging using integrated circuit microluminometry to image directly within a living host in real-time will be introduced and its role in the development of dose/response therapeutic systems will be highlighted.
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Sensors 2012, 12, 732-752; doi:10.3390/s120100732
sensors
ISSN 1424-8220
www.mdpi.com/journal/sensors
Review
The Evolution of the Bacterial Luciferase Gene Cassette (lux)
as a Real-Time Bioreporter
Dan Close 1, Tingting Xu 2, Abby Smartt 2, Alexandra Rogers 2, Robert Crossley 2, Sarah Price 2,
Steven Ripp 2 and Gary Sayler 1,2,*
1 Oak Ridge National Laboratory, The Joint Institute for Biological Sciences, 676 Dabney Hall,
Knoxville, TN 37996, USA; E-Mail: dclose@utk.edu
2 The Center for Environmental Biotechnology,The University of Tennessee, 676 Dabney Hall,
Knoxville, TN 37996, USA; E-Mails: txu2@utk.edu (T.X.); asmartt@utk.edu (A.S.);
aroger24@utk.edu (A.R.); rcrossl1@utk.edu (R.C.); sprice12@utk.edu (S.P.); saripp@utk.edu (S.R.)
* Author to whom correspondence should be addressed; E-Mail: sayler@utk.edu;
Tel.: +1-865-974-8080; Fax: +1-865-974-8086.
Received: 26 November 2011; in revised form: 30 December 2011 / Accepted: 9 January 2012 /
Published: 11 January 2012
Abstract: The bacterial luciferase gene cassette (lux) is unique among bioluminescent
bioreporter systems due to its ability to synthesize and/or scavenge all of the substrate
compounds required for its production of light. As a result, the lux system has the unique
ability to autonomously produce a luminescent signal, either continuously or in response to
the presence of a specific trigger, across a wide array of organismal hosts. While originally
employed extensively as a bacterial bioreporter system for the detection of specific
chemical signals in environmental samples, the use of lux as a bioreporter technology has
continuously expanded over the last 30 years to include expression in eukaryotic cells such
as Saccharomyces cerevisiae and even human cell lines as well. Under these conditions,
the lux system has been developed for use as a biomedical detection tool for toxicity
screening and visualization of tumors in small animal models. As the technologies for lux
signal detection continue to improve, it is poised to become one of the first fully
implantable detection systems for intra-organismal optical detection through direct
marriage to an implantable photon-detecting digital chip. This review presents the basic
biochemical background that allows the lux system to continuously autobioluminesce and
highlights the important milestones in the use of lux-based bioreporters as they have
evolved from chemical detection platforms in prokaryotic bacteria to rodent-based
tumorigenesis study targets. In addition, the future of lux imaging using integrated circuit
OPEN ACCESS
Sensors 2012, 12 733
microluminometry to image directly within a living host in real-time will be introduced and
its role in the development of dose/response therapeutic systems will be highlighted.
Keywords: mammalian cells; bacterial luciferase (lux); bioreporter; biosensor; cell culture;
small animal models
1. Introduction
Bacterial bioluminescence, commonly known as the lux reaction, is the most widely distributed
luminescent mechanism on the planet [1] and, although this process of bacterial light production
has been observed for centuries, it was not until the mid 1900s that it began to be evaluated
scientifically [2,3]. Beginning in the 1980s, after several decades of research, the understanding of this
system became advanced enough that it was possible to exogenously express the full gene cassette,
comprised of five genes (luxCDABE), in alternative host organisms such as Escherichia coli [4]. As
researcher’s understanding of the biochemistry behind the lux reaction continued to be refined, and
genetic manipulation techniques improved, it soon became possible to exploit this cassette as a reporter
system across a wide variety of bacterial species for an extremely diverse set of monitoring objectives.
Following the success of these myriad lux-based bacterial bioreporters, attempts were made to
incorporate the system into eukaryotic organisms in order to expand the lux system’s usefulness as a
reporter. While initially expression of the bacterial genes was unsuccessful, through rearrangement of
the lux cassette gene expression pattern and improvement of expression efficiency via codon-optimization
and the addition of specialized linker regions, these hurdles were overcome and the lux reaction was
demonstrated to occur in the lower eukaryote Saccharomyces cerevisiae [5]. Building upon this early
success of eukaryotic expression, the luxAB genes were then further engineered to express in a human
cell line, leading to the emergence of the lux system as a truly multifunctional reporter system similar
to the more commonly employed firefly luciferase system [6].
Recently the lux system has undergone another substantial improvement, as it has been
demonstrated that the full cassette can be optimized in a similar manner to the luxAB genes in order to
promote fully autonomous bioluminescent production in a human cell line without the need to
exogenously supplement a chemical substrate [7]. This review will highlight the development of the
lux cassette from a curiosity observed in marine bacteria, through its extensive use as a bacterial
bioreporter system and modification for expression in eukaryotic organisms, up to its recent
demonstration as the only fully autonomous, substrate-free bioluminescent reporter system available in
the eukaryotic host background. The unique, autonomous nature of the lux cassette will also be
reviewed in light of the development of advanced photon detection hardware, detailing the future
directions of lux development and its potential for biomedical as well as basic research applications.
Wild-Type lux Background
One does not have to look very far to see the glow of naturally bioluminescent organisms. On land
bioluminescence is most commonly observed in the glow of fungi growing on decaying wood or from
Sensors 2012, 12 734
insects displaying their luminescent signal after dusk, while in marine environments bioluminescence
is most commonly observed in single celled bacteria that are found either living freely or in symbiosis
with larger hosts. It is these bioluminescent bacteria that are the most abundant and widely distributed
of the light emitting organisms on Earth and they can be found in both aquatic (freshwater and marine)
and terrestrial environments. Despite the widespread prevalence of bacterial bioluminescence,
however, the majority of these organisms are classified into just three genera: Vibrio, Photobacterium,
and Photorhabdus (Xenorhabdus) [1]. Although they are viable as free-living bacteria, these organisms
are most commonly observed in symbiosis with a larger host. There is still no consensus as to the
evolutionary benefit of bioluminescent production, however, in general it is theorized that the
production of light can aid in the consumption of free living bacteria by higher trophic organisms,
transferring them to a more controlled, nutrient rich habitat inside the host, or that, likewise, symbiotic
bacteria can aid their hosts through the production of light that attracts prey, aids in camouflage, or
attracts mates, in return for the shelter and nutrients provided by living within the body of the host
organism [8]. Regardless of the reasons, the genetic system employed for the generation of
bioluminescence is well conserved across all known bioluminescent bacteria. The luciferase protein is
a heterodimer formed by the luxA and luxB gene products. The luxC, luxD, and luxE gene products
encode for a reductase, transferase, and synthase respectively, that work together in a single complex
to generate an aldehyde substrate for the bioluminescent reaction. In some species, there is an
additional gene, frp, that functions as a flavin reductase to aid in the regeneration of the required
FMNH2 substrate. Together with molecular oxygen, these components are all that are required to
produce a bioluminescent signal [9] (Figure 1).
Figure 1. The luxCDABEfrp genes work synergistically with endogenous myristic acid,
FMN, and O2 to generate a bioluminescent signal. The frp gene is not found in all
organisms expressing the remaining lux genes. Originally published in and used with
permission from [1].
In addition, some marine species have additional genes that govern the expression of the remainder
of the operon. The luxI and luxR genes function as an autoinducer and transcriptional activator
(Figure 2), allowing the bioluminescent bacteria to participate in quorum sensing, therefore producing
S
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irst recom
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ron [12,13
u
x genes, i
n
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porters th
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esulting b
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ation of v
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]. Althoug
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vestigators
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oughput
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E
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isible ligh
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genes upo
n
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nstration,
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Photorha
b
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these eff
o
began to a
p
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expressin
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and the
f
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ontinuous
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nce is em
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t
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and a
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r
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g
Sensors 2012, 12 736
2.2. Development of lux as a Method for Visualizing Gene Expression
The first use of lux as a biomonitoring technology came soon after its transgenic expression in
E. coli, when Enbreghet et al. [14] fused the lux cassette to an inducible promoter that could be used to
monitor gene expression in vivo. Using this experimental design it became possible to monitor
autonomous bioluminescence as an indicator for the transcriptional activity of a promoter of interest.
Using this method, the first major targets of study were the E. coli lac and ara promoters and it was
discovered that upon IPTG or arabinose induction, light production in hosts expressing lux fusions
increased between 600 to 1,000-fold. Following these reports the lux system was used to monitor
regulation of the lateral flagella genes in Vibrio parahaemolyticus [14,15], providing its first
demonstration in a previously uncharacterized system. These applications represented a significant
shift in the way gene expression was investigated because, unlike traditional biochemical assays using
enzymatic reporters, the bioluminescent signal from the lux genes could be easily detected and
measured with high sensitivity without cell perturbation. This allowed the same sample to be
continuously monitored, thus revealing the dynamics of gene expression through changes in
bioluminescence over time. This new method was therefore capable of generating data that could not
previously be generated.
2.3. lux-Based Bioluminescence as a Tool for Cellular Population Monitoring
While the Lux proteins do not require exogenous substrate addition, their function does require
continued access to the molecular oxygen, FMNH2, and aldehyde co-substrates. For this reason, their
bioluminescence can only be detected in actively growing cells. This knowledge, combined with the
discovery that lux bioluminescent output is proportionally correlated to the number of cells present, has
therefore been used as a simple, sensitive, and non-destructive means for in situ bacterial monitoring.
This was first demonstrated by Shaw et al. [16] in 1986 when constitutively expressed V. fischeri
luxCDABE genes were introduced into the phytopathogen Xanthomonas campestris, and their
subsequent invasion of a cauliflower leaf was visualized. Similarly, de Weger and colleagues [17]
were successfully able to detect luxCDABE-labeled Pseudomonas fluorescens in the rhizosphere of
soybean roots using the same technique. Additionally, through the use of a lux-based system rather
than an enzymatic reporter, it was possible for these researchers to achieve detection limits three orders
of magnitude lower than what was previously possible, leading to improved signal detection. These
early examples highlighted the application of lux-based bioluminescence as a rapid, simple and
sensitive tool for in situ detection of living bacteria and established the foundation for future research
using lux to monitor genetically engineered microorganisms. In perhaps the most notable use of the lux
genes for tracking a cellular population, a P. fluorescens strain was transformed with the lux genes and
used for the first bioremediation-related environmental field release of a genetically engineered
microorganism in 1996.
This release was approved by the Environmental Protection Agency to determine the efficiency of
bioremediation process monitoring through inoculation of the bioluminescent strain directly into
contaminated soil and to determine its ability to monitor the bioremediation of polycyclic aromatic
hydrocarbons [18]. By placing the lux genes under the control of promoters in the naphthalene
Sensors 2012, 12 737
degradation pathway, it was possible to monitor their bioluminescent output as a measure of
naphthalene contamination in the soil [19]. Using a combination of bioluminescent and traditional
culture based detection methods, the release area was monitored for two years after the release of
bioluminescent P. fluorescens. Over this time, regular sampling was performed to track the amount of
bacteria present in the soil, as well as the amount of bioluminescence produced, which were indicative
of organism presence and naphthalene degradation, respectively (Figure 3). Based on culture detection
methods, the bioluminescent P. fluorescens persisted in both contaminated and non-contaminated
soils, decaying at similar rates and producing similar colony counts [18]. The long term nature and
difficulty in remote monitoring of bacterial populations presented in this study illustrates how the
unique properties of the lux operon can provide it with an advantage over its substrate requiring
bioluminescent or UV stimulation requiring fluorescent counterparts. Because of its autonomous
nature, the lux-tagged P. fluorescens could be continually surveyed for bioluminescent production,
without the need for repeated stimulation to induce a reporter signal.
Figure 3. Using bioluminescent bacteria, Ripp et al. were able to track both the presence of
the genetically modified organisms as well as the their effectiveness in degrading
naphthalene over time. Naphthalene concentrations are shown in ppm. Adapted and used
with permission from [18].
2.4. The Use of lux for Exogenous Target Detection
Following the work that demonstrated how the lux cassette could be used as a tool for visualizing
gene expression, it soon became clear that these genes could be adapted for use as a traditional
bioreporter target through activation under specific, predetermined conditions as well. By expressing
the lux cassette under the control of a promoter with a known inducer, the resultant bioluminescent
emission could be used as an indicator for the presence of the given stimulus, and fluctuation of the
bioluminescent signal could be interpreted as changes in the bioavailable concentration of the inducer
compound. Building upon these ideas, the first use of bioluminescence for monitoring metabolic
Sensors 2012, 12 738
activity was demonstrated in Pseudomonas putida by Burlage et al. in 1990 [20]. Here, naphthalene
degradation was monitored using a transcriptional fusion of the salicylate inducible nah promoter and
the luxCDABE genes. Salicylate is an intermediate metabolite of naphthalene, which is eventually
degraded to acetaldehyde and pyruvate in Pseudomonas. Therefore, naphthalene degradation could be
correlated to the light emission upon induction with naphthalene-derived salicylate. The nondestructive
nature of the lux system allowed for this analysis to occur in real time in a growing culture, providing
continuous monitoring of naphthalene metabolism across various stages of growth. It was later
determined by King et al. [19] that the bioluminescent signal was controlled in a dose/response
fashion (Figure 4), therefore demonstrating its usefulness in determining contaminant levels in mixed
environmental samples. This opened the door for a multitude of environmental bioreporters featuring
lux, such as that developed by Applegate and colleagues that was used to monitor for water soluble
benzene, toluene, ethylbenzene, and xylene (BTEX) compounds indicative of petroleum spills. This
reporter, constructed by linking expression of the lux cassette to the toluene dioxygenase promoter,
was capable of detecting as little as 30 µg of toluene/L in as quickly as 2 h and maintained its detection
ability for over 100 generations without antibiotic selection [21].
Figure 4. By treating with naphthalene over 8 h intervals (black boxes), King et al. were
able to demonstrate a corresponding dose/response bioluminescent production (y) curve.
Adapted and used with permission from [19].
Another common target for lux-based environmental sensing has been phenol. Notably,
Abd-El-Haleem et al. [22] constructed one of the first lux-based phenol biosensors by inserting a
mopR-like promoter fused to the V. fischeri lux cassette genes into Acinetobacter sp DF4. This reporter
was capable of demonstrating a lower detection limit of 2.5 ppm in 4 h when exposed to phenol, and
was only responsive to three of the ten phenol derivatives tested, suggesting that it was relatively
specific as well. This is, however, not by any means the only lux-based phenol reporter to be
developed. Davidov et al. [23] made extensive use of recA promoters fused to lux cassettes, with each
of the reporters containing a slight variation in its promoter sequence, that were expressed either in
E. coli or Salmonella typhimurium and using lux genes from either V. fischeri or P. luminescens. The
most sensitive of these reporters was that expressing the V. fischeri lux genes in E. coli, which was
capable of detecting 0.008 mg phenol/L in 2 h. This same construct, when expressed in S. typhimurium
was also capable of detecting phenol in 2 h but required a minimum concentration of 16 mg phenol/L,
demonstrating the differences in host phenol bioavailability.
Tim
e
(
h
)
Bioluminescence
(
µ
A)
Sensors 2012, 12 739
2.5. Further Uses of lux as a Bacterial Bioreporter
As the popularity of the lux system has grown over the years, an increasing number of bacterial
reporters have been leveraged for the detection of a wide variety of contaminants. While this review
focuses only on the seminal examples of lux’s growth as a reporter system, a larger list of target
compounds and detection limits of various bioreporters can be found in recent reviews [24,25] and
Table 1.
Table 1. A representative listing of luxCDABE-based bioreporters.
Analyte Time for induction Concentration Reference
2,3 Dichlorophenol 2 h 50 mg/L [23]
2,4,6 Trichlorophenol 2 h 10 mg/L [23]
2,4-D 20–60 min 0.44 mg/L [26,27]
3-Xylene Hours 3 μM [28]
4-Chlorobenzoate 1 h 380 μM–6.5 mM [29]
4-Nitrophenol 2 h 0.25 mg/L [23]
Alginate production 1 h 50–150 mM NaCl [30]
Ammonia 30 min 20 μM [31]
Androgenic chemicals 3–4 h 109–1010 M [32]
Antibiotic effectiveness
against Staphylococcus aureus
infections in mice
4 h 100 CFU [33]
Antimony (antimonate and
antimonite) 3–4 h 0.1 mg/L [34]
Arsenic 3–4 h 80 µg/L As(V); 8 µg/L As(III) [35]
BTEX (benzene, toluene,
ethylbenzene, xylene) 1–4 h 0.03–50 mg/L [21]
Cadmium 4 h 19 mg/kg [36]
Chlorodibromomethane 2 h 20 mg/L [23]
Chloroform 2 h 300 mg/L [23]
Chromate 1 h 10 μM [37]
Cobalt 9 µM 2.0 mM [38]
Copper 1 h 0.05 mg/L [39]
Dichloromethane 1–2 h ~0.01 mg/L [40]
DNA damage (cumene
hydroperoxide) 50 min 6.25 mg/mL [41]
DNA damage (mitomycin) 1 h
Not specified
0.032 μg/mL
0.31 μg/mL
[42]
[43]
DNA damage and other cell
stressors/activators
A library of luxCDABE-based fusions with 689 E. coli
gene promoters [44]
Estrogenic chemicals 1 h 1011 M [45]
Gamma-irradiation 1.5 h 1.5–200 Gy [46]
Heat shock 20 min Various, depending on chemical
inducer used [47,48]
Heavy metals A multi-bioreporter panel for detecting and identifying
multiple heavy metal contaminants in a single sample [49]
Sensors 2012, 12 740
Table 1. Cont.
Analyte Time for induction Concentration Reference
Hemolysin production Not specified 5 mM cAMP [50]
Hydrogen peroxide 20 min 0.1 mg/L [51]
in vivo monitoring of
Salmonella typhimurium
infections in living mice
4 h 100 CFU [52]
Iron Hours 10 nM–1 μM [53]
Isopropyl benzene 1–4 h 1–100 μM [54]
Lead 1 h 0.33 mg/L [39,55]
Mercury 2 h 0.5 ng/L [56]
N-acyl homoserine lactones
(3-oxo-C6-HSL) 200 min 3 nM [57]
Naphthalene 8–24 min 12–120 μM [58]
Nickel 4–6 h 0.1 µM [38,59]
Nitrate ~1 h 1 mg/L [60]
Organic peroxides 20 min Not specified [51]
Oxidative stress Not specified 0.015 ppm (paraquat) [61]
PCBs 1–3 h 0.8 μM [62]
p-chlorobenzoic acid 40 min 0.06 g/L [29]
p-cymene <30 min 60 ppb [63]
Pentachlorophenol 2 h 0.008 mg/L [23]
Phenol 2 h 16 mg/L [23]
Salicylate 15 min 36 μM [58]
Shiga toxin expression in E. coli Gene expression profiling in enterohemorrhagic E. coli [64]
Tetracycline 40 min 5 ng/mL [65]
Toxicity monitoring Use of multi-bioreporter arrays to survey and identify multiple
chemicals within single samples [66,67]
Trichloroethylene 1–1.5 h 5–80 μM [68]
Trinitrotoluene Not specified Not specified [69]
Ultrasound 1 h 500 W/cm2 [70]
Ultraviolet light (bacterial) 1 h 2.5–20 J/m2 [71]
Ultraviolet light (yeast) 1 h 7 mJ/cm2 [72]
Zinc 4 h 0.5–4 μM [73]
3. Eukaryotic Expression of the lux Cassette
Despite its success as a bacterial bioreporter, widespread use of the lux system faced a major hurdle
in that it was initially believed to be capable of expression only in prokaryotes. Although several
attempts were made to express the lux genes in eukaryotic hosts, none of these made significant
headway [74–76]. It would not be until 2003 that the first major achievement was documented with the
demonstration of autonomous bioluminescence from the yeast Saccharomyces cerevisiae [5].
Following this breakthrough, the lux genes continued to be modified and improved for eukaryotic
expression, later being developed into a reliable yeast-based bioassay tool and, eventually, becoming
capable of expression in a human cell line [7], opening the door for continued development in the future.
Sensors 2012, 12 741
3.1. lux Expression in Yeast
It was the demonstration of lux function in S. cerevisiae by Gupta et al. [5] in 2003 that marked the
first time a eukaryotic organism successfully produced levels of bioluminescence comparable to
prokaryotic lux-based bioreporters (Figure 5). To achieve this, Gupta and colleagues chose to express
the lux genes from the terrestrial bacterium P. luminescens rather than those from the traditional
marine organisms V. harveyi or V. fischeri. This was done because the resulting luciferase proteins
from P. luminescens exhibit a higher thermal stability than those of their marine counterparts. To
mimic the organization and expression of the lux genes found in prokaryotic organisms, the luxA and
luxB genes were expressed from a single promoter and linked by an internal ribosomal entry site
(IRES). Under this expression strategy it was determined that bioluminescence was 20 times greater
than that reported for fused luciferases upon exposure to an n-decanal substrate. Building upon these
findings, the remainder of the lux genes were incorporated using the same strategy, with a pair of genes
linked by an IRES element and driven by a unique promoter. When expressed concurrently this design
was capable of producing an easily detectable bioluminescent signal.
Figure 5. Comparison of (A) S. cerevisiae and (B) E. coli expressing the luxCDABEfrp
genes. Used with permission from [5].
Using the lessons learned from creation of the bioluminescent yeast strain, work was then begun to
develop the eukaryotic lux system into a functional bioreporter for the detection of estrogenic
compounds—a task that was not possible using prokaryotic hosts. Sanseverino et al. [45] built upon
the lux plasmids developed by Gupta, creating a second set that constitutively expressed the luxC,
luxD, luxE, and frp genes while regulating expression of the remaining luxA and luxB genes through
insertion of human estrogen response elements (Figure 6). Upon exposure to estrogenic compounds,
yeast expressing these regulated lux genes would produce a bioluminescent signal in as quickly as 1 h.
This improved significantly over the colorimetric yeast estrogen screen, which could take as long
as five days to produce results under identical conditions. Within two years of this successful
demonstration of lux-based bioluminescent yeast as estrogen reporters, the same group had expanded
S
t
h
a
p
o
J
e
3
fu
t
h
b
i
s
t
h
t
h
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r
c
f
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t
r
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r
p
t
h
h
C
a
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m
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n
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ensors 201
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e functio
n
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o
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r
Continui
n
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rther refi
n
h
e lux sys
t
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iolumines
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s
capable
o
h
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h
erefore pr
o
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r
eporters s
u
c
ellular bac
k
Despite
t
f
irst publis
h
D
espite the
r
ansfect h
u
P
atterson et
r
equired op
t
p
reference.
T
h
e probabil
h
uman ribo
s
C
raney et
a
a
llowed fo
r
u
nlike the
w
r
eplacemen
t
m
proved e
x
n
corporati
o
2, 12
n
ality of th
e
e
d by repl
androgen
r
c
and andr
o
o
re, the lux
-
e
ening met
h
e
6. Sanse
v
r
aldehyde-3
the contro
l
n
the prese
n
r
ession in
M
n
g the dev
e
n
ed and opt
i
t
em posses
c
ent reporte
r
o
f producin
g
luminesce
n
o
duce real
-
it is not s
u
u
ch as GF
P
k
ground.
t
he potenti
a
h
ed report
o
success ac
h
u
man cells
a
l
. [6] we
r
t
imization
o
T
his optim
i
ity that the
s
ome (Tab
l
al
. [79], wh
r
improved
w
ork of C
t
of the ye
a
x
pression o
o
n of these
m
e
assay by
i
acing the
r
esponse el
o
genic com
p
-
b
ased syst
e
h
ods [32].
v
erino et al.
-phosphate
l
of estrog
e
n
ce of estro
g
M
ammalian
e
lopment o
f
i
mized for
e
ses some
u
r
systems t
h
g
continuo
u
n
t reporter a
v
-
time data
i
u
bject to t
h
P
[78]. Th
e
a
l benefits
o
o
f human o
p
h
ieved in e
x
with the
r
e the first
o
f the DN
A
i
zation ser
v
full length
l
e 2). Thes
o reported
expressio
n
raney et a
a
st IRES s
e
f the down
m
odificatio
i
ncorporati
n
estrogen r
e
ements. H
o
p
ounds wh
e
e
m was sh
o
placed the
dehydrog
e
e
n response
g
enic comp
o
Cells
f
the lux s
y
e
xpression
i
u
nique cha
r
h
at are ava
i
u
s biolumi
n
v
ailable fo
r
i
n vivo [7,
7
h
e same hi
g
e
se traits
m
o
f human
c
p
timizatio
n
x
pressing t
h
same cons
to demons
t
A
sequenc
e
v
ed to incre
a
of the ope
n
e results
m
that codon
n
in GC-ri
c
l
., Patterso
e
quence wi
t
stream lux
B
ns, Patters
o
n
g detectio
n
e
sponse el
e
o
wever, thi
s
e
n combine
d
o
wn to be
m
alcohol de
h
e
nase pro
m
elements
(
o
unds. Ada
p
y
stem as a
i
n human c
e
r
acteristics
i
lable for u
s
n
escence wi
r
human cel
7
7]. In add
i
g
h levels
o
m
ake it an
c
ell line ex
p
n
until full
c
h
e full lux
c
tructs and
t
rate that e
f
e
of each g
e
a
se the effi
c
n
reading fr
a
m
irrored th
o
modificati
o
c
h bacteria
n had to
i
t
h a mam
m
B
gene in t
h
o
n et al. w
e
n
of andro
g
e
ments th
a
s
simple ch
a
d
with the e
m
ore effec
t
h
ydrogena
s
m
oter (P
GPD
)
ERE) to p
r
p
te
d
and us
e
functional
e
ll lines. T
compared
s
e in huma
n
thout addit
i
l imaging t
h
i
tion,
b
eca
u
o
f backgro
u
attractive
o
p
ression, it
c
assette ex
p
c
assette in
S
produce
a
f
ficient exp
r
e
ne to mo
r
c
iency of tr
a
a
me would
b
o
se demons
t
o
n of the
A
such as
S
i
nclude ad
d
m
alian opti
m
h
e
m
amma
l
e
re able to
d
g
enic comp
o
a
t controll
e
a
nge allow
e
xisting lux
-
t
ive, faster,
s
e 1 promot
)
controlli
n
r
ovide con
d
e
d with per
m
eukaryotic
h
is process
w
to the alt
e
n
cell lines.
i
on of an e
x
h
at can fun
c
u
se it is bi
o
u
nd produc
e
o
ption for
would stil
l
p
ression w
a
S
. cerevisia
a
biolumin
e
r
ession of t
h
r
e closely
m
a
nscription
/
b
e recogni
z
t
rated in t
h
A
T-rich P.
S
treptomyc
e
d
itional mo
m
ized IRES
l
ian cellula
r
d
emonstrat
e
o
unds as w
e
e
d lux exp
r
e
d for para
l
-
b
ased estr
o
and more
er (P
ADH1
)
a
n
g luxA an
d
itional exp
m
ission fro
m
reporter s
y
w
as undert
a
e
rnative flu
o
Because t
h
x
ogenous l
u
c
tion indep
e
o
luminesce
n
e
d by som
e
imaging i
n
l
be five y
e
a
s finally r
e
e, it was n
o
e
scent sign
h
e luxA an
d
m
atch the
h
/
translation
z
ed and exp
r
h
e prokary
o
luminescen
e
s coelicol
o
difications
element t
o
r
environm
e
e
constituti
v
7
4
e
ll. This w
a
r
ession wi
t
l
lel detecti
o
o
gen report
e
specific th
a
a
nd the
d luxB
ression
m
[45].
y
stem, it w
a
a
ken becau
s
o
rescent a
n
h
e lux syste
m
u
ciferin, it
e
ndently, a
n
n
t rather th
a
e
fluoresce
n
n
the hum
a
e
ars from t
h
e
ported [6,
7
o
t possible
t
al. In 200
5
d
luxB gen
e
h
uman cod
o
and increa
s
r
essed by t
h
o
tic arena
b
s lux oper
o
or
. Howev
e
such as t
h
o
provide f
o
e
nt. Throu
g
v
e expressi
o
4
2
a
s
t
h
o
n
e
r.
a
n
a
s
s
e
n
d
m
is
n
d
a
n
n
t
a
n
h
e
7
].
t
o
5
,
e
s
o
n
s
e
h
e
b
y
o
n
e
r,
h
e
o
r
g
h
o
n
S
o
c
t
o
o
g
e
o
c
e
t
h
S
ensors 201
o
f the lux
l
c
assette an
d
o
firefly lu
c
Table
(http:/
/
genes
Used
w
Gene
luxA (wt)
luxA (op)
luxB (wt)
luxB (op)
Followin
o
ptimized f
o
g
enes to m
o
e
lements, a
n
o
riginal co
n
c
ontaining
t
e
longation
f
h
e control
o
Fi
g
ur
e
codon
simul
t
2, 12
l
uciferase
h
d
creating a
c
iferase, on
l
2. Transc
r
/
genes.mit.
e
in a huma
n
w
ith permi
s
Type B
e
1
6
1
1
1
a Initiaion si
g
g the inro
a
o
r expressi
o
o
re closely
m
n
d then di
v
n
struct cre
a
t
he luxC a
n
f
actor 1-α p
o
f the cyto
m
e
7. For e
x
-optimized
,
t
aneously e
x
h
eterodime
r
substrate
d
l
y with add
i
r
iption and
e
du) for e
x
n
host. Sc
o
s
sion from
[
e
gin En
d
6
1 1,08
1 1,08
1 98
4
1 98
4
g
nal; b Termi
n
a
ds made b
y
o
n in huma
n
m
atch the
h
v
iding thei
r
a
ted by P
a
n
d luxE ge
n
romoter an
d
m
egalovirus
x
pression o
f
,
separated
b
x
pressed w
i
r
, providin
g
d
ependent l
u
i
tion of the
translation
x
pression o
f
o
re interpre
t
[
6].
d
Lengt
h
3 1,023
3 1,083
4
984
4
984
n
ation; c Codi
n
y
Patterso
n
n
cell lines.
h
uman cod
o
r
expressio
n
a
tterson w
a
n
es (separat
e
d
the luxD
immediate
f
the full l
u
b
y IRES e
l
i
thin the ho
s
g
the fram
e
u
x reporter
s
inexpensiv
e
prediction
f
wild type
t
ation: 0–5
0
h
I a
45
66
51
66
n
g region sco
r
n
et al., it
w
This was
a
o
n preferen
c
n
across th
r
a
s retaine
d
e
d by an I
R
and frp ge
n
early pro
m
u
x cassette
l
ements, an
d
s
t. Adapted
e
work for
f
s
ystem tha
t
e
aldehyde
n
scores fro
m
(wt) and
o
0
weak, 50
T b
42
42
38
41
r
e; d Probabili
t
w
as not lo
n
ccomplish
e
c
e, separati
n
r
ee sequen
c
d
, but co-
e
R
ES eleme
n
n
es (also se
p
m
oter (Figur
e
in a huma
n
d
divided a
and used
w
f
uture opti
m
t
could be
u
n
-decanal r
a
m
the GE
N
o
ptimized (
o
100 mode
CodRg c
791
1,910
585
1,952
t
y of an exon
;
n
g before t
h
e
d by re-en
g
n
g them by
c
es in two
e
xpressed
w
n
t) under t
h
p
arated by
a
e
7).
n
cell, all
o
cross two
p
w
ith permis
s
m
ization o
f
u
sed in a m
a
ather than
D
N
SCAN al
g
o
p) luxA a
n
rate, >100
P d
T
0.7
0.88
0.97
0.99
;
e Exon score
h
e full lux
g
ineering e
a
human opt
separate p
l
w
ith a sec
o
h
e control
o
a
n IRES el
e
o
f the gen
e
p
lasmids th
a
s
ion from [
7
7
4
f
the full l
u
a
nner simil
a
D
-luciferin.
g
orith
m
n
d luxB
strong.
T
ranslated
e
67.01
181.78
46.37
185.60
.
cassette w
a
a
ch of the l
u
imized IR
E
l
asmids. T
h
o
nd plasm
i
o
f the hum
a
e
ment) und
e
e
s were
a
t were
7
].
4
3
u
x
a
r
e
a
s
u
x
E
S
h
e
i
d
a
n
e
r
Sensors 2012, 12 744
It is possible that this two plasmid expression system could itself contribute to the production of
bioluminescence in human cells since Yagur-Kroll and Belkin [80] have recently reported that splitting
the five lux genes into two smaller units (luxAB and luxCDE) resulted in improved bioreporter
performance in E. coli. This increased bioluminescent production is hypothesized to be due to the
associated enhanced transcriptional and/or translational efficiency achieved through the expression of
smaller open reading frames, which theoretically could serve the same function in eukaryotic cells
as well.
Using this expression strategy, it was demonstrated that these changes were both necessary and
sufficient for autonomous production of a bioluminescent signal when expressed in a human kidney
cell line (Figure 8) [7]. It should be noted that bioluminescent production from the human-optimized
lux cassette was demonstrated to be several orders of magnitude lower than that of the more common
firefly luciferase reporter and therefore greater numbers of bioluminescent cells were required to
produce a significantly detectable signal in both cell culture (15,000 lux-expressing cells vs. 50 firefly
luciferase-expressing cells) and small animal imaging experiments (25,000 lux-expressing cells
vs. 2,500 firefly luciferase cells). However, due to the autonomous nature of the lux system,
bioluminescent production was maintained over a longer period and produced less variability than did
the firefly luciferase system [78].
Figure 8. The optimization process employed by Close et al. was both necessary and
sufficient to induce bioluminescent production from a human cell line upon expression.
Adapted and used with permission from [7].
In one interesting experiment that took advantage of the autonomous nature of lux bioluminescence,
constitutively bioluminescent human cells expressing the lux genes were used to evaluate the
cytotoxicity of the aldehyde n-decanal [77]—the same aldehyde that was used by Patterson et al. [6] to
stimulate bioluminescent production in cell extracts containing optimized luxA and luxB genes. By
monitoring the changes in bioluminescent production following aldehyde treatment, it was possible to
evaluate not only which concentrations were cytotoxic to the cells, but also at what time following
exposure the effects began to take place, how long the cells were able to continue functioning under a
diminished capacity following introduction of the aldehyde, and at what point cells succumbed to
treatment and died. It was demonstrated that treatment with 0.00001%, 0.0001%, and 0.001% volumes
of aldehyde did not show any changes in bioluminescence [77], despite the fact that this compound has
been shown previously to function as a substrate for the lux reaction [6]. However, while treatment
with a 0.1% volume of aldehyde quickly diminished bioluminescent production, treatment with 0.01%
S
a
o
d
o
w
i
t
4
d
w
b
t
h
F
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p
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s
o
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t
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(
S
ensors 201
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llowed the
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scillated
be
d
eparture t
o
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ere taken
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e
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With th
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o
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ill most l
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ackground
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t
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ptimized
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g
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xpressing
t
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ircuit tran
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n
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o
e
tween a si
g
o
significan
t
c
hanges hig
h
a
t set time
p
v
e been pos
s
e
9. While
t
d
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n
o
l cells (red
significant
l
0
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n
-d
e
o
lism of c
e
e
, autonom
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f
lux Ima
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n in euka
r
i
kely revol
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Close et
a
reagent in
t
which red
u
n
the availa
b
however,
. It is also
i
t
he eukary
o
t
inues to b
e
a
nd biolumi
g
ive it a br
o
from the
s
nature alr
e
s
. In sever
a
t
he full lu
x
s
ducers. Th
e
n
provide f
o
o
rs to vis
u
g
nificant d
e
t
ly reduced
h
lights the
p
oints rathe
r
s
ible to co
m
t
reatment o
f
n
d above 0
boxes) and
l
y diminis
h
e
canal prov
e
lls in real
o
us nature
o
i
n
g
use of l
u
r
yotes, the
m
ve around
al
. [7] have
t
he lux rea
c
u
ces FMN t
o
b
ility of th
e
not to as
g
i
mportant t
o
o
tic-optimiz
e
used an
d
nescent ou
t
o
ader appe
a
biological
e
ady presen
t
a
l instances
,
x
cassette o
n
e
se miniat
u
o
r the dete
c
u
alize a 3.
5
e
cline and
t
biolumine
s
utility of t
h
r
than the n
e
m
pletely o
m
f
constituti
v
.001% did
all time po
i
h
ed in bio
l
ided the o
p
time. This
o
f the lux re
u
x as a bi
o
m
ajority of
its further
reported th
a
c
tion in hu
m
o
FMNH
2
,
c
e
aldehyde
c
g
reat an e
x
o
note that,
ed lux sys
t
d
is expose
t
put can b
e
a
l than it ha
s
modificati
o
t
s unique o
p
,
investigat
o
n
to small
f
u
rized devi
c
c
tion of the
b
5
h period
t
he negati
v
s
cent produ
c
h
e autonom
o
e
ar continu
o
m
it this peri
o
v
ely biolum
i
not show
a
i
nts survey
e
l
uminesce
n
p
portunity
t
type of a
n
action. Use
o
reporter i
n
developme
n
optimizati
a
t the avail
a
m
an cells, a
n
c
ould lead t
o
c
o-substrat
e
x
tent and
c
unlike alte
r
t
em has on
l
d to a wi
d
e
improved.
s
currently.
o
ns that c
o
p
portunitie
s
o
rs lead by
f
ootprint (
~
c
es, called
b
b
ioreporter
where the
e control
v
c
tion (Figu
r
o
us lux sys
t
o
us monito
r
o
d of activi
t
i
nescent hu
m
a
ny signific
a
e
d from cel
l
n
t producti
o
t
o view c
h
n
alysis is
m
d with per
m
n
bacteria,
n
t that rem
a
o
n and ex
p
a
bility of t
h
n
d increase
s
o
biolumin
e
e
were als
o
c
ame with
r
nate repor
t
l
y recently
d
er variety
This wou
l
o
uld be e
n
s
for mergi
n
Sayler et
a
~
1 c
m
3
), lo
w
b
iolumines
c
optical sig
n
biolumine
s
v
alue, follo
w
r
e 9) [77].
T
t
em, becau
s
r
ing perfor
m
t
y from the
r
m
an cells
w
a
nt differe
n
l
s treated w
i
o
n (green
h
anges in c
m
ade possi
b
m
ission fro
m
and the r
e
a
ins for thi
s
p
ansion in
h
e FMNH
2
c
s
in the effi
c
e
scent incre
a
o
shown to
e
possible c
t
er systems
become a
v
of investi
g
l
d enhance
n
gineered i
n
n
g it directl
y
al
. [81–83]
h
w
power (
3
c
ent biore
p
n
al, the dis
t
s
cent outp
u
w
ed by a fi
T
he monit
o
s
e if indivi
d
m
ed in the
i
r
esultant d
a
w
ith concen
t
n
ce from u
n
i
th 0.1%
n
-
d
boxes), tr
e
ellular hea
l
b
le because
m
[77].
e
cent proo
f
s
unique re
p
the eukar
y
c
o-substrat
e
c
iency of e
x
a
ses as larg
e
e
nhance bi
o
oncerns o
v
such as G
F
v
ailable fo
r
g
ators, it c
a
the usabili
t
n
to the lu
x
y
with exist
h
ave integ
r
3
.3 milliwa
t
p
orter integ
r
t
inguishing
7
4
u
t from ce
l
nal, consta
n
o
ring of the
s
d
ual readin
g
i
nvestigatio
n
a
ta.
t
rations
n
treated
d
ecanal
e
atment
l
th and
of the
f
-in-princip
p
orter syste
m
y
otic cellul
a
e
is current
l
x
pressing t
h
e
as 151-fol
d
o
luminesce
n
v
er increas
e
F
P and fire
fl
r
use. As t
h
a
n be furth
e
t
y of the l
u
x
system,
i
ing detecti
o
r
ated bacter
t
t) integrat
e
r
ated circu
i
of this sign
4
5
l
ls
n
t
s
e
g
s
n
,
al
m
a
r
l
y
h
e
d
.
n
t
e
d
fl
y
h
e
e
r
u
x
i
ts
o
n
ia
e
d
i
ts
al
Sensors 2012, 12 746
from noise, the digital processing of the signal, and local communication of the result within a single,
self contained package (Figure 10).
Figure 10. Despite its small size, the BBIC chip contains all of the necessary circuitry for
the detection and reporting of bioluminescent cells. By imaging directly on the chip prior
to photons passing through host tissue, signal collection will be greatly simplified. Used
with permission from [84].
When bioluminescent bacterial cells are interfaced to the BBIC, as few as 5,000 can be detected and
distinguished from background [81] and it is sensitive enough to differentiate bioluminescent output
levels stemming from changes in the concentration of the exposed stimulating analyte [81,82]. These
devices could be paired with lux-expressing eukaryotic cells and then implanted into small animal
models for direct internal imaging of reporter signals without the need to anesthetize or remove the
animal from its natural habitat, offering unparalleled opportunities for studying changes in physiology
and compound bioavailability under a wide range of conditions. Similarly, these cells could be
complexed with microcircuitry capable of initiating hormone or therapeutic compound dosage. In this
fashion, the lux-expressing cells could be programed to continuously monitor the body for specific
target compounds, acting as real-time biosentinels that detect changes in physiology and whose
resultant changes in bioluminescent output could trigger the release of counteractive compounds. This
would allow the development of fully autonomous, implantable dose/response therapeutic devices.
5. Conclusions
While the use of bacterial bioluminescence as a reporter system has been employed for quite a long
time, it is still a continually developing reporter system. There are many examples from the recent
literature that demonstrate new and creative lux-based bacterial biosensors that are employed for
myriad sensing applications, and there are also recent examples showing how modification of these
genes has expanded their usage to new applications that were previously thought impossible. The
unique ability of the lux system to produce a bioluminescent signal without exogenous substrate input
has ensured that it will continue to find use in basic and applied scientific research for years to come.
Whether or not it continues to be improved for function in eukaryotic cells may well decide the true
extent of lux usage in the future, however, for the time being it remains both an interesting and
practical example of the benefits available from an optical reporter system.
Sensors 2012, 12 747
Acknowledgements
Portions of this review reflecting work by the authors was supported by the the National Institutes of
Health, National Cancer Institute, Cancer Imaging Program, award number CA127745-01, the University
of Tennessee Research Foundation Technology Maturation Funding program, National Science
Foundation Division of Chemical, Bioengineering, Environmental, and Transport Systems (CBET) under
award number CBET-0853780, and the Army Defense University Research Instrumentation Program.
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Chapter
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