Note: Descriptions are shown in the official language in which they were submitted.
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DESCRIPTION
IN VIVO BIOSENSOR APPARATUS AND METHOD OF USE
1.1 FIELD OF THE INVENTION
The invention generally relates to the field of implantable diagnostic devices
(i.e.
devices deployed within the body of an animal) for monitoring one or more
target
substances, analytes, or metabolites in the animal. More particularly, the
invention
provides implantable biosensor devices for monitoring and regulating the level
of
analytes in the tissues and circulatory system of a human. In illustrative
embodiments, the
apparatus comprises a biosensor that is utilized to monitor the level of blood
glucose in a
diabetic or hypoglycemic patient. The disclosed sensors may also be used to
control or
regulate the delivery of a drug or other pharmaceutical agent from an external
or an
implantable drug delivery system. For example, the device may form part of an
artificial
pancreas to regulate insulin dosage in response to the level of glucose
detected in situ.
1.2 DESCRIPTION OF RELATED ART
1.2.1 BIOSENSORS
Biosensors are hybrid devices combining a biological component with an
analytical measuring element. The biological component reacts and/or interacts
with the
analytes of interest to produce a response measurable by an electronic,
optical, or
mechanical transducer. The most common configurations presently available
utilize
immobilized macromolecules such as enzymes or antibodies to form the
biological
component. Examples of analytes and immobilized macromolecules include:
glucose and
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immobilized glucose oxidase (e.g., Wilkins et al., 1990: nitrate and
immobilized nitrate
reductase (Wu et al., 1997); hydrogen peroxide and 2,3-dichlorophenoxyacetic
acid and
immobilized horseradish peroxidase (Rubtsova et al.. 1998); and aspartate and
immobilized L-aspartase (Campanella et al., 1995).
1.2.2 WHOLE-CELL BIOSENSORS
A further refinement for biosensors has been developed in recent years that
utilizes
intact living cells, such as a microorganism, or an eukan~otic cell or cell
culture as an
alternative to immobilized enzymes. Microbial cells are especially well suited
for
biosensor technologies; they are physically robust, capable of existing under
extremely
harsh and widely fluctuating environmental conditions, they possess an
extensive
repertoire of responses to their environment, and they can be genetically
engineered to
generate reporter systems that are highly sensitive to these environmental
responses.
Polynucleotide sequences that comprise specific promoter sequences are
operably linked
to a gene or a plurality of genes that encode the desired reporter enzymes)
and then
introduced into and maintained within the living cell. 'Vhen the target
analyte is present,
the reporter genes are expressed, generating the enzymes) responsible for the
production
of the measured signal. Commonly used reporter systems have utilized either
the ~i-
galactosidase (lack or catechol-2,3-dioxygenase (xyl.E~ enzymes (Kricka,
1993).
Unfortunately, a limitation of these systems has been that following exposure
to
the target substance(s), the cells must be destructively Ivsed and the
enzymes) isolated.
This lysis is then followed bs the addition of one or more secondary
metabolites to yield a
colorimetric signal that is proportional to the concentration of enzyme{s) in
solution,
providing a means to quantiy the concentration of the original target
substance.
A more recent improvement in such sensors utilizes green fluorescent protein
as a
reporter system, with the significant advantage that cells do not require
destructive assay
techniques to produce colorimetric signals. Because a substrate must be added
to the
green fluorescent protein constructs to first initiate the light response,
however, these
systems are quite complicated and offer little advantage for detection of
analytes in situ
(Prasher, 1995).
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1.2.3 Irv Vwo SENSORS
The development of an integrated in vivo implantable glucose monitor was first
reported by Wilkins and Atanasov (1995). This system utilizes glucose oxidase
immobilized within a micro-bioreactor. This enzyme catalyzes the oxidation of
(3-D-
glucose by molecular oxygen to yield gluconolactone and hydrogen peroxide,
with the
concentration of glucose being proportional to the consumption of O~ or the
production of
Hz02. Unfortunately, the presence of a glucose oxidase inhibitor molecule in
the human
bloodstream tended to offset proportionality constants, and made the device
unsatisfactorily inaccurate for precise glucose monitoring and control (cough
et al.,
1997). Also limiting was the device's relatively large size (~5 x 7 cm), which
negated its
usefulness as an implantable device.
Although several smaller needle-type and microdialysis glucose sensors have
since
been developed to circumvent size limitations (cough et al., 1997, Selam,
1997), their
reliance on a glucose oxidase enzyme-based system limits their overall
effectiveness and
reliability.
Several nonspecific electrochemical sensors have also been investigated as
potential in vivo glucose sensors (e.g., Yao et al., 1994; Larger et al.,
1994), but problems
including limited sensitivity. instability, and limited long-term reliability
have prevented
their wide-spread utilization (Patzer et al., 1995). According to Atanasov et
al. (1997),
continuously functioning implantable glucose biosensors with long-term
stability have yet
to be achieved.
1.3 DEFICIENCIES IN THE PRIOR ART
Despite a significant miniaturization of biosensors during the past decade,
they are
still relatively large and obtrusive to serve as ideal implantable devices.
Current
methodologies using mammalian bioluminescent reporter cells require cell lysis
and
addition of an exogenous substrate to generate a measurable response.
Consequently,
these cells cannot serve as continuous on-line monitoring devices.
Therefore, there remains a need for the development of a small implantable
monolithic (i.e. containing both biological and electrical components
constructed on a
single substrate layer) bioelectronic monitor that is durable, inexpensive,
wireless, and
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that can communicate remotely to a drug delivery system to provide the
controlled
delivery of a therapeutic agent such as insulin.
2.0 Summary of the Invention
The present invention overcomes these and other inherent limitations in the
prior
art by providing implantable apparatus and methods for detecting and
quantitating
particular analytes in the body of an animal. In particular, the invention
provides devices
for the in vivo detection and quantitation of metabolites, drugs, hormones,
toxins, or
microorganisms such as viruses in a human or animal. In illustrative
embodiments, the
invention provides a BBIC device useful for the detection of glucose in a
human. Such
devices provide for the first time an accurate on-line detector for glucose
monitoring, and
offer the ability to control the administration of pharmaceutical agents via
an external or
implantable drug delivery system. Also disclosed are BBIC devices for
detecting the
concentration of signature molecules (i. e. proteins released from cancer
cells, etc. ),
clotting factors, enzymes and the like, and other analytes present in the
bloodstream or
interstitial fluid. In the area of oncology, the biosensor devices find
utility in both initial
and remission monitoring, on-line measurement of the effectiveness of
chemotherapy, and
stimulation/activity of the immune system. Likewise, the biosensor devices are
useful in
other areas of medicine, including on-line monitoring for enzymes associated
with the
occurrence of blood clots (strokes, heart attacks, etc. ), detection and
quantitation of
clotting factors (maintain level), hormone replacement, continuous drug
monitoring
(testing for controlled substances in prisoners, military personnel, etc. ),
monitoring of
soldiers exposure to sub-lethal exposure to nerve agents and other
debilitating agents,
monitor levels of compounds affecting mental illness, and the like.
In one embodiment there is provided an implantable monolithic bioelectronic
device for detecting at least one analyte within the body of an animal, said
device
comprising:
an integrated circuit including at least one transducer for generating an
electrical
signal in response to light incident thereon; and
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a bioreporter, said bioreporter for emitting light when exposed to said
analyte, said
bioreporter positioned so that at least a portion of said emitted light
reaches said
transducer.
In a further embodiment there is provided an implantable monolithic
bioelectronic
device for detecting an analyte within the body of an animal. In a general
sense this
device comprises a bioreporter that is operably positioned above a substrate
that is on an
integrated circuit. The bioreporter is capable of metabolizing the target
analyte and emits
light consequent to this metabolism when in contact with the analyte. The
device further
comprises a sensor closely positioned to the integrated circuit that detects
the emitted
light and generates an electrical signal in proportion to the amount of light
generated by
the bioreporter. Preferably the entire implantable device is contained within
a
biocompatible
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container that is implanted within the body of the animal in which the analyte
detection is
desired.
The biocompatible container may be comprised of silicon nitride, silicon
oxide, or
a suitable polymeric matrix, with exemplary matrices such as polyvinyl
alcohol, poly-L-
lysine, and alginate being particularly preferred. The polymeric matrix may
also further
comprise a microporous, mesh-reinforced or a filter-supported hydrogel.
In certain embodiments, it may also be desirable to provide a transparent,
biocompatible, bioresistant separator that is operably positioned between the
phototransducer and the bioreporter.
The bioreporter preferably comprises a plurality of eukaryotic or prokaryotic
cells
that produce a bioluminescent reporter polypeptide in response to the presence
of the
target analyte. Prokaryotic cells such as one or more strains of bacteria, and
eukaryotic
cells such as mammalian cells are particularly preferred. Exemplary mammalian
cells are
human cells such as islet (3-cells, immortal stem cells, or hepatic cells,
with immortal stem
I S cells being particularly preferred.
These cells preferably comprise one or more nucleic acid segments that encode
a
luciferase polypeptide or a green fluorescent protein that is produced by the
cells in
response to the presence of the analyte. Preferably the nucleic acid segment
encodes an
Aqueorea Victoria, Renilla reniformis, or a humanized green fluorescent
protein, or more
preferably, a bacterial Lux polypeptide, such as the LuxA. LuxB, LuxC, LuxD,
or LuxE
polypeptide, or the LuxAB or LuxCDE fused polypeptides described herein.
Exemplary bacterial hrx gene sequences that may be employed to prepare the
genetic constructs include the Vibrio fischerii or more preferably, the
Xenorhabdus
luminescens IuxA, IuxB, IuxC, luxD, IuxE, IuxAB, or IuxCDE genes.
Exemplary lux gene sequences that may be employed for preparation of the
genetic constructs as described herein include the gene sequences disclosed in
SEQ ID
NO:I. Exemplary Lux polypeptide sequences are disclosed in SEQ ID N0:2, SEQ ID
N0:3, SEQ ID N0:4, SEQ ID NO:S and SEQ ID NO:6.
The Lux polypeptides preferably comprise at least a 10 contiguous amino acid
sequence from one or more of the polypeptide sequences disclosed in SEQ ID
N0:2
through SEQ ID N0:6. More preferably the Lux polypeptides comprise at least a
15
contiguous amino acid sequence from one or more of the polypeptide sequences
disclosed
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in SEQ ID N0:2 through SEQ ID N0:6, and more preferably still. comprise at
least a 20
contiguous amino acid sequence from one or more of the polypeptide sequences
disclosed
in SEQ ID N0:2 through SEQ ID N0:6.
Such polypeptides are preferably encoded by a nucleic acid sequence that
comprises at least 20, at least 25, at least 30, at least 3~. at least 40, or
at least 45 or more
contiguous nucleotides from SEQ ID NO: 1.
The expression of the Lux-encoding nucleic acid segments is preferably
regulated
by a nucleic acid regulatory sequence operably linked to the Lux-encoding
segment.
Preferably this regulatory sequence comprises a cis-acting element that is
responsive to
the presence of the target analyte. Exemplary cis-acting response elements are
selected
from the group consisting of an S14 gene sequence. a hepatic L-pyruvate kinase
gene
sequence, a hepatic 6-phosphofructo-2-kinase gene sequence, a (3-islets
insulin gene
sequence, a mesangial transforming growth factor-(3 gene sequence, and an
acetyl-
coenzyme-A carboxylase gene sequence.
In an illustrative embodiment, the cis-acting response element comprises a
contiguous nucleotide sequence from a (3-islets insulin gene sequence or a
hepatic L-
pyruvate kinase gene sequence. Expression of the nucleic acid sequence is
preferably
regulated by a promoter sequence such as the one derived from an L-pyruvate
kinase-
encoding gene described herein.
The device may further comprise a wireless transmitter, an antenna, and a
source
of nutrients capable of sustaining the bioreporter cells. Likewise the
biocompatible
container enclosing the bioreporter may further comprise a membrane that is
permeable to
the analyte but not to the bioreporter cells themselves. Such a semi-permeable
membrane
permits analytes to flow freely from the bodily fluid into the detector
device, but restricts
the migration of bioreporter cells from the device into the surrounding
tissues or
circulatory system of the body in which the device is implanted.
In one embodiment, the integrated circuit is a complementary metal oxide
semiconductor (CNiOS) integrated circuit. The integrated circuit may comprise
one or
more phototransducer, that themselves may be comprised of one or more
photodiodes.
Likewise, the integrated circuit may also further comprise a photodiode, a
current-to-
frequency converter. a digital counter, and/or a transmitter that is capable
of transmitting
either digital or analog data.
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The invention also provides an implantable controlled drug delivery system
that
comprises both the bioluminescent bioreporter integrated circuit (BBIC) device
and an
implantable drug delivery pump that is capable of being operably controlled by
the BBIC
and that is capable of delivering the drug to the body of the animal in
response to controls
by the device. The invention also concerns a method of providing a controlled
supply of
a drug to a patient in need thereof. The method generally involves implanting
within the
body of the patient the controlled drug delivery system.
The invention also provides a method of determining the amount of a drug
required by a patient in need thereof, such as in the case of giving a
diabetic patient an
appropriate amount of insulin. The method generally involves implanting within
the body
of the diabetic patient one or more BBIC devices that are responsive to either
glucose,
glucagons, insulin, or another glucose metabolite, and determining the amount
of insulin
required by the patient based upon the levels of the analyte detected in the
body fluids by
the device. When the device indicates that higher levels of insulin are
required, the
appropriate control signal can be sent to the drug delivery system and more
insulin is
injected into the body. When the device indicates that lower levels of insulin
are required,
then the appropriate control signal can be sent to the drug delivery system
and less insulin
can be administered. Such "real-time" monitoring of glucose in the body of the
animal
permits for controlled release of insulin throughout the day, and obviates the
need for
daily or more frequent injections of insulin that may either be too much or
too little for
the particular time of administration. This affords a more cost-effective
administration of
the drug, and also provides a more stable dosing of the insulin to the patient
on an "as
needed" basis.
The invention also provides a kit for the detection of an analyte, and such
kits
generally will include one or more of the disclosed BBIC devices in
combination with
appropriate instructions for using the detection device. Such kits may also
routinely
contain one or more standardized reference solutions for calibrating the
device, and may
also include suitable storage or nutrient medium for sustaining the
bioreporter cells either
during storage or during use once implanted within the body of the animal. In
the case of
therapeutic kits, such kits will also generally include one or more controlled
delivery
systems for administration of the drug to the body of the animal.
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The invention also provides a method of regulating the blood glucose level of
an
animal in need thereof. This method generally comprises monitoring the level
of glucose
in the bloodstream or inerstitial fluid of the patient using the BBIC device,
and
administering to the patient an effective amount of an insulin composition
sufficient to
regulate the blood glucose level.
This new type of bioluminescence-based bioreporter is capable of monitoring
target substances without the disadvantageous requirement that cells be
destroyed to
produce the measurable signal. This allows for monitoring to occur
continuously, on-line
and in real-time (Simpson et al., 1998a, 1998b). These cells rely on
luciferase genes
(designated lux in prokaryotes and luc in eukaryotes) for the reporter enzyme
system. Intl.
Pat. Appl. Ser. No. PCT/LJS98/25295, published as W099/27351 on June 3, 1999
discloses a self contained miniature bioluminescence bioreporter integrated
circuit
("BBIC") that was designed to detect specific molecular targets ex situ or ex
vivo.
The present invention concerns an implantable, or an in situ or an in vivo
BBIC
device that is capable of being implanted within the body of an animal, and
that is capable
of detecting the concentration of one or more analytes present within the
animal. The
implantable monolithic bioelectronic device of the present invention generally
comprises
a substrate, a bioreporter capable of responding to a particular substance by
the emission
of light, a container affixed to the substrate capable of holding the
bioreporter, an
integrated circuit on the substrate including a phototransducer operative to
generate an
electrical signal in response to the light wherein the signal indicates the
concentration of
the substance ; and a biocompatible housing that is capable of being implanted
within the
body of an animal, with that portion of the housing covering the bioreporter
container
comprising a semi-permeable membrane that permits passage of the analyte from
the
body of the animal to contact the biosensor, but restricts the bioreporter
molecules from
diffusing into the body of the animal that contains the implanted device. The
bioreporter
may be in solution, that is a cell suspension, and entrapped in the container
by the
semipermeable membrane, or alternatively the bioreporter may be encapsulated
in a
selectively permeable polymer matrix that is capable
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The apparatus may further comprise a layer of bioresistantJbiocompatible
material
between the substrate and the container, such a layer of silicon nitride. The
integrated
circuit is preferably a CMOS integrated circuit, and the phototransducer is
preferably a
photodiode.
The integrated circuit may also include a current to frequency converter
and/or a
digital counter. Additionally, the integrated circuit may also include one or
more
transmitters. Such transmitters may be wireless, or conventionally wired. In
preferred
embodiment, the apparatus also includes a drug delivery device capable of
receiving
transmissions from the transmitter.
A further embodiment of the invention is an implantable apparatus for
detecting a
selected substance in solution, which comprises an integrated circuit
including a
phototransducer adapted to input an electrical signal into the circuit in
response to light, a
bioreporter capable of responding to selected substance in solution by
emitting light, the
reporter adapted to contact the substance; and a transparent, biocompatible,
and
bioresistant separator positioned between the phototransducer and the
bioreporter to
enable light emitted from the bioreporter to strike the phototransducer. In a
preferred
embodiment of the present invention, the selected substance is glucose. The
bioreporter
may be a mammalian cell that contains a nucleotide sequence that encodes one
or more
luminescent reporter molecules. Such a nucelotide sequence may comprise one or
more
lux genes. In a preferred embodiment the lux genes comprise both IuxCDE genes
and
fused IuxAB genes. In one embodiment, these lux genes are derived from
Xenorhabdus
luminescens. The lux genes may be regulated by a nucleic acid sequence
comprising one
or more cis-acting glucose response elements. In an illustrative embodiment,
the glucose
response element may be derived from the ~i-islets or hepatic L-pyruvate
kinase gene. In
a highly preferred embodiment the p.LPK.LucFF plasmid is used to provide one
or more
glucose response elements and the L-pyruvate kinase promoter to drive the
expression of
one or more lux genes. The cells constituting the bioreporter may be in
suspension,
entrapped in place on the IC by a semi-permeable membrane. Alternatively the
cells
constituting the bioreporter may be encapsulated in a polymer matrix affixed
to IC. Such
a matrix may be permeable to the selected substance in solution.
A further embodiment of the invention concerns an implantable monolithic
bioelectronic device for detecting a selected substance in body fluid. This
device
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generally comprises a biocompatible housing; a bioreporter capable of
responding to a
selected substance by emitting; and, a sensor capable of generating an
electrical signal in
response to the reception of the emitted light. Such a device may also include
a
transparent, bioresistant and biocompatible separator positioned between the
bioreporter
and the sensor and a semi-permeable membrane positioned in the biocompatible
housing
so that the selected substance can access the bioreporter.
A standard integrated circuit (IC) is coated with a layer of insulating
material such
as silicon dioxide or silicon nitride. This process is called passivation and
serves to
protect the surface of the chip from moisture, contamination, and mechanical
damage.
BBICs require a second coating that must be biocompatible and bioresistant,
must protect
the OASIC from chemical stresses, must be optically tuned to efficiently
transmit the light
from the material under test, must adhere to an oxide coating, must be pin-
hole free, and
must be able to be patterned in order to form openings over the bonding pads
and
whatever structures that might be needed to maintain the bioreporter or
collect a sample.
I S The present invention contemplates that the components of the biosensor
may be
packaged in kit form. Kits may comprise, in suitable container means, one or
more
bioreporters and an integrated circuit including a phototransducer. Kits may
further
comprise a drug delivery device.
2O 3.O BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included
to
further demonstrate certain aspects of the present invention. The invention
may be better
understood by reference to one or more of these drawings in combination with
the detailed
description of specific embodiments presented herein. Illustrative embodiments
of the
25 present invention are depicted in the drawings, with like numerals being
used to refer to
like and corresponding parts of the various drawings.
FIG. 1 shows a perspective view of one illustrative embodiment of the
invention.
FIG. 2 shows a side view of an illustrative embodiment of the present
invention.
FIG. 3 shows a block diagram of an illustrative embodiment of the integrated
30 circuit.
FIG. ~1A shows a high-quality photodetector that can be made using a standard
N-
well CMOS process.
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FIG. 4B shows two photodetector structures fabricated in a silicon-on-
insulator
CMOS process: on the left, a lateral PIN detector; on the right, a device
similar to left
except that the junction is formed with a Schottky junction.
FIG. 5A shows a simple photodiode consisting of a P-diffusion layer, an N-
well,
and a P-substrate.
FIG. 5B shows a circuit using a large area photodiode for efficient light
collection,
and a small-area diode in a feedback loop to supply the forward bias current
that cancels
out the photocurrent.
FIG. SC shows a circuit using correlated double sampling (CDS) to minimize the
effects of low frequency (flicker) amplifier noise as well as time or
temperature dependent
variations in the amplifier offset voltage.
FIG. 6 shows the current-to-frequency converter architecture of the apparatus.
FIG. 7 shows a prototype BBIC biosensor.
FIG. 8 shows a minimum detectable concentration of toluene as a function of
integration time for the prototype BBIC employing the bioreporter Pseudomonas
putida
TVAB.
FIG. 9 shows the schematic representation of a peritoneal glucose biosensor
and
insulin pump.
FIG. 10A shows a schematic representation of an implantable biosensor
containing t<vo separate photodetectors with the bioreporters responding to
either an
increase or decrease in glucose concentrations.
FIG. lOB shows a side view of biosensor showing silastic covering.
FIG. lOC shows a schematic representaion of the utilization of a seleetably
permeable membrane to protect bioreporters from the immune response.
4.O DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
The luciferase system has been adapted for use in biosensors in vivo. In
prokaryotes, the lux system consists of a luciferase composed of two subunits
coded for
by the genes luxA and IuxB that oxidize a long chain fatty aldehyde to the
corresponding
fatty acid resulting in a blue-green light emission at an approximate
wavelength of 490 nm
(Tu and Mager, 1995). The system also contains a multienzyme fatty acid
reductase
consisting of three proteins, a reductase encoded by Ia~xC, a transferase
encoded by IuxD,
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and a synthetase encoded by luxE that convert and recycle the fatty acid to
the aldehyde
substrate. The genes are contained on a single operon, allowing for the
cloning of the
complete lux gene cassette downstream from user-specific promoters for the
utilization of
bioluminescence to monitor gene expression. The majority of bioluminescent
bioreporters consist of Gram-negative organisms engineered to detect and
monitor
critically important chemical and environmental stressors (Ramanathan et al.,
1997,
Steinberg et al., 1995). Luciferase fusions in Gram-positive bacteria, as well
as in yeast
cell lines, are also being successfully perfornned (Andrew and Roberts, 1993.
Srikantha et
al., 1996).
Eukaryotic luciferase genes cloned into bacterial reporters include the
firefly
luciferase (luc) producing light near 560 nm and the click beetle luciferase
(lucOR)
emitting light near 595 nm (Cebolla et al., 1995, Hastings, 1996). Eukaryotic
bioreporters
have been designed to monitor glucose concentrations in rat islet ~3-cells
(Kennedy et al.,
1997), steroid activity in HeLa cells (Gagne et al., 1994}, ultraviolet light
effects in mouse
fibroblast cells (Filatov et al., 1996), toxicity effects in human liver
cancer cells
(Anderson et al., 1995), estrogenic and antiestrogenic compounds in breast
cancer cell
lines (Demirpence et al., 1995), and erythropoiten gene induction in human
hepatoma cell
lines (Gupta and Goldwasser, 1996). To date, most eukaryotic bioluminescent
reporters
require cell destruction and the addition of an exogenous substrate, usually
luciferin, to
generate a measurable luminescent response.
Green fluorescent protein ("GFP") is also routinely used as a reporter system,
with
the significant advantage that cells do not require destructive assay
techniques to produce
colorimetric signals (Hanakam et al., 1996; Grygorczyk et al., 1996; Siegel
and Isacoff,
1997; Biondi et al., 1998). However, a substrate must be added to the GFP
constructs to
first initiate the light response (Prasher, 1995). Humanized GFP cDNA has been
developed which is specifically adapted for high-level expression in mammalian
cells,
especially those of human origin (Zolotukhin 1996). Humanized GFP can be
efficiently
inserted into mammalian cells using viral vectors (Levy et al., 1996; Gram er
al., 1998).
Detection of the bioluminescent signal from the reporter organisms is achieved
through the use of optical transducers, including photomultiplier tubes,
photodiodes,
microchannel plates, photographic films, and charge-coupled devices. Light is
collected
and transferred to the transducer through lenses, fiber optic cables. or
liquid light guides.
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However, applications requiring small volumes, remote detection, or multiple
parallel
sensing necessitate a new type of instrumentation that is small and portable,
yet maintains
a high degree of sensitivity.
S 4.1 OVERVIEW OF THE SYSTEM
The present invention describes an implantable BBIC that detects selected
substances. The bioreporter is a genetically engineered cell line in which the
nucleic acid
sequence contains a cis-activating response element that is responsive to the
selected
substance. In preferred embodiments, the selected substance is glucose.
Exposure of the
bioreporter to the selected substances causes the response element to up-
regulate a nucleic
acid sequence that encodes one or more polypeptides that generate a
luminescent
response. In a preferred embodiment, the luminescent response is generated by
a
prokaryotic lux system.
The function of the IC portion of the BBIC is to detect, filter, amplify,
digitize,
I S and report the bioluminescent signal. In effect, the IC serves as a
complete laboratory
instrument-on-a-chip: a microluminometer.
Silicon-based ICs can detect optical signals in the near ultraviolet, visible,
and near
infrared regions using the PN junctions normally used to form transistors
(Simpson et al.,
1999a). Using an n-well/p-substrate photodiode in a 0.~-um bulk CMOS IC
process, an
~66% quantum efficiency has been measured at the 490-nm bioluminescent
wavelength
(Simpson et al., 1999b). :~ variety of signal-processing schemes can be
employed.
However, counting the pulses from a current-to-frequency converter circuit
forms a long
time-constant integrator and is the causal portion of the matched filter for a
low-level
bioluminescent signal in white noise. Using the photodiode mentioned above
with this
2S signal-processing scheme, an rms noise level of 17S electrons/second was
measured for a
13-minute integration time, corresponding to a detection limit of 500
photons/second
(Simpson et al., 1999b).
A prototype BBIC was constructed by placing the toluene sensitive bioreporter,
P.
putida TVAB, above a custom integrated microluminometer. FIG. 6 shows the
prototype
BBIC (including the bioreporter enclosure) as used in the characterization
studies
(Simpson et al., 1998b: Simpson et al., 1998c; Simpson et al., 1998d).
13
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With no luminescent signal coming from the cells, multiple measurements were
taken with the integration time set to 1-minute. Leakage currents produced a
signal of ~6
counts/minute .with a standard deviation (a) of 0.22 counts/minute. As
expected. the a
decreased with the square root of the integration time. Longer integration
times were
produced off line by summing 1-minute measurements.
Bioltuninescence was induced in the BBIC cells and a control sample of cells
by
exposure to toluene vapor. From the control sample measurements, we estimate
that the
toluene concentration was no more than 1 ppm. A signal of 12 counts/minute (6
counts/minute above background) was measured. From previous measurements, P.
putida
TVA8 is known to have a linear response to toluene concentration until
saturating when
the concentration reaches a level of approximately 10 ppm. The minimum
detectable
toluene concentration for this BBIC as a function of integration time is shown
in FIG. 8.
In general. the minimum detectable concentration is also a function of the
number of
bioreporter cells and the area of the photodiode.
A naphthalene-sensitive BBIC was produced using the microluminometer
described above and the bioreporter P. fluorescens SRL. Using the same
experimental
procedure described above, this BBIC was exposed to naphthalene vapor with a
concentration of approximately 10 ppm. A signal of 240 counts/minute was
recorded.
To eliminate the need for the addition of exogenous substrate, cells must
themselves supply the appropriate substrate for the luciferase. In the
bacterial system the
substrate is generated by a fatty acid reductase complex coded for by the
luxCDE genes.
This enzyme complex reduces short chain fatty acids to the corresponding
aldehyde. The
lueiferase then oxidizes the aldehyde to the corresponding fatty acid. The
preferred fatty
acid for this reaction is myristic acid, which is present in eukaryotic
organisms (Rudnick
et al., 1993). Myristic acid is usually involved in the myristoylation of the
amino
terminus that is associated with membrane attachment (Borgese et al., 1996,
Brand et al.,
1996).
In a preferred embodiment, the bioreporter for glucose monitoring will be a
mammalian bioluminescent reporter cell line that has been genetically
engineered to
express luminescence in response to glucose concentrations on a continuous
basis, without
the need for cell destruction and exogenous substrate addition. Current
methodologies
using mammalian bioluminescent reporter cells require cell lysis and addition
of an
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WO 00/33065 PCT/US99/28733 _
exogenous substrate to generate a measurable response. Consequently, these
cells cannot
serve as continuous on-line monitoring devices. In a preferred embodiment,
this new cell
line is constructed with a bioluminescent reporter utilizing the IuxAB and
IuxCDE genes
from X. luminescens incorporated into a plasmid-based system designated
p.LPK.Luc~
which contains a eukaryotic luc gene able to respond to glucose
concentrations.
Replacement of the luc gene with the IuxAB gene will allow for bioluminescence
measurements to occur in real-time with glucose concentrations, negating the
requirement
for cell destruction and substrate addition.
To form an implantable, glucose-monitoring BBIC, the bioreporters may be
entrapped in a container behind a semi-permeable membrane that keeps them in
place
over the IC photodetector. Alternatively the bioreporter may be encased in a
polymer
matrix. The BBIC is enclosed in a biocompatible housing with a semi-permeable
membrane covering the bioreporter region. This membrane allows glucose to pass
to the
bioreporters, yet stops the passage of larger molecules that could interfere
with the
glucose measurement. When the glucose reaches the bioreporter, it is
metabolized and the
cells emit visible light. The IC detects this light, amplifies and filters
this signal, and then
reports this measurement. This measurement could be reported to the patient
(e.g., to a
wristwatch receiver) or could be reported to an insulin pump in a closed-loop
system that
functions much like the pancreas.
FIG. 1 shows a perspective view of the present invention. Glucose 10 that is
being detected enters the BBIC 11 through the semi-permeable membrane 12 that
covers
the bioreporter.
FIG 2 shows a side view of the present invention. The BBIC is enclosed in a
biocompatible housing 20 with a semi-permeable membrane 21 covering the
bioreporter
held in a container 22. The cells constituting the bioreporter may be in
suspension or
encapsulated in a polymer matrix. The bioreporter is separated from a
photodetector 23
by a protective coating 24. A single substance 25 contains the photodetector
as well as
additional circuits 26 that process and transmits the signal.
FIG. 3 shows a block diagram of one embodiment of the integrated circuit
("IC").
The photodetector is a photodiode 33 connected to a current to frequency
converter 30.
The photodiode responds to light by sinking a current. The current is
converted to a series
of pulses that are accumulated in a digital counter 31. The number of counts
in the
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counter in a fixed amount of time is directly proportional to the amount of
light collected
by the photodiode, which is directly proportional to the concentration of
glucose. Digital
processing circuitry in the digital counter would determine the appropriate
next step for
an insulin pump based on the measured glucose levels. The measured
concentration or
next instruction for the insulin pump could be reporting via the wireless
transmitter 32.
All these circuits (photodiode, signal processing, and wireless transmission)
can be
fabricated on one IC.
FIG. 4 shows the bioreporter being supplied with water and nutrients. A fluid
and
nutrient reservoir 141 is connected to a microfluidic pump 142 so that
nutrient and fluid
144 may flow through the polymer matrix 143 enclosing the bioreporter. Each of
these
components can be constructed on a single substrate 140.
FIG. 4A shows a high-quality photodetector made using a standard N-well CMOS
process. The photodetector consists of two reverse biased diodes in parallel.
The top
diode is formed between the P+ active layer 45 and the N-well 46, and the
bottom diode
is formed between the N-well 46 and the P-substrate 47. The top diode has good
short
wavelength light sensitivity (400 - 550 nm), while the bottom diode provides
good long
wavelength sensitivity (500 - 1100 nm). Thus, the complete diode is sensitive
over the
range from 400 to 1100 nm. The luminescent compound under test 41 is separated
from
the photodetector by a layer 40 of Si3N4 and a layer 42 of SiOz.
FIG. 10A, FIG. 10B, and FIG. lOC show schematic representations of an
implantable
biosensor containing two separate photodetectors with the bioreporters
responding to
either an increase or decrease in glucose concentrations.
FIG. lOB shows a side view of biosensor showing silastic covering.
FIG. lOC shows a schematic representaion of the utilization of a selectably
permeable
membrane to protect bioreporters from the immune response.
4.2 PHOTODETECTOR
The first element in the micro-luminometer signal processing chain is the
photodetector. The key requirements of the photodetector are:
~ Sensitivity to wavelength of light emitted by the bioluminiscent or
chemiluminiscent compound under test;
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Low background signal (i.e. leakage current) due to parasitic reverse biased
diodes;
~ Appropriate coating to prevent the materials in the semiconductor devices
from interfering with the bioluminescent or chemiluminescent process
S under study and to prevent the process under study from degrading the
performance of the micro-luminometer; and.
~ Compatibility with the fabrication process used to create the micro-
luminometer circuitry.
Two photodetector configurations that satisfy these requirements are described
below. It should be understood, however, that alternative methods of
constructing such a
photodetector can be used by one skilled in the art without departing from the
spirit and
scope of the invention as defined in the claims.
In the first embodiment, the photodetector is fabricated in a standard N-well
CMOS process. Shown in FIG. 5A, this detector is formed by connecting the PN
junction between the PMOS active region and the N-well in parallel with the PN
junction
between the N-well and the P-type substrate. The resulting detector is
sensitive to light
between approximately 400 nm and approximately 1100 nm, a range that
encompasses the
450 to 600 nm emission range of most commonly used bioluminescent and
chemiluminescent compounds or organisms. In order to meet the requirement that
the
device have a low background signal, the device is operated with a zero bias,
setting the
operating voltage of the diode equal to the substrate voltage. The photodiode
coating may
be formed with a deposited silicon nitride layer or other material compatible
with
semiconductor processing techniques.
In the second photodeteetor embodiment, the detector is fabricated in a
silicon-on
insulator (SOI) CMOS process. The internal leakage current in an SOI process
is two to
three orders of magnitude lower than in standard CMOS due to the presence of a
buried
oxide insulating layer between the active layer and the substrate. Two
photodetector
structures are envisioned in the SOI process. The first structure, shown on
the left of FIG.
5B, consists of a lateral PIN detector where the P-layer is formed by the P+
contact layer,
the I (intrinsic) region is formed by the lightly doped active layer, and the
N region is
formed by the N+ contact layer of the SOI CMOS process. The spectral
sensitivity of this
17
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lateral detector is set by the thickness of the active layer, which may be
tuned for specific
bioluminescent and chemiluminescent compounds.
The second structure, shown on the right side of FIG. 4B, is similar to the
first
except that the junction is formed with a Schottky junction between a
deposited cobalt
silicide (CoSi2) or other appropriate material layer and the lightly doped
active layer.
The inventors contemplate that other photodetector configurations may be
envisioned in silicon or other semiconductor processes meeting the criteria
set forth
above.
4.3 LOW NOISE ELECTRONICS
The low noise electronics are the second element in the micro-luminometer
signal
processing chain. The requirements for the low noise electronics are:
~ Sensitivity to very low signal levels provided by the photodetector;
~ Immunity to or compensation for electronic noise in the signal processing
chain;
~ Minimum sensitivity to variations in temperature;
~ Minimum sensitivity to changes in power supply voltages (for battery powered
applications);
~ For some applications the electronics must have sufficient linearity and
dynamic
range to accurately record the detected signal level; and,
~ In other applications the electronics must simply detect the presence of a
signal
even in the presence of electronic and environmental noise.
Three embodiments that satisfy these requirements are described below. It
should
be understood, however, that alternative methods of detecting small signals
while
satisfying these requirements may be used without departing from the spirit
and scope of
the invention as defined in the claims.
FIG. 5A schematically shows the first approach to the detection of very small
signals. This device uses a P-diffusion/N-well photodiode, a structure
compatible with
standard CMOS IC processes, in the open circuit mode with a read-out amplifier
(fabricated on the same IC with the photodiode). The luminescent signal
generates
electron-hole pairs in the P-diffusion and the N-well. The photo-generated
electrons in the
P-diffusion are injected into the N-well, while the photo-generated holes in
the N-well
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are injected into the P-diffusion. The N-well is tied to ground potential so
that no charge
builds up in this region. However, since the P-diffusion is only attached to
the input
impedance of a CMOS amplifier (which approaches infinity at low frequencies),
a
positive charge collects in this region. Thus, the voltage on the P-diffusion
node begins to
rise.
As the P-diffusion voltage begins to rise, the P-diffusion/N-well photodiode
becomes forward biased, thereby producing a current in a direction opposite to
the photo-
generated current. The system reaches steady state when the voltage on the P-
diffusion
node creates a forward bias current exactly equal in magnitude (but opposite
in polarity)
to the photocurrent. If this PN junction has no deviations from the ideal
diode equation,
then the output voltage is given by the following equation:
Ir"", = V. ln(Ip l (A Ir) + 1 ), (Eq. I )
where V, is the thermal voltage (approximately 26 mV at room temperature), Ip
is
the photo-current, A is the cross-sectional area of this PN junction, and Is
is the reverse
1 S saturation current for a PN junction with unit cross-sectional area. The
value of Is depends
greatly on the IC process and material parameters.
Two major error currents are present in PN junctions operating at low current
density: recombination current and generation current. Except at very low
temperatures,
free carriers are randomly created in the PN junction space charge region.
Since this
region has a high field, these thermally excited earners are immediately swept
across the
junction and form a current component (generation current) in the same
direction as the
photocurrent. Carriers crossing the space-charge region also have a finite
chance of
recombining. This creates another current component (recombination current) in
the
opposite direction of the photocurrent. Therefore, taking into account these
error currents,
Eq. I becomes:
you = Y~ ln((lo + IR- I~) l {A Is) + 1 ). {Eq. 2)
This output voltage is a function of parameters that are generally beyond the
inventors' control. However, the inventors do have control over the junction
area. A.
Unfortunately, to make the inventors' output signal larger, the inventors want
a small A,
while the inventors want a large A for a high quantum efficiency (QE).
19
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FIG. 5B shows a second microluminometer embodiment that satisfies both of
these needs. This circuit uses a large area photodiode for efficient light
collection, but
uses a small-area diode in a feedback loop to supply the forward bias current
that cancels
out the photocurrent. Once again, the amplifier and feedback diodes are
fabricated on the
same IC as the photodiode. For this circuit:
Vouc = 3 Vt ln((Ip + Ig - I,) ~ (Ag, IS) + 1), (Eq. 3)
where A,b is the small cross-sectional area of the feedback diode. More than
one diode is
used in the feedback path to make the output signal large compared to the DC
offset of
any subsequent amplifier stages. This technique allows efficient collection of
the light
with a large-area photodiode, yet produces a large output voltage because of
the small-
area diodes in the feedback path.
The feedback circuit of FIG. 5B maintains the photodiode at zero bias. With no
applied potential, the recombination and generation currents should cancel.
Eq. 3
becomes:
1 s vout = 3 Vt ln((Ip i (A~ IS)) + I ) (Eq. 4)
if the smaller recombination and generation currents in the smaller feedback
diodes are
neglected.
The principal advantages of the second micro-luminometer embodiment shown in
FIG. 5B include:
~ The SNR is totally determined by the photodiode; noise from the small
diode and amplifier are negligible;
~ Diodes can be added in the feedback path until the signal level at the
output of the amplifier is significant compared to offset voltages (and
offset voltage drift) of subsequent stages;
~ This method is completely compatible with standard CMOS processes
with no additional masks, materials, or fabrication steps;
~ This detection scheme can be fabricated on the same IC with analog and
digital signal processing circuits and RF communication circuits; and,
~ Measurement can be made without power applied to the circuit. Power
must be applied before the measurement can be read, but the measurement
can be obtained with no power.
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A third microluminometer implementation shown in FIG. SC uses correlated
double sampling (CDS) to minimize the effects of low frequency (flicker)
amplifier noise
as well as time or temperature dependent variations in the amplifier offset
voltage. As
shown in FIG. SC, a photodiode with capacitance Cd and noise power spectral
density S;
is connected to an integrating preamplifier with feedback capacitance Cf and
input noise
power spectral density S,, through a set of switches that are controlled by
the logical level
of a flip-flop output. When the flip-flop output is low, the switches are
positioned so that
the photocurrent flows out of the preamplifier, causing the output voltage of
the integrator
to increase. When the low-pass filtered integrator output voltage exceeds a
threshold,
V,", the upper comparator "fires" setting the flip-flop and causing its output
to go high.
The detector switches change positions, causing current to flow into the
integrating
amplifier, which in turn causes the amplifier output voltage to decrease. When
the
integrator output goes below a second threshold, VLO, the lower comparator
"fires"
resetting the flip-flop and causing the output to go low again. The process
repeats itself
as long as a photocurrent is present.
The average period of the output pulse, dt, is given by the following
equation:
Ot= 2Cf~VNr -yco
I , (Eq.S)
where YH, and V~o are the threshold voltages of the comparators and Ip is the
diode
photocurrent. Two noise sources contribute to error in the measured value of
dt. S; is the
input noise current power spectral density associated primarily with the
photodiode, and
S" is the input noise voltage power spectral density associated primarily with
the
preamplifier. The diode noise is given by the equation:
z
S; =2q~2Is+Ip Hz~, (Eq.6)
where IS is the photodiode reverse saturation current and Ip is the
photocurrent. As the
photocurrent approaches zero, the noise power spectral density approaches a
finite value
of 4yISA2/Hz. The noise voltage SV of the preamplifier is determined by its
design and has
units of VZ/Hz.
The transfer function from the point where the diode noise is introduced to
the
output of the integrator is given approximately by the equation:
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H' (~ ) ~ sC Cs+w ) ' (Eq. 7)
I t
where w~ is the corner frequency of the integrating amplifier and s = jw.
Ignoring for the
moment the effect of the switches, the transfer function from the point where
the amplifier
noise is introduced to the output of the integrator is given approximately by
the equation:
Hv(~) ~ C C Cd ~s+w ~ ~ (Eq. 8)
I t
The switches perform a correlated double sampling function that attenuates the
noise that appears below the switching frequency of the output pulse string.
The transfer
function of a correlated double sampling circuit is approximated to first
order by the
equation:
N(w ) ~ 2 , (Eq. 9)
s+/Or
where Ot is the average period of the output pulse string. Thus, taking into
account the
switches, the transfer function from the point where the amplifier noise is
introduced to
the output of the integrator is approximately given by the equation:
_CI+Cd Cwtl s
H"(w) ~ CI s+w t s+ 2/- (Eq. 10)
~I
This is an important result because the effective zero introduced in the noise
voltage transfer function reduces the effect of the flicker noise of the
amplifier. This is
particularly useful in CMOS implementations of the micro-luminometer where
flicker
noise can have a dominant effect.
The mean squared output noise at the output of the integrator is given by the
equation:
v"z = JS~~H~*Hv)+S; (H;*H;)dc~, (Eq. 11)
and the Ru'IS noise voltage is then given by the equation:
6,, - v"-' . (Eq. 12)
22
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The RMS error in the measured period is determined by the slope of the
integrated
signal and the noise at the output of the integrator following the
relationship:
~ (Eq. 13)
"~~r
or, approximately, by the equation:
S
(Eq. 14)
~ LVHI -~co) .
or
The error in measuring Ot may be reduced by collecting many output pulses and
obtaining an average period. The error in the measured average pulse period
improves
proportionately to the square root of the number of pulses collected, such
that
_ a" 1
~~ ~ ~VHr -I'io~ ~ (Eq~ 15)
nr
or
6" rmro's (Eq. 16)
~~Hi -y~o~ ~r
or
where tme~ is the total measurement time.
Thus, implementation of the micro-luminometer has the following advantages:
~ The low frequency "flicker" noise of the amplifier is reduced by a
correlated double sampling process; and,
~ Ideally, the accuracy of the measured photocurrent may be improved
without limit by acquiring data for increasing periods of time.
Of course, practical limitations imposed by the lifetime and stability of the
signals
produced by the luminescent compound under test will ultimately determine the
resolution
of this implementation.
4.~ READ-OUT ELECTRONICS
Several methods of communicating data from the BBIC to external receivers or
in
aivo drug delivery systems are envisaged. In a preferred embodiment the
communication
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WO 00/33065 PCT/US99/28733 _ ~--
method is an on-chip wireless communication system that reports the level of
the
photocurrent to computing circuitry contained within in vivo drug delivery
system or an
external receiver. In a closed-loop system, this computing circuitry would
determine the
amount of drug to be delivered by the in vivo drug delivery system. If an
external receiver
were used, the data from the BBIC along with the user inputs would be used to
determine
the amount of drug to be administered. The external receiver may include
wireless
transmission circuitry for communication with the in vivo drug delivery system
or the
drugs may be administered manually. Other methods of communicating BBIC data
include;
~ Generation of a DC voltage level proportional to the photocurrent with a
hardwire connection to an in vivo drug delivery system;
~ Generation of a DC current level proportional to the photocurrent with a
hardwire cannection to an in vivo drug delivery system;
~ Generation of a logical pulse string whose rate is proportional to the
photocurrent with a hardwire connection to an in vivo drug delivery
system;
~ On-chip implementation of an analog to digital converter that reports a
numerical value proportional to the photocurrent with a hardwire
connection to an in vivo drug delivery system;
~ On-chip implementation of a serial or parallel communications port that
reports a number proportional to the photocurrent with a hardwire
connection to an in vivo drug delivery system;
~ Generation of a logical flag when the photocurrent exceeds a predefined
level with a hardwire connection to an in vivo drug delivery system; and,
~ Generation of a radio-frequency signal or beacon when the photocurrent
exceeds a predefined level.
Wireless communication in vivo may be limited by signal attenuation by body
fluids, tissues, and health-related limits on RF signal levels. This may
require the BBIC
and in vivo drug delivery system to be closely spaced. which may not be the
optimum
configuration for all cases. In such cases, the BBIC could communicate to an
external
receiver located ex vivo but closer to the BBIC. This receiver could be
connected
24
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(hardwired or wirelessly) to a transmitter located ex vivo but closer to the
in vivo drug
delivery system.
Numerous algorithms are envisioned for controlling an in vivo drug delivery
system with a BBIC. These include, but are not limited to
~ a simple look-up table that administers a prescribed drug level that is
determined
only by a single BBIC data point;
~ a simple look-up table that administers a prescribed drug level when a
predetermined number of data points exceed a preset threshold;
~ an algorithm that determines drug dosage by rate of increase or decrease of
BBIC
signal
~ an algorithm that determines drug dosage by matching BBIC data points to
data
point patterns stored in memory
~ learning algorithms that use BBIC data point history and user inputs to
predict
correct drug dosage to achieve desired results.
Some of these algorithms may require two-way communication between the BBIC
and in
vivo drug delivery system. In this case, a receiver would be included on the
BBIC.
4.5 BIOCOMPATIBLE HOUSING AND SEMI-PERMEABLE MEMBRANE
The BBIC is enclosed in a biocompatible housing with a semi-permeable
membrane covering the bioreporter region. The preparation of biocompatible
coverings
for implants and prosthetic devices so as to minimize capsule formation and
physiological
rejection has been an area of extensive investigation. For example, U.S.
Patent 5,370,684
and U.S. Patent 5,387,247, describe the application of a thin biocompatible
carbon film to
prosthetic devices. A biocompatible implant material comprising a three-
dimensionally
woven or knitted fabric of organic fibers is disclosed in U.S. Patent
5,711,960. Other
coverings for implants constructed to present a biocompatible surface to the
body are
described in U.S. Patent 5,653,755, U.S. Patent 5,779,734, and U.S. Patent
5,814,091. In
addition collagen coating and albumin coating have been shown to improve the
biocompatibilty of implants and prosthetic devices (Marios et al., 1996;
Ksander, 1988).
The present invention contemplates the use of any suitable biocompatible
material to
either coat or form the housing.
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A semi-permeable membrane comprises that part of the BBIC housing that covers
the bioreporter and entraps them on the integrated circuit. This membrane
allows the
selected substance. such as glucose, to pass to the bioreporter, yet prevents
the passage of
larger molecules. Membranes designed for use with glucose-oxidase based
biosensors
may also used in the preferred embodiments of the present invention. Membranes
investigated and designed for use with glucose-oxidase based biosensors
include, but are
not limited to: polytetrafluoroethylene membranes (Vaidaya and Wilkins, 1993);
perfluorinated ionomer membranes (Moussy et al., 1994); charged and uncharged
polycarbonate membranes (Vadiya and Wilkins 1994); and cellulose acetate
membranes
(Wang and Yuan, 1995; Sternberg et al., 1988). In addition, other membranes
have been
developed for the use transplantation of islets or other cells bioengineered
to produce
insulin. The membranes must be permeable to glucose and other metabolites
while
exclude elements of the host immune system. Such membranes may be adapted for
use
with the present invention and include, but are not limited to: asymmetric
polyvinyl
alcohol) membranes (Young et al., 1996); poly(L-lysine) membranes (Tziampazis
and
Sambanis, 1995); polyurethane (Zondervan et al., 1992); nucleopore membranes
(Ohgawara et al., 1998); and agarose gel (Taniguchi et al., 1997).
Biocompatible
semipermeable membranes for encapsulation of cells to form an artificial organ
are
described in U.S. Patent 5,795,790 and U.S. Patent 5,620,883. A biocompatible
semi-
permeable segmented block polyurethane copolymer membrane and its use for
permeating molecules of predetermined molecular weight range are disclosed in
U.S.
Patent No. 5,428,123. The present invention contemplates the use of any
suitable semi-
permeable membrane that allows the selected substance access to the
bioreporter yet
prevents the passage of larger molecules.
4.6 DRUG DELIVERY DEVICES
Numerous drug delivery devices, implantable and external, have been previously
described which can be controlled by radio telemetry. For example, U.S. Patent
4,944,659
describes an implantable piezoelectric pump for drug delivery in ambulatory
patients.
U.S. Patent 5,474,552 describes an implantable pump for use in conjunction
with a
glucose sensor that can deliver multiple active agents, such as glucose,
glucagon, or
insulin as required. Separate pumps may be used for delivering each of the
agents or a
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single pump that is switchable between them may be used. U.S. Patent 5,569,186
describes a closed loop infusion pump system controlled by a glucose sensor.
U.S. Patent
4,637,391 describes a remote controlled implantable micropump for delivery of
pharmaceutical agents. The use of external drug delivery systems is
contemplated in other
embodiments of the present invention. For example, U.S. Patent 5,800,420
discloses a
pump position topically against the skin surface that delivers a liquid drug,
such as
insulin, via a hollow delivery needle extending into the dermis. In other
embodiments of
the present invention, the drug delivery system may be interfaced with the
biosensor
device and controlled directly, as opposed to remote telemetry control, from
the BBIC.
The pump delivery systems described above are examples to facilitate the use
of
the present invention. Drug delivery devices other than pump systems are also
contemplated by the present invention. For example, U.S. Patent 5,421,816
describes an
ultrasonic transdermal drug delivery system. Ultrasonic energy is used to
release a stored
drug and forcibly move the drug through the skin of an organism into the blood
stream.
Thus the invention contemplates the use of any suitable drug delivery system
that can be
controlled by the BBIC glucose monitor. The factors dictating the choice of
such a drug
delivery system and its use with the BBIC glucose monitor use will be known to
those
skill in the art in light of the present disclosure.
4.7 BIOLUMINESCENT BIOREPORTERS
In a preferred embodiment of the invention, the bioreporter for glucose
monitoring
will be a mammalian bioluminescent reporter cell line that has been
genetically
engineered to express luminescence in response to glucose concentrations on a
continuous basis. An implantable bioluminescent sensor requires a
bioluminescent
reporter that can function without the exogenous addition of substrate for the
luciferase
reaction. Current
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eukaryotic luciferase systems used in molecular biology require the addition
of exogenous
substrate because of the complex nature for the production of eukaryotic
luciferins. Cells
must be either permeabilized or lysed and then treated with an assay solution
containing
luciferin. Thus current eukaryotic luciferases systems are not preferred
candidates for on
line monitoring.
The requirement for the addition of exogenous substrate can be obviated by the
use of bacterial lux genes. In a preferred embodiment of the present invention
the lux
genes of X. luminescens, IuxAB and luxCDE, are used as the bioluminescent
reporter
system. The X. luminescens luxAB gene encodes the a- and /3-subunits of a
luciferase
enzyme that exhibits greatest thet~rnostability at 37°C, while other
bacterial iuciferases
lose significant activity above 30°C. The IuxCDE genes are required to
eliminate the need
for the addition of exogenous substrate. The aldehyde substrate of the
luciferase encoded
by the IuxAB genes is generated by a fatty acid reductase complex coded for by
the
IuxCDE genes. The preferred fatty acid for this reaction is myristic acid,
which is present
in eukaryotic organisms (Rudnick et al., 1993), and thus eukaryotic cells are
suitable host
cells for this reporter. The enzyme complex reduces the fatty acid to the
corresponding
aldehyde. The luciferase then oxidizes the aldehyde to back to the fatty acid.
Other bioluminescence nucleic-acid segments may include the lux genes of
Vibrio
fischerii, luxCDABE, or luciferases from other organisms capable of
bioluminescence that
can be adapted so not as to require the addition of exogenous substrate. In
other
embodiments of the invention. nucleic acid segment encodes green fluorescent
protein of
Aqueorea victoria or Renilla reniformis.
4.H RECOMBINANT VECTORS EXPRESSING BIOLUi~fINESCENCE GENES
One important embodiment of the invention is a recombinant vector that
comprises one or more nucleic-acid segments encoding one or more
bioluminescence
polypeptides. Such a vector may be transferred to and replicated in a
eukaryotic or
prokaryotic host.
It is contemplated that the coding DNA segment will be under the control of a
recombinant, or heterologous promoter. As used herein. a recombinant or
heterologous
promoter is intended to refer to a promoter that is not normally associated
with a DNA
segment encoding a crystal protein or peptide in its natural environment.
Naturally, it will
28
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be important to employ a promoter that effectively directs the expression of
the DNA
segment in the cell type, organism, or even animal, chosen for expression. The
use of
promoter and cell type combinations for protein expression is generally known
to those of
skill in the art of molecular biology (see e.g., Sambrook et al., 1989). In a
preferred
embodiments of this. such promoters are directed by cis-acting glucose
response elements.
In one preferred embodiment, the glucose response element is the L4 box which
directs
the L-pyruvate kinase ("L-PK") promoter in liver and islet ~-cells. The L4 box
consists of
a tandem repeat of non-canonical E-boxes (Kennedy et al.. 1997). Glucose
enhances the
hepatic and pancreatic (3-cell by modifying the transactivating capacity of
upstream
stimulatory factors ("USF") bound to the L4 box (Kennedy et al., 1997; Doiron
et al.,
1996).
The exact mechanism by which glucose controls the transactivational capacity
of
USF proteins is unclear. One possibility is the reversible phosphorylation of
USF
proteins. Glucose may alter the phosphorylation status through the pentose
phosphate
shunt via xyulose ~-phosphate (Dorion et al., 1996). An alternative mechanism
is via the
intracellular concentration of glucose 6-phosphate (Foufelle et al., 1992).
Other glucose
metabolites may also be implicated. Phosphorylated glucose metabolites
include, but are
not limited to, fructose 6-phosphate, 6-phosphogluconic acid, 6-phosphoglucono-
8-
lactone, ribulose ~-phosphate. ribose S-phosphate, erythrose 4-phosphate.
sedoheptulose
7-phosphate, glyceraldehyde 3-phosphate and dihdyro~yacetone phosphate. Non-
phosphorylated glucose metabolites include, but are not limited to, citric
acid, cis-aconitic
acid, threo-isocitric acid, succinic acid, fumaric acid, malic acid,
oxaloacetic acid, pyruvic
acid and lactic acid.
Another glucose response element, similar in arrangement to the L-PK gene L4
box, is the regulatory sequence involved in the transcriptional induction of
the rat S14
gene (Shih et al., 1995). Other glucose response elements that have been
described
include, but are not limited to, the hepatic 6-phosphofructo-2-kinase gene
(Dupriez and
Rousseau, 1997), the ~i-islets insulin gene (German and Wang, 1994), the
mesangial
transforming growrth factor-beta gene (Hoffman et al., 1998), and the gene for
acetyl-
coenzyme-A carboxvlase (Girard et al., 1997). The present invention
contemplates the
use any glucose response element that can effectively direct a promoter or
otherwise
control the expression of the reporter protein in response to glucose.
29
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In a preferred embodiment, the recombinant vector comprises a nucleic-acid
segment encoding one or more bioluminescence polypeptides. Highly preferred
nucleic-
acid segments are the lux genes of X. luminescens luxAB and luxCDE. Bacterial
luciferases may have to be modified to optimize expression in eukaryotic
cells.
Almashanu et al. (1990) fused the luxAB genes from Y'. harveyi by removal of
the TAA
stop codon from IuxA, the intervening region between the two genes, and the
initial
methionine from IuxB without disrupting the reading frame. The fusion was
successfully
expressed in Saccharomyces cerevisiae and Drosophila melanogaster. The same
strategy
was used with IuxAB from X. luminescens. The resultant construct has been
sequenced to
verify the genetic changes to generate the fusion and they were confirmed. The
sequence
of the fusion region is as follows:
5'-tacctagggagaaagagaatg-3' (SEQ ID N0:7)
(end of luxA underlined) (start of IuxB underlined)
The fusion successfully expresses fused protein in E.coli and has been
successfully cloned
into the mammalian vector as described in Section 5.1.2.
In a further embodiment, the inventors contemplate a recombinant vector
comprising a nucleic-acid segment encoding one or more enzymes that are
capable of
producing a reaction that yields a luminescent product or a product that can
be directly
converted to a luminescent signal. For example, substrates of the commonly
used (3-
galactosidase and alkaline phosphates enzymes are commercially available that
are
luminescent (chemiluminescence) when converted by the respective enzyme.
In another important embodiment, the biosensor comprises at least a forst
transformed host cell that expresses one or more of recombinant expression
vectors. The
host cell may be either prokaryotic or eukaryotic. In a preferred embodiment,
the host cell
is a mammalian cell. Host cells may include stem cells, (3-islets cells or
hepatocyte cells.
In a preferred embodiment the host cells are homologous cells, i.e. cells
taken from the
patient that are cultured, genetically engineered and then incorporated in the
BBIC.
Particularly preferred host cells are those which express the nucleic-acid
segment or
segments comprising the recombinant vector which encode the lux genes of X.
luminescens, IuxAB and luxCDE. These sequences are particularly preferred
because the
transcribed proteins of the X. luminescens lux system have the ability to
function at 37°C
(ambient human body temperature).
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A wide variety of ways are available for introducing a nucleic-acid segment
expressing a polypeptide able to provide bioluminescence or chemiluminescence
into the
microorganism host under conditions that allow for stable maintenance and
expression of
the gene. One can provide for DNA constructs which include the transcriptional
and
translational regulatory signals for expression of the nucleic-acid segment,
the nucleic-
acid segment under their regulatory control and a DNA sequence homologous with
a
sequence in the host organism, whereby integration will occtu or a replication
system
which is functional in the host, whereby integration or stable maintenance
will occur or
both.
The transcriptional initiation signals will include a promoter and a
transcriptional
initiation start site. In preferred instances, it may be desirable to provide
for regulative
expression of the nucleic-acid segment able to provide bioluminescence or
chemiluminescence, where expression of the nucleic-acid segment will only
occur after
release into the proper environment. This can be achieved with operators or a
region
binding to an activator or enhancers, which are capable of induction upon a
change in the
physical or chemical environment of the microorganisms. For translational
initiation, a
ribosomal binding site and an initiation codon will be present.
Various manipulations may be employed for enhancing the expression of the
messenger RNA, particularly by using an active promoter, as well as by
employing
sequences, which enhance the stability of the messenger RNA. The
transcriptional and
translational termination region will involve stop codon or codons, a
terminator region,
and optionally, a polyadenylation signal (when used in an Eukaryotic system).
In the direction of transcription, namely in the 5' to 3' direction of the
coding or
sense sequence, the construct will involve the transcriptional regulatory
region, if any, and
the promoter, where the regulatory region may be either 5' or 3' of the
promoter, the
ribosomal binding site, the initiation codon, the structural gene having an
open reading
frame in phase with the initiation codon, the stop codon or codons, the
polyadenylation
signal sequence, if any, and the terminator region. This sequence as a double
strand may
be used by itself for transformation of a microorganism host, but will usually
be included
with a DNA sequence involving a marker, where the second DNA sequence may be
joined to the expression construct during introduction of the DNA into the
host.
31
CA 02352571 2005-O1-20
-32-
By "marker" the inventors refer to a structural gene that provides for
selection of
those hosts that have been modified or transformed. The marker will normally
provide for
selective advantage, for example, providing for biocide resistance (e.g.,
resistance to
antibiotics or heavy metals); complementation, so as to provide prototrophy to
an
auxotrophic host and the like. One or more markers may be employed in the
development
of the constructs, as well as for modifying the host.
Where no functional replication system is present, the construct will also
include a
sequence of at least 50 basepairs (bp), preferably at least about 100 bp, more
preferably at
least about 1000 bp, and usually not more than about 2000 by of a sequence
homologous
with a sequence in the host. In this way, the probability of legitimate
recombination is
enhanced, so that the gene will be integrated into the host and stably
maintained by the
host. Desirably, the nucleic-acid segment able to provide bioluminescence or
chemiluminescence will be in close proximity to the gene providing for
complementation
as well as the gene providing for the competitive advantage. Therefore, in the
event that
the nucleic-acid segment able to provide bioluminescence or chemiluminescence
is lost,
the resulting organism will be likely to also have lost the complementing
gene, and the
gene providing for the competitive advantage, or both.
A large number of transcriptional regulatory regions are available from a wide
variety of microorganism hosts, such as bacteria, bacteriophage,
cyanobacteria, algae,
fungi, and the like. Various transcriptional regulatory regions include the
regions
associated with the trp gene, lac gene, gal gene, the 7~L and ~,R promoters,
the tac
promoter. See for example, U.S. Patent 4,332,898; U.S. Patent 4,342,832; and
U.S. Patent
4,356,270. The termination region may be the termination region normally
associated
with the transcriptional initiation region or a different transcriptional
initiation region, so
long as the two regions are compatible and functional in the host. In a
preferred
embodiment of the present invention, a fragment of the L-pyruvate kinase gene
is used
that contains the L-PK promoter and the L4 box glucose responsive elements as
described
by Kennedy et al. (1997). In a highly preferred embodiment, the p.LPK.LucFF
plasmid is
used (Kennedy et al., 1997), with the exception that the luc gene coding for
the firefly
luciferase is removed and replaced with the fused X. luminescens luxAB genes.
Where stable episomal maintenance or integration is desired. a plasmid will be
employed which has a replication system that is functional in the host. The
replication
CA 02352571 2005-O1-20
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system may be derived from the chromosome, an episomal element normally
present in
the host or a different host, or a replication system from a virus that is
stable in the host.
A large number of plasmids are available, such as pBR322, pACYC 184, RSF 1010,
pR01614, and the like. See for example, Olsen et al., 1982; Bagdasarian et
al., 1981, and
U.S. Patent 4,356,270, U.S. Patent 4,362,817, U.S. Patent 4,371,625, and U.S.
Patent
5,441,884.
The desired gene can be introduced between the transeriptional and
translational
initiation region and the transcriptional and translational termination
region, so as to be
under the regulatory control of the initiation region. This construct will be
included in a
plasmid, which will include at least one replication system, but may include
more than
one, where one replication system is employed for cloning during the
development of the
plasmid and the second replication system is necessary for functioning in the
ultimate
host. In addition, one or more markers may be present, which have been
described
previously. Where integration is desired, the plasmid will desirably include a
sequence
homologous with the host genome.
The transformants can be isolated in accordance with conventional ways,
usually
employing a selection technique, which allows for selection of the desired
organism as
against unmodified organisms or transfernng organisms, when present. The
transformants
then can be tested for bioluminescence or chemiluminescence activity. If
desired,
unwanted or ancillary DNA sequences may be selectively removed from the
recombinant
bacterium by employing site-specific recombination systems, such as those
described in
U.S. Patent 5,441,884.
4.9 ASSEMBLY AND STORAGE OF THE IN VIYO BIOSENSOR
When the biosensor consists of bioengineered cells entrapped in suspension
behind a semi-permeable membrane, as opposed to encapsulated in a matrix, the
cells
may be added to the BBIC any time from immediately to several h before
implantation of
the biosensor. The biosensor may alternatively consist of cells encapsulated
in a
polymeric matrix. Matrices will include materials previously shown to be
successful in
the encapsulation of living cells, including polyvinyl alcohol, sol-gel, and
alginate
(Cassidy et
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al., 1996). Prior to encapsulation, prokaryotic cell lines may be lyophilized
in a freeze dry
system (e.g., Savant) following the manufacturer's protocol. Lyophilization
allows cells
to undergo periods of long-term storage (several years) with a simple
rehydration protocol
being required for cell resuscitation prior to BBIC use (Malik et al.. 1993).
S. cerevisiae
eukaryotic cells may be similarly lyophilized. Eukaryotic cell lines,
preferably consisting
of islet (3-cells, stem cells, or hepatic cells, may be encapsulated on the IC
within
polyvinyl alcohol mesh-reinforced or microporous filter supported hydrogels,
which have
previously been successfully implemented in these types of cell encapsulations
(Baker et
al., 1997; Burczak et al., 1996; Gu et al., 1994; moue et al., I 991 ).
In the case of the mammalian cell lines, lyophilization, however, is not an
alternative. In such cases, mammalian cells may be encapsulated in a sol gel
or another
immobilization matrix as previously described and attached to the BBIC. The
completed
BBIC in its enclosure would then be stored in serum or another appropriate
maintenance
medium and maintained until use. The advantage of using an immortal stem cell
line is
apparent for both long-term use and storage. Implantation may be performed
according to
the specific application. In the case of glucose detection, an area where
interstitial fluid is
accessible would be most appropriate. However an implantable device with the
specific
application of detecting hormones or other blood borne molecules would have to
be
accessible to the bloodstream. A synthetic vein or catheter system may need to
be
employed to allow continuous monitoring of the blood levels of the target
molecule. A
specific example other than glucose would be the use of the in vivo biosensor
device to
detect molecules associated with colon cancer. In this case the biosensor
would be
implanted in the colon.
Integrated circuits may be individually packaged in sterile, static-proof
bags.
Prokaryotic-based and yeast eukaryotic biosensors consisting of lyophilized
cells may be
individually stored in sterile, static-proof, vacuum sealed bags for time
periods
approaching several years. Cells typically undergo rehydration in a minimal
nutrient
medium prior to use. Mammalian cell systems will remain frozen for long-term
storage
(up to 7 years at -I50°C) or refrigerated for short-term storage
(several days), either
separately or, if entrapped. frozen or refrigerated in situ on the BBIC. In
all cases, cell
viability may be checked be exposing the BBIC to a known concentration of the
analyte
of interest, thus producing a quantitative bioluminescent signal of known
magnitude. One
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WO 00/33065 PCT/US99/28733 _ --
or more control vials of analyte(s) or reference "standards" may be included
as part of a
diagnostic kit, or may be supplied for proper calibration of the implantable
device.
4.10 INIPLANTATION AND USE OF THE BIOSENSOR DEVICES
In a preferred embodiment of the present invention, the BBIC analyte biosensor
is
implanted such that it is contact with the interstitial fluid of the animal.
For example, in
the case of glucose biosensors, it has been shown that glucose kinetics in
interstitial fluid
can be predicted by compartmental modeling (Gastaldelli et al., 1997). In
particular the
subcutaneous placement of glucose sensors has been demonstrated (Schmidt et
al., 1993;
Poitout et al., 1993; Ward et al., 1994; Stenberg et al., 1995; Bantle and
Thomas, 1997).
Other potential analyte biosensor tissue implant sites include the peritoneum,
pleura and
pericardium (Wolfson et al., 1982). In fact, the inventors contemplate that
depending
upon the particular analyte or metabolite that is being detected, the
implantable biosensor
may be placed in any convenient location throughout the body using
conventional surgical
I S and implant methodologies. For example, the device may be implanted in
such as way as
to be in contact with interstitial fluid, lymph fluid, blood, serum, synovial
or cerebrospinal
fluid depending upon the particular analyte to be detected.
In certain embodiments the implantable device msy be placed in contact with
particular tissues, organs, or particular organ systems. Likewise, it may be
desirable to
implant the biosensor such that it contacts particular intracellular fluids,
intercellular
fluids, or any other body fluid in which the target substance can be
monitored.
The present invention also contemplates the use of multiple biosensors for the
detection of a plurality of different analytes. For example, in the case of
glucose
monitoring, one or more devices may be used to monitor various glucose or
glucose
metabolites, glucagons, insulin, and the like. Likewise, one or more biosensor
devices
may be employed in controlled drug delivery systems. As such, the device may
be
operably connected to a drug delivery pump or device that is capable of being
controlled
by the biosensor and that is able to introduce into the body of the animal an
amount of a
particular drug, hormone, protein, peptide, or other pharmaceutical
composition
determined by the concentration of one or more anaiytes detected by the BBIC
device.
Thus, controlled drug delivery systems are contemplated by the inventors to be
particularly desirable in providing long-term administration of drugs to an
animal such as
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in the case of chronic or life-long medical conditions or where symptoms
persist for a
long period of time. The long term controlled delivery of drags such as pain
medications,
heart or other cardiac regulators, diuretics, or homones or peptides such as
insulin, or
metabolites such as glucagon or glucose can be facilitated by such
biosensor/pump
systems. In cases where it is necessary to deliver more than one drug or
metabolite to the
animal, multiple drug delivery systems or a single switchable drug delivery
system is
contemplated to be particularly useful.
Host-rejection effects can be minimized through immunoisolation techniques.
Previous studies have shown that living non-host cells enclosed in hydrogel
membranes
are protected from immune rejection after transplantation (Baker et al., 1997;
Burczak et
al., 1996; moue et al., 1991 ). The hydrogels block access by the humoral and
cellular
components of the host's immune system but will remain permeable to the target
substance glucose. A mesh-reinforced polyvinyl alcohol hydrogel bag developed
by Gu
et al. (1994) may be used to fully encapsulate the BBIC. allowing for
transplantation void
of immunosuppressive responses.
Host rejection of the implanted biosensor is not an issue if cells from the
host are
used for the biosensor construction. However if other cell lines are used it
may be
necessary to provide a barrier between the cells and the appropriate body
fluid that
permits passage of the signature molecules or analytes but not bioreporter
cells or body
cells (white blood cells, etc.). Immunosuppressed patients are not affected,
as the implant
does not contain any kind of pathogenic agent that would affect the patient.
In all cases,
the surgical methods involved in implantation of the disclosed BBIC devices
are well
known to one of skill in the surgical arts.
In an illustrative embodiment, the BBIC glucose sensor may be used for
monitoring glucose in diabetic patients. However, such a sensor can also be
used in other
conditions where glucose concentrations are of concern. such as in endurance
athletes or
other condition involving either hypo- or hyperglycemia. Such measurements may
be the
end point for investigative or diagnostic purposes or the sensors may be
linked via
telemetry or directly to a drug delivery system.
The use of implantable BBICs for substances other than glucose can be used in
a
range of therapeutic situations. With the incorporation of an appropriate cis-
activating
response element. BBICs could monitor a number of substances and could find
use in
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WO 00/33065 PCT/US99/287~3 _ :-
chronic pain treatment, cancer therapy, chronic immunosuppression, hormonal
therapy,
cholesterol management, and lactate thresholds in heart attack patients. For
example,
Section 5.7 describes the use of the BBICs in the detection and diagnosis of
cancer.
Individual biosensors can be calibrated to check for viability of the cells as
well as
performance. The calibration is performed by exposing the sensor to solutions
containing
varying concentrations of the analyte(s) of interest. The bioreporter may be
calibrated by
a series of standard analyte concentrations for the specific application after
its initial
construction. The overall on-line performance can be monitored using
microfluidics with
a reservoir of the analyte, which would systematically provide a known
concentration to
the cells this would allow both calibration and test for viability.
The luminescence response is then correlated to concentration and the
parameters
set. Viability can also be continuously monitored by bioengineered cells in
which the
reporter exhibits continuous luminescence. Loss of viability results in
decreased
luminescence. This technique has been used to detect the viability of
prokaryotic cells.
Thus the BBIC would contain two bioreporters, the bioreporter detecting the
selected
substance and the second bioreporter exhibiting a luminescence proportional to
cell
viability. iVieasurement of the ratio of the signals from the two bioreporters
would give a
detection method that would automatically correct for any loss in viability.
Once prepared the bioreporters can be stored in the appropriate maintenance
medium (e.g., standard tissue culture media, sera, or other suitable growth or
nutrient
formulations), and then calibrated prior to implantation. The viability of the
devices may
be checked by bioluminescence using microfluidics, or by the quantiation of
known
standards or other reference solutions to ensure viability and integrity of
the system prior
to, or after implantation..
In certain embodiments of the invention, the monolithic biosensor devices may
be
used external to the body of the monitored individual. In some clinical
settings the
monitor may be used to monitor glucose in body fluids in an extracorporeal
fashion. The
device may even be used in the pathological or forensic arts to detect the
quantity of
particular analytes in body tissues or fluids and the like. Likewise, the
present invention
also contemplates use of the biosensor devices in the veterinary arts.
Implantation of such
devices in animals for the monitoring of hormone levels in the blood (i. e.
for optimizing
milk production), monitoring the onset of estrous (heat) in numerous animals
to maximize
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artificial insemination efficiency, and monitoring hormone levels in the milk
produced on-
line (in the udder) etc. is contemplated to provide particular benefits to
commercial
farming operations, livestock industries and for use by artisans skilled in
veterinary
medicine.
4.11 DIAGNOSTICS KITS COMPRISING IN VIYO BIOSENSORS
While the individual components of the invention described herein may be
obtained and assembled individually, the inventors contemplate that, for
convenience, the
components of the biosensor may be packaged in kit form. Kits may comprise, in
suitable
container means, one or more bioreporters and an integrated circuit including
a
phototransducer. The kit will also preferably contain instructions for the use
of the
biosensor apparatus, and may further, optionally comprise a drug delivery
device or a
second biosensor apparatus. The kit may comprise a single container means that
contains
one or more bioreporters and the integrated circuit including a
phototransducer and drug
delivery device. Alternatively, the kits of the invention may comprise
distinct container
means for each component. In such cases, one container would contain one or
more
bioreporters, either in an appropriate medium or pre-encapsulated in a polymer
matrix,
another container would include the integrated circuit, and another conatiner
would
include the drug delivery device. When the bioreporter is pre-encapsulated,
the kit may
contain one or more encapsulation media. The use of distinct container means
for each
component would allow for the modulation of various components of the kits.
For
example, several bioreporters may be available to choose from, depending on
the
substance one wishes to detect. By replacing the bioreporter, one may be able
to utilize
the remaining components of the kit for an entirely different purpose, thus
allowing reuse
of components.
The container means may be a container such as a vial, test tube, packet,
sleeve,
shrink-wrap, or other container means, into which the components of the kit
may be
placed. The bioreporter or any reagents may also be partitioned into smaller
containers or
delivery vehicles, should this be desired.
The kits of the present invention also may include a means for containing the
individual containers in close confinement for commercial sale, such as, e.
g., injection or
blow-molded plastic containers into which the desired components of the kit
are retained.
38
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WO 00133065 PCT/US99/28733 -_
Irrespective of the number of containers, the kits of the invention also may
comprise, or be packaged with, an instrument for assisting with the placement
of the
bioreporter upon the integrated circuit. Such an instrument may be a syringe,
pipette,
forceps, or any other similar surgical or implantation device. The kit may
also comprise
one or more stems, catheters, or other surgical instrument to facilitate
implantation within
the body of the target animal. Such kits may also comprise devices for remote
telemetry
or devices for data storage or long term recordation of the data obtained from
the
monitoring device. Likewise, in the case of controlled drug delivery systems,
the kits may
comprise one or more drug delivery pumps as described above, and may also
comprise
one or more pharmaceutical agents themselves for administration. As an
example, in the
case of a glucose monitoring system, the system would typically comprise a
glucose-
sensitive BBIC device, a drug delivery pump, instructions for the implantation
and/or use
of the system, and optionally, reference standards or pharmaceutical
formulations of
insulin, glucagon or other pharmaceutical composition. The system may also
optionally
comprise growth and/or storage medium to support the nutritive needs of the
bioreporter
cells comprised within the BBIC device.
S.O EXAMPLES
The following examples are included to demonstrate preferred embodiments of
the
invention. It should be appreciated by those of skill in the art that the
techniques
disclosed in the examples which follow represent techniques discovered by the
inventor to
function well in the practice of the invention, and thus can be considered to
constitute
preferred modes for its practice. However, those of skill in the art should,
in light of the
present disclosure, appreciate that many changes can be made in the specific
embodiments
which are disclosed and still obtain a like or similar result without
departing from the
spirit and scope of the invention.
S.1 EXAMPLE 1 -- CONSTRUCTION OF A BIOLUMINESCENCE REPORTER FOR
MAMMALIAN CELL LINES
To facilitate the construction of an implantable bioluminescent glucose sensor
it
will be necessary to create a bioluminescent reporter system that can function
without the
exogenous addition of substrate for the luciferase reaction. This exogenous
addition is
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due to the complex nature of the production of luciferins for the various
eukaryotic
luciferases. Cells must be either permeablized or lysed and then treated with
an assay
solution containing luciferin. Therefore, the present state of bioluminescence
reporters
used in eukaryotic molecular biology makes them unsuitable for "on-line"
monitoring.
The firefly luciferase has been used in examining the regulation of L-pyruvate
kinase
promoter activity in single living rat islet ~i-cells (Kennedy et al., 1997).
However, these
cells had to be perfused with Beetle luciferin in order to generate a
luminescence
response.
To alleviate this limitation, a preferred bioluminescent reporter system for
the
present invention is one that does not require the addition of exogenous
substrate. In the
case of bacterial luciferase-based detection systems, this may be accomplished
using the
bioluminescent genes from X. luminescens. In this organism, IuxA and luxB
genes (or a
single fused IuxAluxB gene encode the a- and ~3-subunits, respectively, of the
luciferase
enzyme (Meighen et al., 1991). This luciferase exhibits greatest
thermostability at 37°C
while other bacterial luciferases lose significant activity above 30°C.
Therefore, these
bacterial luciferases can be expressed in eukaryotic cells with slight
modification.
Almashanu et al. (1990) fused the IuxAB genes from V. harveyi by removal of
the TAA
stop codon from IuxA, the intervening region between the two genes, and the
initial
methionine from luxB without disrupting the reading frame. The fusion was
successfully
expressed in S. cerevisiae and D. melanogaster. Using the same strategy a
fused IuxAB
gene sequence was developed using the genes from X. luminescens.
To eliminate the need for the addition of exogenous substrate, cells must
themselves supply the appropriate substrate for the luciferase. In the
bacterial system the
substrate is generated by a fatty acid reductase complex encoded by the IuxCDE
genes.
This enzyme complex reduces short chain fatty acids to the corresponding
aldehyde. The
luciferase then oxidizes the aldehyde to the corresponding fatty acid. The
preferred fatty
acid for this reaction is myristic acid, which is present in eukaryotic
organisms (Rudnick
et al., 1993). Myristic acid is usually involved in the myristoylation of the
amino
terminus that is associated with membrane attachment (Borgese et al., 1996,
Brand et al.,
1996). Thus, to obviate the need for an exogenous supply of the luciferase
substrate, the
biosensor also preferably comprises a nucleic acid sequence that encodes the
three IuxC,
IuxD, and hrrE-encoded subunits. As in the case of the luxAluxB gene fusion,
the IuxC,
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IuxD, and luxE genes have been fused to produce a single IuxCDE gene fusion
that
encodes the three subunits of the enzyme complex. The methods of preparing
such gene
fusions are described below:
S j.1.1 FUSION OF THE LUXAB AND LUXCDE GENES
The IuxAB genes may be fused using conventional molecular biology techniques.
For example, the polymerase chain reaction may be routinely employed for this
purpose.
By synthesizing a S'-primer whose sequence begins with ATG for the start codon
for the
IuxA gene juxtaposed by a 3'-primer ending with the codon immediately
preceding the
ATT stop codon. These primers may then be used in amplification reactions and
the
product gel purified. The IuxB gene may also be amplified as above using
primers that
eliminate the ATG initial methionine codon but preserve the reading frame. The
PCRTM
reactions employ a thermostable polymerase such as the PfuTM polymerase of
Stratagene
(La Jolla, CA), which does not have terminal deoxytransferase activity and
therefore
1 S generates a blunt end. The resultant PCRTM products are blunt-end ligated,
and the
ligation is then subjected to PCRTM using the S'-primer from IuxA and the 3'-
primer from
IuxB using Taq polymerase to facilitate TA cloning (Invitrogen, San Diego,
CA). Only
ligations with the correct orientation of fragments are amplified. The IuxAB
amplicon is
then gel purified and TA cloned into a suitable vector (such as the PCRIITM
vector) and
transformed into E. coli using standard manufacturer's protocols.
Transformants are screened for light production by the addition of n-decanal
which, when oxidized by the luciferase, generates bioluminescence. Only
colonies
emitting light are selected since they are in the proper orientation for
further genetic
manipulation. The IuxCDE fusion is generated using the same strategy as above
except
2S transformants are screened by minipreps followed by restriction digestion
analysis to
determine orientation. Plasmids are amplified in E. coli, recovered and
purified twice on
CsCI gradients.
5.L2 EXPRESSION OF LUYAB AND LUXCDE IN HELA CELLS
To determine the relative activity of the fused bacterial luciferase
components,
cloned fragments containing IuxAB are cloned into a suitable mammalian
expression
vector (such as pcDNA 3.1 and IuxCDE-containing fragments are cloned into a
suitable
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mammalian expression vector (such as pcDNA/Zeo 3.1 ) (Invitrogen, Faraday.
CA). Both
vectors constitutively express inserted genes. HeLa cells are then transfected
with IuxAB
or both IuxAB and luxCDE and selected using appropriate antibiotics following
the
manufacturer's protocol (Promega, Madison, WI). Cells receiving the IuxAB
fusion are
S exposed to n-decanal and checked for bioluminescence. These cells
cotransfected with
IuxCDE are then examined for bioluminescence to ascertain the relative
expression of the
IuxCDE fusion. This permits the comparison of bioluminescence via the addition
of
exogenous aldehyde versus aldehyde that is produced endogenously.
An alternate strategy to enhance bioluminescent expression involves
engineering a
vector that would contain three copies of the eukaryotic expression machinery
contained
in pcDNA3.1 (Stratagene, La Jolla, CA). This allows for the expression of the
individual
components of IuxCDE since it has already been shown that the fused luciferase
is
expressed in eukaryotic cell lines (Almashanu et al., 1990).
1S S.2 EXAMPLE 2 -- CONSTRUCTION OF A GLUCOSE BIOLUMINESCENT BIOSENSOR
The firefly Iuciferase has been used in examining the regulation of L-pyruvate
kinase promoter activity in single living islet (3-cells (Kennedy et al.,
1997). A glucose
response element designated the L4 box has been determined to be in the
proximal
promoter. A 200-by fragment containing this region was cloned in front of the
firefly
luciferase (laic) in plasmid pGL3Basic resulting in a glucose reporter plasmid
designated
p.LPK.LucFF. Results resulted in the detection of single cells that were
exposed to 16 mM
glucose but not 3 mM glucose. However, these cells had to be perfused with
Beetle
Iuciferin making it unacceptable for an on-Line biosensor. Therefore, a
bioluminescent
sensor for glucose was constructed by replacing the firefly luciferase in
p.LPK.Luc~ with
2S the fused IuxAB gene as described below.
S.3 EXAMPLE 3 -- BIOLUMINESCENT REPORTER CONSTRUCTION AND
TRANSFECTION OF RAT ISLET ~-CELLS
The bioluminescent reporter plasmid was constructed by removing the luc gene
coding for the firefly luciferase from p.LPK.Luc~ and replacing it with the
fused IuxAB
gene. This was accomplished by cleaving the luc gene from p.LPK.Luc~ and
cloning in
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the IuxAB gene. The resultant plasmid was amplified in E. coli and the plasmid
DNA
extracted and double purified on CsCI gradients.
Islet cells were prepared as previously described (German et al., 1990) and
transfected by electroporation with the bioluminescent reporter construct and
the plasmid
containing the constitutively expressed IuxCDE construct. This configuration
causes the
cells to maintain a pool of the aldehyde substrate that is available to the
reporter genes
(luxAB). Cells were screened for light production in a range of glucose
concentrations
from 3 mM to 30 mM. Transfected cells were washed, concentrated, and placed in
a
microwell in a light-tight cell that is then affixed to the integrated
circuit. Different
concentrations of glucose and assay media (Kennedy et al., 1997) were added to
the cells
to examine sensitivity and response time of the glucose BBIC.
S.4 EXAMPLE 4 - PREPARATION OF BIOLUMINESCENT REPORTER CONSTRUCTS
The use of reporter gene technology is widespread in studying gene regulation
in
both eukaryotic and prokaryotic systems. Various genes are used depending on
the cell
lines being investigated. However with the BBIC technology the use of reporter
genes
that result in the emission of light is required. Therefore, reporter genes
coding for
bioluminescence are utilized. All previously developed reporters utilizing
other reporter
genes for example the gene coding for ~3-galactosidase (lack may be converted
to the
bioluminescent version using standard molecular techniques and the reporter
genes
utilized in this specific application (modified lux system). Therefore, any
currently
existing reporter cell line for testing gene expression in mammalian cell
lines may be
adapted for use as a bioreporter when converted to the lux reporter. The
implantable
system simply contains the appropriate reporter cell line. Table 1 shows a
list of examples
of eukaryotic reporter cell lines that may be exploited in an implantable
biosensor.
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TABLE 1
Reporter Gene Application Reference
Fusion
ADH4-LUC Monitors expression of alcoholEdenberg et al.,
dehydrogenase 1999
to increasing concentrations
of alcohol
TH-lacZ Shows increased gene expressionBoundy et al., 1998
in mice
subjected to chronic cocaine
or morphine
exposure
Estrogen regulated-LUCDetects estrogens and xenoestrogensBalaguer et al.,
by there 1999
effect on the estrogen response
element
IGFBP-5-LUC Detects the presence of progesteroneBoonyaratanakomkit
by the et al., 1999
upregulation of the reporter
construct
CYPIA-IacZ Detects compounds that causeCampbelleraL, 1996
an upregulation
of cvtochrome P450 (potential
carcinogens)
S.S EXAMPLE S -- CONSTRUCTION AND IMPLANTATION OF A
GLUCOSE BIOSENSOR AND INSULIN DELIVERY PUMP
In one embodiment, a pair of bioluminescent reporters may be utilized that are
in
tandem and that specifically respond to deviations in glucose concentrations.
One
bioreporter utilizes the IuxAB and IuxCDE genes from X. luminescens
incorporated into a
plasmid-based system designated p.LPK.Luc~, which contains a eukaryotic luc
gene able
to respond to glucose concentrations (increasing bioluminescence corresponds
to
increasing glucose concentrations). The second bioreporter utilizes a plasmid
construct
containing the promoter for the phosphoenolpyruvate carboxylase gene (PEPCK)
that also
responds to glucose concentrations, except increased bioluminescence
corresponds to
decreased levels of glucose. The incorporation of the IuxAB and IuxCDE genes
into each
construct allow for bioluminescence measurements to occur in real-time with
deviations
in glucose concentrations, negating the requirement for cell destruction and
substrate
addition.
In this embodiment, the integrated circuit comprises separate photodetector
units
for each bioreporter (FIG. 9A, FIG. 9B, and FIG. 9C). Bioluminescent responses
from
each construct can be independently monitored, allowing for the signal
processing
'?0 circuitry to differentiate between one bioreporter's response to increased
glucose
concentrations and the second bioreporter's response to decreased glucose
concentrations.
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The signal processing circuitry processes the signals from the photodetectors,
converts it
to a digital format and relays the information to the implanted insulin pump
(FIG. 10).
The tandem set of bioreporters allows a more accurate signal as well as
redundancy in the
detector. Due to the often-fatal outcome of hypoglycemia, this tandem system
also allows
for more careful monitoring and warning of the onset of hypoglycemia.
The cells used in the tandem bioreporter system may be affixed to each of the
photodetectors either directly by attachment or encapsulated in hydrogel
(Prevost et al.,
1997). It may be necessary to isolate the bioreporters using a semi-permeable
membrane
to allow the transport of small molecules such as glucose and insulin across
the membrane
and prohibit the influx of immune effector cells and antibodies (Monaco et
al., 1993,
Suzuki et al., 1998). However small molecules such as cytokines can still
enter the
selective membranes and interfere with the bioluminescent reporter cell lines.
This
approach has been used extensively by those of skill in the art.
When applicable, bioluminescent reporter cell lines may be constructed from
cells
taken directly from the patient to receive the implant. This approach is
particularly
desirable in cases of long-term implants such as implantable insulin delivery.
Cells may
be obtained from the patient, genetically engineered for the appropriate
monitoring
function, grown in cell culture, evaluated and then preserved for long-term
storage. The
use of cell lines developed from the patient's own cells, is particularly
desirable as it
reduces the chance of host rejection and creation of an immune response to the
implanted
device. Preferably, stem cells (immortal stem cells, if attainable) are used
when
appropriate, and may be maintained and nourished in suitable culture medium.
Such
pluripotent, totipotent, or otherwise immortal cell lines provide particular
advantage in the
creation of suitable long-term implantable devices.
Before implantation the biosensor may be calibrated injecting the chamber
containing the cells with various concentrations of glucose delivered from an
auxiliary
pump and reservoir on the insulin delivery pump (FIG. 10). This permits
determination of
the appropriate parameters to allow the proper dosage of insulin to be
delivered. Once the
parameters are set, the pump may be evaluated for insulin delivery.
Systematically the
glucose biosensor is recalibrated in the patient utilizing the glucose
standard contained in
the delivered pump.
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In the case of drug delivery systems, the glucose biosensor may be operabiy
connected to the delivery pump via a hardwire or wireless connection. The
biochip
provides digital data that may be input directly to the signal processing
circuitry of the
pump to proportionally dispense the insulin. Alternatively, the digital data
may be
converted into analog data and used to control the pump. When a wireless
capability is
added to the bioreporter device, remote monitoring of the sensor is possible.
For example,
in this configuration, the patient may place a radio transmitter/receiver
outside the body
near the implanted device to communicate the data from the implanted device
toa remote
station. In some applications, the radio transmitter/receiver may be linked to
a computer
programmed to forward the data to a remote station over a network such as a
local area
network, a wide area netword, or even the Internet. Such wireless applications
allow
remote monitoring and maintenance of the patients. There are several pumps
currently on
the market, which are candidates for interfacing with the biosensor. In one
embodiment,
the Medtronic Synchronized infusion system may be used as it has extensively
used in
drug delivery and utilizes a portable computer to allow programming of the
pump from
outside the body (www.asri.edu/neuro/brochure/pain6.htm). The pump can also be
refilled through the skin via a self sealing septum. The pump is one inch
thick and three
inches in diameter and weighs approximately six ounces. The biosensor can be
integrated
into the preexisting electronic circuitry to take advantage of the out-of body
programming
by a portable computer. The chip can be powered utilizing the battery that
powers the
delivery pump.
The biosensor/insulin pump apparatus may be surgically implanted using local
anesthesia in the abdominal cavity. Both the sensor and the pump may be
implanted in
the peritoneal space of the abdomen both for simplicity and to avoid the
complications of
direct catheter placement in the blood stream. Glucose concentrations are
monitored and
the insulin delivered peritoneally as required by the patient (FIG. 10).
S.6 EXAMPLE 6 -- BIOLUI'IINESCENT REPORTER CONSTRUCTION AND
TRANSFECTION OF RAT ISLET ~-CELLS AND H~IIE iIEPATOMA CELLS
The regulation of the PEPCK gene will be exploited in the construction of the
bioluminescent reporter for detecting decreased glucose concentrations. This
system is
highly regulated as the phosphoenolpyruvate carboxylase is the rate-limiting
enzyme in
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gluconeogenesis. PEPCK gene expression is increased in the presence of
glucocorticoids
and cAMP and decreased in the presence of insulin (Sasaki et al., 1984; Short
et al.,
1986). In both rat liver and H4IIE hepatoma cells the insulin effect is
dominant and the
glucocorticoids and cAMP is additive. The promoter region of the PEPCK will be
cloned
in front of the fused IuxAB. The resultant construct will then produce
increased
bioluminescence in the presence of low glucose concentrations.
The bioluminescent reporter plasmid for detecting increased glucose
concentration
may be constructed by removing the luc gene coding for the firefly luciferase
from
p.LPK.Luc~ and replacing it with the fused luxAB. This is accomplished by
cleaving the
luc gene from p.LPK.Luc~ and cloning in the IuxAB gene. The bioluminescent
reporter
plasmid for the detection of low glucose concentrations is constructed by
replacing the
chloramphenicol transferase (CAT) gene in the previously constructed PEPCK
promoter
CAT fusion (Petersen et al., 1988; Quinn et al., 1988) with the IuxAB gene.
The resultant
plasmid is amplified in E. coli and the plasmid DNA extracted and double
purified on
CsC1 gradients.
Islet and hepatoma cells may be prepared as previously described (German et
al.,
1990; Petersen et al., 1988) and co-transfected with the bioluminescent
reporter construct
and the plasmid containing the constitutively expressed IuxCDE gene
constructed in
objective one. This configuration causes the cells to maintain a pool of
aldehyde substrate
that will be available to the reporter genes (luxAB). Cells are screened for
light production
in a range of glucose concentrations from 3 mM to 30 mM. Transfected cells are
washed,
concentrated, and placed in a microwell in a light-tight chamber that is then
affixed to the
integrated circuit. Different concentrations of glucose and assay media
(Kennedy et al.,
1997) are added to the cells to examine sensitivity and response time of the
glucose BBIC.
After initial characterization. the bioluminescent glucose reporters may also
be tested in a
flow cell. Cells are placed in an encapsulation medium on the integrated
circuit and
media containing different concentrations of glucose (3-to-30 mM) is then
perfused across
the cells to examine dynamic responses.
S.7 EXAMPLE 7 - BBICS IN THE DIAGNOSIS AND DETECTION OF CANCER
Colon cancer is the second leading cause of cancer death after lung cancer in
the
United States, and the incidence increases with age in that 97% of colon
cancer occurs in
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persons greater than 40 (Coppola and Karl, 1998). Although most cases of colon
cancer
are sporadic, in 15% of the patients there is a strong familial history of
similar tumors in
first-degree relative relatives (Coppola and Karl, 1998). These familial
cancers such as
hereditary nonpolyposis colon cancer (HNPCC) and familial adenomatous
polyposis
(FAP) result from autosomal dominant inheritable genetic mutations in putative
tumor
suppressor genes, and a spectrum of lesions occurs from hyperplasia-dysplasia-
adenoma
earcinoma (Coppola and Karl, 1998). Because much of the early molecular
lesions are
known about inherited colonic cancer, they represent a useful model for
development of a
novel biosensor strategy for early clinical detection. Biosensors are hybrid
devices
combining a biological component with a computerized measuring transducer.
This example describes the adaptation of the implantable biosensor device to
permit early detection of cancers, and to permit means for monitoring
remission and
recurrence of cancer. Because the miniaturized biosensors of the present
invention are
small enough to be implantable, and can be combined with a reporter system
engineered
to produce light without the need for cellular lysis or additional substrate,
a powerful tool
for early diagnosis of colon cancer in the form of an implantable device is
now possible
for the first time.
As described above for glucose and other metabolite biosensors, the inducible
reporter system utilized is based on the IuxAB and IuxCDE genes from X.
luminescens
placed in a eukaryotic reporter cell so that expression of certain genes or
their products
can be detected by expression of bioluminescence by the BBICdevice. The
eukaryotic
reporter cell is treated with mitomycin C so it is unable to divide, but is
still able to
respond metabolically and produce a quantitative bioluminescent signal.
Colon cancer is the second leading cause of cancer death in the United Sates,
with
at least 50% of the population developing a colorectal tumor by the age of 70
(Kinzler and
Vogelstein, 1996). Although most cases of colorectal cancer are sporadic, 1 S%
are the
result of heritable cancer syndromes, familial adenomatous polyposis (FAP) and
hereditary nonpolyposis colorectal cancer (HNPCC) (Kinzler and Vogelstein,
1996).
Familial adenomatous polyposis is a syndrome characterized by the development
of
hundreds to thousands of adenomas or polyps in the colon and rectum, only a
small
number of which develop into invasive cancer (Kinzler and Vogelstein, 1996).
Loss of
function of both alleles of the adenomatous polyposis toll (APC) tumor
suppressor gene
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predisposes persons to develop malignant cancer (Coppola and Marks, 1998). In
addition.
most sporadic colon cancers are also found to contain mutations in the APC
gene (Kinzler
and Vogelstein, 1996). In hereditary nonpolyposis colorectal cancer. there is
marked
microsatellite instability secondary to mutations in DNA mismatch repair genes
such as
hMSH2 and hMSHI; single, high grade tumors develop at a young age and are
usually
confined to the right colon (Coppola and Karl, 1996; Smyrk, 1994). Whereas
cells with
mutations in APC are generally aneuploid from loss of whole sections of
chromosomes,
cells with mutations in hMSH2 or hMSHI are euploid (Lengauer et al., 1998).
The molecular events leading to the development of colonic neoplasia are
fairly
well understood (Kinzler and Vogelstein, 1996). Persons with complete loss of
APC
develop lesions in the colon called dysplastic aberrant crypt foci that
progress to early
adenomas (Kinzler and Vogelstein, 1996). Other mutations begin to accumulate,
such as
those in K-Ras or p~3, and the tumors progress to late adenomas, carcinomas,
and
metastatic carcinomas (Kinzler and Vogelstein, 1996). A similar progression is
seen in
HNPCC as well. Because the sequence of genetic events is fairly well
understood for
both these types of cancer, they represent excellent models for development of
sensitive
and specific diagnostic tests that can be used to detect one or more altered
cells in vitro.
The APC gene encodes a cytoplasmic protein that localizes to the ends of
microtubules at focal adhesion complexes (Kinzler and Vogelstein, 1996). As
cells
migrate up through the crypts, expression of APC increases until the
terminally
differentiated and located colonic epithelial cells undergo apoptosis (Kinzler
and
Vogelstein, 1996). Cadherins are transmembrane proteins that are localized to
focal
adhesion plaques in most epithelial cells (Aplin et al., 1998). The carboxy
terminus of
each cadherin interacts with cytoplasmic structural proteins known as catenins
(Aplin et
al., 1998). There are three types of catenins: ~3-catenin binds to the
cytoplasmic domain of
cadherin; -catenin binds to (3-catenin and the actin cytoskeleton via -
actinin; -catenin
functions in place of ~3-catenin in some cell types (Aplin et al., 1998). (3-
Catenin also is
part of a signal transduction pathway involving the secreted glycoprotein Wnt
and
glycogen synthase kinase 3 (GSK3) (Aplin et al., 1998). APC interacts with
several
components of the W nt-(3-catenin-GSK3 pathway, including [3- and y-catenins,
GSK3, and
tubulin (Aplin et al.. 1998). Most of the mutations in colorectal cancer are
in the carboxy
terminal region of APC so that it can no longer bind (3-catenin (Aplin et al.,
199$). In
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fact, -catenin lies downstream of APC and is critical for its function as a
tumor suppressor
gene (Aplin et al., 1998). When the Wnt pathway is inactivated, GSK3
phosphorylates
the N-terminus of ~i-catenin, targeting it for degradation by the ubiquitin
pathway
(Munemitsu et al., 1996). When ~3-catenin accumulates, it activates gene
transcription via
the transcription factor Lef 1/TCF (Morin et al., 1997). APC works in concert
with GSK3
to inhibit -catenin-mediated transcriptional activity (Kinzler and Vogelstein,
1996).
In hereditary nonpolyposis colon cancer, microsatellite instability is the
result of
mutations in one or more DNA-mismatch repair genes (Jiricny 1998; Nicholaides
et al.,
1994). At least 90% of HNPCC tumors have microsatellite instability (Karran
1996;
Smyrk 1994). One potential marker for microsatellite instability in colorectal
tumors is
inactivation of the type II receptor for TGF-~i (Markowitz et al., 1995). Loss
of function
of ~iRII is associated with loss of growth regulation and tumor progression in
colorectal
adenomas in HNPCC (Wang et al., 1995). Other signaling components of the TGF-
~i
pathway that are involved in colorectal tumorigenesis include mutations in
Smad 3 and
1 S Smad 4, both of which result in the development of colorectal
adenocarcinomas in mice
(Zhu et al., 1998; Takaku et al., 1998). Loss of function of (3RII is a useful
marker for
early lesions in HNPCC (Markowitz et al., 1995).
Because mutations in .APC are the most common mutations in colorectal cancer,
a
reporter construct for T cell transcription factor (Tcf) was devised to screen
multiple colon
cancer cell lines for activation of transcriptional activity. Mutations in
either APC or (3
catenin result in activation of Tcf responsive transcription through the
accumulation of
unphosphorylated cvtoplasmic (3-catenin (Morin et al., 1997) and detecting
activation of a
reporter construct is useful as a marker for mutations in either of these
genes. The vector
pDISPLAY (Invitrogen) permits expression of the promoter for Tcf on the
surface of the
bioreporter cell; this construct consists of a tandem set of Tef promoters:
one upstream of
the genes for luxAB, the other upstream of the luxCDE. In the presence of
excess J3-
catenin the promoter constructs will stimulate activity of the reporter and
bioluminescence
will result.
Once the HepG2 and HeLa cells have been transfected with pcDNA3 encoding the
luxAB genes, the cells are attached to the biosensor chip. It is necessary to
insure that
these cells are incapable of dividing, so after transfection and selection,
the cells are
irradiated with 6,000 rads -r-radiation from a G°Co source (UT College
of Veterinary
CA 02352571 2001-05-28
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Medicine). In some embodiment it may be necessary to attach the cells to the
biochip
prior to irradiation so that efficient attachment can occur. ~n alternative is
to treat the
cells with mitomycin C to prevent further mitosis. Biochips may be coated with
Matrigel,
a basement membrane material that promotes attachment of epithelial cells. An
alternative approach suspends the cells in Matrigel and allows it to form a
gel on the
surface of the biochip. The cells are then immobilized in the basement
membrane
material and are not subject to dislodgement by friction. Optionally, the
surface of the
chip may be altered by adding a net charge (e.g., poly-L-lysine), coating the
surface with
surgical tissue glue, or by adding some other surface modification that allows
the
biopolymers to adhere tightly to the surface. Because mutations in APC are the
most
common mutations in colorectal cancer, a reporter construct for T cell
transcription factor
(Tcf) may be devised to screen multiple colon cancer cell lines for activation
of
transcriptional activity.
The present invention also provides a biosensor that may be used for
endoscopic
screening of the colonic mucosa to detect the presence of mutated cells prior
to the onset
of gross morphological alterations. It may be necessary to attempt detection
of more than
one abnormality at a time for the degree of sensitivity needed to detect small
foci of
malignant transformation. For example, many colonic tumors, especially those
with
mutations in APC, overexpress cyclooxygenase-2 (COX-2) and secrete large
amounts of
prostaglandins (Kutchera et al., 1996; Sheng et al., 1997; Coffey et al.,
1997; Kinzler and
Vogelstein, 1996). Cyclooxygenase-2 is an early response gene that not
constitutively
expressed, but is turned on in colonic epithelial cells by growth factors and
tumor
promoters (Kutchera et al., 1996; Sheng et al., 1997; Coffey et al., 1997). It
may be
possible to bioengineer reporter cells to bioluminesce in the presence of
increased levels
of prostaglandins in the intestinal lumen. Prostaglandins freely pass the cell
membrane
and would be able to enter the cytoplasm of the reporter cell to activate a
reporter
construct. Engineering a reporter cell to detect increased levels of
prostaglandins through
the use of the cyclooxygenase-2 promoter fused to the hcrAB genes could also
be of
benefit in early detection of colon cancer. Because the levels of
prostaglandins may be
elevated in inflammation as well as neoplasia, this approach lacks appropriate
specificity
for diagnosing cancer. It would, however, be useful in determining which
patients would
benefit from treatment with specific cyclooxygenase inhibitors.
51
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6.0 REFERENCES
The following references provide exemplary procedural or other details
supplementary to
those set forth herein.
U. S. Patent 4,332,898, issued Jun l, 1982.
U. S. Patent 4,342,832, issued Aug 3, 1982.
U. S. Patent 4,356,270, issued Oct 26, 1982.
U. S. Patent 4,362,817, issued Dec 7, 1982.
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All of the compositions, methods, devices, apparatus and systems disclosed and
claimed herein can be made and executed without undue experimentation in light
of the
present disclosure. While the methods, devices, apparatus and systems of this
invention
have been described in terms of preferred embodiments, it will be apparent to
those of
skill in the art that variations may be applied to the methods, devices,
apparatus and
systems and in the steps or in the sequence of steps of the methods described
herein
without departing from the concept, spirit and scope of the invention. More
specifically,
1 S it will be apparent that certain agents which are both chemically and
physiologically
related may be substituted for the agents described herein while the same or
similar results
would be achieved. All such similar substitutes and modifications apparent to
those
skilled in the art are deemed to be within the spirit, scope and concept of
the invention as
defined by the appended claims. Accordingly, the exclusive rights sought to be
patented
are as described in the claims below.
63
CA 02352571 2005-O1-20
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SEQUENCE LISTING
<110> UT-BATTELLE, LLC
THE UNIVERSITY OF TENNESSEE RESEARCH CORPORATION
SAYLER, GARY S.
SIMPSON, MICHAEL L.
APPLEGATE, BRUCE M.
RIPP, STEVEN A.
<120> IN VIVO BIOSENSOR APPARATUS AND METHOD OF USE
<130> 4300.004310
<140> PCT/US99/28733
<141> 1999-12-02
<140> 60/110,684
<141> 1998-12-02
<160> 7
<170> PatentIn Ver. 2.0
<210> 1
<211> 7669
<212> DNA
<213> Xenorhabdus luminescens
<220>
<221> CDS
<222> (1215)..(2657)
<223> LUXC
<220>
<221> CDS
<222> (2671)..(3594)
<223> LUXD
<220>
<221> CDS
<222> (3776) . . (4858)
<223> LUXA
<220>
<221> CDS
<222> (4873)..(5847)
<223> LUXB
<220>
<221> CDS
<222> (6160)..(7272)
<223> LUXE
<400> 1
gaattctcag actcaaatag aacaggattc taaagactta agagcagctg tagatcgtga 60
CA 02352571 2005-O1-20
ttttagtacg atagagccaa cattgagaaa ttatggggca acggaagcac aacttgaaga 120
cgccagagcc aaaatacaca agcttaacca agaacagagg ttatacaaat gacagttaat 180
acagaggcac taataaacag cctaggcaag tcctaccaag aaatttttga tgaagggcta 240
attccttata ggaataagcc aagtggttct cctggggtgc ctaatatttg tattgacatg 300
gtgaaagagg ggattttttt gtcgtttgaa cggaatagta aaatattaaa cgaaattact 360
ttaagattgc ttagagacga taaagctttg tttatatttc caaatgaatt gccatcaccg 420
ttgaagcatt ctatggatag gggatgggtt agagaaaatt taggtgatct gattaaatca 480
ataccaccga gacaaatttt aaaaaggcag tttggttgga aagatctata tcgttttacg 540
gatgaaatca gtatgcagat ttcttatgat ttacgtgaac aggttaattc agtgactttc 600
ttgcttacat cagacgtgag ttggtaattt aatatatata cccttcatcc ttcaagttgc 660
tgctttgttg gctgctttct ctcaccccag tcacatagtt atctatgctc ctggggattc 720
gttcacttgc cgccgcgctg caacttgaaa tctattgggt atatgctatt ggtaattatg 780
gaaaattgcc tgatttatat ataacttaac ttgtaaacca gataataatt tacatgaata 840
ttatcacgta taaaaaaatt gcgattcttt taatttgaaa tagttcaatt taattgaaac 900
tttttattaa caaatcttgt tgatgtgaaa attttcgttt gctattttaa cagatattgt 960
taaacggaga aggcagcatg ttgatgattc actcagccag actgacagtt ttaagcggaa 1020
aattgcagag tatgatcgca ttctgataaa ggttacaggt cactcgcaac cagaatttca 1080
tctttgtata ttttgttttg ttatttacgt tgcagcaaga caaaaataga agaaacaaat 1140
atttatacaa cccgtttgca agagggttaa acagcaattt aagttgaaat tgccctatta 1200
aatggatggc aaat atg aac aaa aaa att tca ttc att att aac ggt cga 1250
Met Asn Lys Lys Ile Ser Phe Ile Ile Asn Gly Arg
1 5 10
gtt gaa ata ttt cct gaa agt gat gat tta gtg caa tcc att aat ttt 1298
Val Glu Ile Phe Pro Glu Ser Asp Asp Leu Val Gln Ser Ile Asn Phe
15 20 25
ggt gat aat agt gtt cat ttg cca gta ttg aat gat tct caa gta aaa 1346
Gly Asp Asn Ser Val His Leu Pro Val Leu Asn Asp Ser Gln Val Lys
30 35 40
aac att att gat tat aat gaa aat aat gaa ttg caa ttg cat aac att 1394
Asn Ile Ile Asp Tyr Asn Glu Asn Asn Glu Leu Gln Leu His Asn Ile
45 50 55 60
atc aac ttt ctc tat acg gta ggg caa cga tgg aaa aat gaa gaa tat 1442
Ile Asn Phe Leu Tyr Thr Val Gly Gln Arg Trp Lys Asn Glu Glu Tyr
65 70 75
CA 02352571 2005-O1-20
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tcaagacgc aggacatat attcgtgat ctaaaaaga tatatggga tat 1490
SerArgArg ArgThrTyr IleArgAsp LeuLysArg TyrMetGly Tyr
80 85 90
tcagaagaa atggetaag ctagaggcc aactggata tctatgatt ttg 1538
SerGluGlu MetAlaLys LeuGluAla AsnTrpIle SerMetIle Leu
95 100 105
tgctctaaa ggtggcctt tatgatctt gtaaaaaat gaacttggt tct 1586
CysSerLys GlyGlyLeu TyrAspLeu ValLysAsn GluLeuGly Ser
110 115 120
cgccatatt atggatgaa tggctacct caggatgaa agttatatt aga 1634
ArgHisIle MetAspGlu TrpLeuPro GlnAspGlu SerTyrIle Arg
125 130 135 140
gettttccg aaaggaaaa tccgtacat ctgttgacg ggtaatgtg cca 1682
AlaPhePro LysGlyLys SerValHis LeuLeuThr GlyAsnVal Pro
145 150 155
ttatctggt gtgctgtct atattgcgt gcaatttta acaaagaat caa 1730
LeuSerGly ValLeuSer IleLeuArg AlaIleLeu ThrLysAsn Gln
160 165 170
tgcattata aaaacctca tcaactgat ccttttacc getaatgca tta 1778
CysIleIle LysThrSer SerThrAsp ProPheThr AlaAsnAla Leu
175 180 185
gcgctaagt tttatcgat gtggaccct catcatccg gtaacgcgt tct 1826
AlaLeuSer PheIleAsp ValAspPro HisHisPro ValThrArg Ser
190 195 200
ttgtcagtc gtatattgg caacatcaa ggcgatata tcactcgca aaa 1874
LeuSerVal ValTyrTrp GlnHisGln GlyAspIle SerLeuAla Lys
205 210 215 220
gagattatg caacatgcg gatgtcgtt gttgettgg ggaggggaa gat 1922
GluIleMet GlnHisAla AspValVal ValAlaTrp GlyGlyGlu Asp
225 230 235
gcgattaat tgggetgta aagcatgca ccacccgat attgacgtg atg 1970
AlaIleAsn TrpAlaVal LysHisAla ProProAsp IleAspVal Met
240 245 250
aagtttggt cctaaaaag agtttttgt attattgat aaccctgtt gat 2018
LysPheGly ProLysLys SerPheCys IleIleAsp AsnProVal Asp
255 260 265
ttagtatcc gcagetaca ggggcgget catgatgtt tgtttttac gat 2066
LeuValSer AlaAlaThr GlyAlaAla HisAspVal CysPheTyr Asp
270 275 280
cagcaaget tgtttttcc acccaaaat atatattac atgggaagt cat 2114
GlnGlnAla CysPheSer ThrGlnAsn IleTyrTyr MetGlySer His
285 290 295 300
CA 02352571 2005-O1-20
67
tatgaagag tttaagcta gcgttgatagaa aaattgaac ttatatgcg 2162
TyrGluGlu PheLysLeu AlaLeuIleGlu LysLeuAsn LeuTyrAla
305 310 315
catatatta ccaaacacc aaaaaagatttt gatgaaaag gcggcctat 2210
HisIleLeu ProAsnThr LysLysAspPhe AspGluLys AlaAlaTyr
320 325 330
tccttagtt caaaaagaa tgtttatttget ggattaaaa gtagaggtt 2258
SerLeuVal GlnLysGlu CysLeuPheAla GlyLeuLys ValGluVal
335 340 345
gatgttcat cagcgctgg atggttattgag tcaaatgcg ggtgtagaa 2306
AspValHis GlnArgTrp MetValIleGlu SerAsnAla GlyValGlu
350 355 360
ctaaatcaa ccacttggc agatgtgtgtat cttcatcac gtcgataat 2354
LeuAsnGln ProLeuGly ArgCysValTyr LeuHisHis ValAspAsn
365 370 375 380
attgagcaa atattgcct tatgtgcgaaaa aataaaacg caaaccata 2402
IleGluGln IleLeuPro TyrValArgLys AsnLysThr GlnThrIle
385 390 395
tctgttttt ccttgggag gccgcgcttaag tatcgagac ttattagca 2450
SerValPhe ProTrpGlu AlaAlaLeuLys TyrArgAsp LeuLeuAla
400 405 410
ttaaaaggt gcagaaagg attgtagaagca ggaatgaat aatatattt 2498
LeuLysGly AlaGluArg IleValGluAla GlyMetAsn AsnIlePhe
415 420 425
cgggttggt ggtgetcat gatggaatgaga cctttacaa cgattggtg 2546
ArgValGly GlyAlaHis AspGlyMetArg ProLeuGln ArgLeuVal
430 435 440
acatatatt tcccatgaa agaccatcccac tatactget aaagatgtt 2594
ThrTyrIle SerHisGlu ArgProSerHis TyrThrAla LysAspVal
445 450 455 460
gcggtcgaa atagaacag actcgattcctg gaagaagat aagttcctg 2642
AlaValGlu IleGluGln ThrArgPheLeu GluGluAsp LysPheLeu
465 470 475
gtatttgtc ccataataggtaaaag aatatg gaa aaa tccagatat 2691
aat
ValPheVal Pro Met Glu SerArg
Asn Tyr
Lys
480 485
aaaaccatc gaccatgtt atttgtgttgaa gaaaataga aaaattcat 2739
LysThrIle AspHisVal IleCysValGlu GluAsnArg LysIleHis
490 495 500
gtctgggag acgctgcca aaagaaaatagt ccaaagaga aaaaatacc 2787
ValTrpGlu ThrLeuPro LysGluAsnSer ProLysArg LysAsnThr
505 510 515 520
ctt att att gcg tcg ggt ttt gcc cgc agg atg gat cat ttt gcc ggt 2835
CA 02352571 2005-O1-20
68
LeuIleIleAla SerGly PheAlaArgArg MetAspHis PheAla Gly
525 530 535
ctggcagagtat ttgtcg cagaatggattt catgtgatc cgctat gat 2883
LeuAlaGluTyr LeuSer GlnAsnGlyPhe HisValIle ArgTyr Asp
540 545 550
tctcttcaccac gttgga ttgagttcaggg acaattgat gaattt aca 2931
SerLeuHisHis ValGly LeuSerSerGly ThrIleAsp GluPhe Thr
555 560 565
atgtccatagga aaacag agtttattagca gtggttgat tggtta aat 2979
MetSerIleGly LysGln SerLeuLeuAla ValValAsp TrpLeu Asn
570 575 580
acacgaaaaata aataac ctcggtatgctg gettcaagc ttatct gcg 3027
ThrArgLysIle AsnAsn LeuGlyMetLeu AlaSerSer LeuSer Ala
585 590 595 600
cggatagettat gcaagt ctatctgaaatt aatgtctcg ttttta att 3075
ArgIleAlaTyr AlaSer LeuSerGluIle AsnValSer PheLeu Ile
605 610 615
accgcagtcggt gtggtt aacttaagatat actctcgaa agaget tta 3123
ThrAlaValGly ValVal AsnLeuArgTyr ThrLeuGlu ArgAla Leu
620 625 630
ggatttgattat ctcagc ttacctattgat gaattgcca gataat tta 3171
GlyPheAspTyr LeuSer LeuProIleAsp GluLeuPro AspAsn Leu
635 640 645
gattttgaaggt cataaa ttgggtgetgag gtttttgcg agagat tgc 3219
AspPheGluGly HisLys LeuGlyAlaGlu ValPheAla ArgAsp Cys
650 655 660
tttgattctggc tgggaa gatttaacttct acaattaat agtatg atg 3267
PheAspSerGly TrpGlu AspLeuThrSer ThrIleAsn SerMet Met
665 670 675 680
catcttgatata ccgttt attgettttact gcaaataat gacgat tgg 3315
HisLeuAspIle ProPhe IleAlaPheThr AlaAsnAsn AspAsp Trp
685 690 695
gtaaagcaagat gaagtt attacattacta tcaagcatc cgtagt cat 3363
ValLysGlnAsp GluVal IleThrLeuLeu SerSerIle ArgSer His
700 705 710
caatgtaagata tattct ttactaggaagc tcacatgat ttgggt gag 3411
GlnCysLysIle TyrSer LeuLeuGlySer SerHisAsp LeuGly Glu
715 720 725
aacttagtggtc ctgcgc aatttttatcaa tcggttacg aaagcc get 3459
AsnLeuValVal LeuArg AsnPheTyrGln SerValThr LysAla Ala
730 735 740
atcgcgatggat aatggt tgtctggatatt gatgtcgat attatt gag 3507
IleAlaMetAsp AsnGly CysLeuAspIle AspValAsp IleIle Glu
CA 02352571 2005-O1-20
69
745 750 755 760
ccg tca ttc gaa cat tta acc att gcg gca gtc aat gaa cgc cga atg 3555
Pro Ser Phe Glu His Leu Thr Ile Ala Ala Val Asn Glu Arg Arg Met
765 770 775
aaa att gag att gaa aat caa gtg att tcg ctg tct taa aacctatacc 3604
Lys Ile Glu Ile Glu Asn Gln Val Ile Ser Leu Ser
780 785
aatagatttc gagttgcagc gcggcggcaa gtgaacgcat tcccaggagc atagataact 3664
ctgtgactgg ggtgcgtgaa agcagccaac aaagcagcaa cttgaaggat gaagggtata 3724
ttgggataga tagttaactc tatcactcaa atagaaatat aaggactctc t atg aaa 3781
Met Lys
790
ttt gga aac ttt ttg ctt aca tac caa ccc ccc caa ttt tct caa aca 3829
Phe Gly Asn Phe Leu Leu Thr Tyr Gln Pro Pro Gln Phe Ser Gln Thr
795 800 805
gag gta atg aaa cgg ttg gtt aaa tta ggt cgc atc tct gag gaa tgc 3877
Glu Val Met Lys Arg Leu Val Lys Leu Gly Arg Ile Ser Glu Glu Cys
810 815 820
ggt ttt gat acc gta tgg tta ctt gag cat cat ttc acg gag ttt ggt 3925
Gly Phe Asp Thr Val Trp Leu Leu Glu His His Phe Thr Glu Phe Gly
825 830 835
ttg ctt ggt aac cct tat gtg get get get tat tta ctt ggc gca acc 3973
Leu Leu Gly Asn Pro Tyr Val Ala Ala Ala Tyr Leu Leu Gly Ala Thr
840 845 850 855
aag aaa ttg aat gta ggg act gcg get att gtt ctc ccc acc get cat 4021
Lys Lys Leu Asn Val Gly Thr Ala Ala Ile Val Leu Pro Thr Ala His
860 865 870
cca gtt cgc cag ctt gaa gag gtg aat ttg ttg gat caa atg tca aaa 4069
Pro Val Arg Gln Leu Glu Glu Val Asn Leu Leu Asp Gln Met Ser Lys
875 880 885
gga cga ttt cga ttt ggt att tgt cgg ggg ctt tac aat aaa gat ttt 4117
Gly Arg Phe Arg Phe Gly Ile Cys Arg Gly Leu Tyr Asn Lys Asp Phe
890 895 900
cgc gta ttt ggc aca gat atg aat aac agt cgt gcc tta atg gag tgt 4165
Arg Val Phe Gly Thr Asp Met Asn Asn Ser Arg Ala Leu Met Glu Cys
905 910 915
tgg tat aag ttg ata cga aat gga atg act gag gga tat atg gaa get 4213
Trp Tyr Lys Leu Ile Arg Asn Gly Met Thr Glu Gly Tyr Met Glu Ala
920 925 930 935
gac aac gaa cat att aag ttc cat aag gta aaa gtg ctg ccg acg gca 4261
Asp Asn Glu His Ile Lys Phe His Lys Val Lys Val Leu Pro Thr Ala
940 945 950
CA 02352571 2005-O1-20
tatagtcaa ggtggtgca cctatttatgtc gttgetgaa tccgettcc 4309
TyrSerGln GlyGlyAla ProIleTyrVal ValAlaGlu SerAlaSer
955 960 965
acgactgaa tgggccget caacatggttta ccgatgatt ttaagttgg 4357
ThrThrGlu TrpAlaAla GlnHisGlyLeu ProMetIle LeuSerTrp
970 975 980
attataaat actaacgaa aagaaagcacaa attgagctt tataacgag 4405
IleIleAsn ThrAsnGlu LysLysAlaGln IleGluLeu TyrAsnGlu
985 990 995
gtcgetcaa gaatatgga cacgatattcat aatatcgac cattgctta 4453
ValAlaGln GluTyrGly HisAspIleHis AsnIleAsp HisCysLeu
1000 1005 1010 1015
tcatatata acatcggta gaccatgactca atgaaagcg aaagaaatt 4501
SerTyrIle ThrSerVal AspHisAspSer MetLysAla LysGluIle
1020 1025 1030
tgccggaat tttctgggg cattggtatgat tcctatgtt aatgccaca 4549
CysArgAsn PheLeuGly HisTrpTyrAsp SerTyrVal AsnAlaThr
1035 1040 1045
accattttt gatgattca gacaaaacaaag ggctatgat ttcaataaa 4597
ThrIlePhe AspAspSer AspLysThrLys GlyTyrAsp PheAsnLys
1050 1055 1060
ggacaatgg cgcgacttt gtcttaaaagga cataaaaat actaatcgt 4645
GlyGlnTrp ArgAspPhe ValLeuLysGly HisLysAsn ThrAsnArg
1065 1070 1075
cgcgttgat tacagttac gaaatcaatccg gtgggaacc ccgcaggaa 4693
ArgValAsp TyrSerTyr GluIleAsnPro ValGlyThr ProGlnGlu
1080 1085 1090 1095
tgtattgat ataattcaa acagacattgac gccacagga atatcaaat 4741
CysIleAsp IleIleGln ThrAspIleAsp AlaThrGly IleSerAsn
1100 1105 1110
atttgttgt gggtttgaa getaatggaaca gtagatgaa attatctct 4789
IleCysCys GlyPheGlu AlaAsnGlyThr ValAspGlu IleIleSer
1115 1120 1125
tccatgaag ctcttccag tctgatgtaatg ccgtttctt aaagaaaaa 4837
SerMetLys LeuPheGln SerAspValMet ProPheLeu LysGluLys
1130 1135 1140
caacgttcg ctattatat tagctaaggaaaa tgaaatgaaa tttggcttg 4887
GlnArgSer LeuLeuTyr MetLys PheGlyLeu
1145 1150 1155
ttcttcctt aactttatc aattcaacaact attcaagag caaagtata 4935
PhePheLeu AsnPheIle AsnSerThrThr IleGlnGlu GlnSerIle
1160 1165 1170
CA 02352571 2005-O1-20
71
getcgcatgcag gaaataaca gaatatgtc gacaaattgaat tttgag 4983
AlaArgMetGln GluIleThr GluTyrVal AspLysLeuAsn PheGlu
1175 1180 1185
cagattttggtg tgtgaaaat catttttca gataatggtgtt gtcggc 5031
GlnIleLeuVal CysGluAsn HisPheSer AspAsnGlyVal ValGly
1190 1195 1200
getcctttgact gtttctggt tttttactt ggcctaacagaa aaaatt 5079
AlaProLeuThr ValSerGly PheLeuLeu GlyLeuThrGlu LysIle
1205 1210 1215
aaaattggttca ttgaatcat gtcattaca actcatcatcct gtccgc 5127
LysIleGlySer LeuAsnHis ValIleThr ThrHisHisPro ValArg
1220 1225 1230 1235
atagcggaagaa gcgtgctta ttggatcag ttaagcgaagga agattt 5175
IleAlaGluGlu AlaCysLeu LeuAspGln LeuSerGluGly ArgPhe
1240 1245 1250
attttaggattt agtgattgc gagagaaag gatgaaatgcat tttttc 5223
IleLeuGlyPhe SerAspCys GluArgLys AspGluMetHis PhePhe
1255 1260 1265
aatcgccctgaa caataccag cagcaatta tttgaagaatgc tatgac 5271
AsnArgProGlu GlnTyrGln GlnGlnLeu PheGluGluCys TyrAsp
1270 1275 1280
attattaacgat getttaaca acaggctat tgtaatccaaat ggcgat 5319
IleIleAsnAsp AlaLeuThr ThrGlyTyr CysAsnProAsn GlyAsp
1285 1290 1295
ttttataatttc cccaaaata tccgtgaat ccccatgettat acgcaa 5367
PheTyrAsnPhe ProLysIle SerValAsn ProHisAlaTyr ThrGln
1300 1305 1310 1315
aacgggcctcgg aaatatgta acagcaaca agttgtcatgtt gttgag 5415
AsnGlyProArg LysTyrVal ThrAlaThr SerCysHisVal ValGlu
1320 1325 1330
tgggetgetaaa aaaggcatt cctctaatc tttaagtgggat gattcc 5463
TrpAlaAlaLys LysGlyIle ProLeuIle PheLysTrpAsp AspSer
1335 1340 1345
aatgaagttaaa catgaatat gcgaaaaga tatcaagccata gcaggt 5511
AsnGluValLys HisGluTyr AlaLysArg TyrGlnAlaIle AlaGly
1350 1355 1360
gaatatggtgtt gacctggca gagatagat catcagttaatg atattg 5559
GluTyrGlyVal AspLeuAla GluIleAsp HisGlnLeuMet IleLeu
1365 1370 1375
gttaactatagt gaagacagt gagaaaget aaagaggaaacg cgtgca 5607
ValAsnTyrSer GluAspSer GluLysAla LysGluGluThr ArgAla
1380 1385 1390 1395
ttt ata agt gat tat att ctt gca atg cac cct aat gaa aat ttc gaa 5655
CA 02352571 2005-O1-20
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PheIleSerAsp TyrIle LeuAlaMetHis ProAsnGlu AsnPheGlu
1400 1405 1410
aagaaacttgaa gaaata atcacagaaaac tccgtcgga gattatatg 5703
LysLysLeuGlu GluIle IleThrGluAsn SerValGly AspTyrMet
1415 1420 1425
gaatgtacaact gcgget aaattggcaatg gagaaatgt ggtgcaaaa 5751
GluCysThrThr AlaAla LysLeuAlaMet GluLysCys GlyAlaLys
1430 1435 1440
ggtatattattg tccttt gaatcaatgagt gattttaca catcaaata 5799
GlyIleLeuLeu SerPhe GluSerMetSer AspPheThr HisGlnIle
1445 1450 1455
aacgcaattgat attgtc aatgataatatt aaaaagtat cacatgtaa 5847
AsnAlaIleAsp IleVal AsnAspAsnIle LysLysTyr HisMet
1460 1465 1470 1475
tataccctat ggatttcaag gtgcatcgcg acggcaaggg agcgaatccc cgggagcata 5907
tacccaatag atttcaagtt gcagtgcggc ggcaagtgaa cgcatcccca ggagcataga 5967
taactatgtg actggggtaa gtgaacgcag ccaacaaagc agcagcttga aagatgaagg 6027
gtatagataa cgatgtgacc ggggtgcgtg aacgcagcca acaaagaggc aacttgaaag 6087
ataacgggta taaaagggta tagcagtcac tctgccatat cctttaatat tagctgccga 6147
ggtaaaacag gt atg act tca tat gtt gat aaa caa gaa atc aca gca agt 6198
Met Thr Ser Tyr Val Asp Lys Gln Glu Ile Thr Ala Ser
1480 1485
tcagaaattgat gatttgatt ttttcgagt gatccatta gtctgg tct 6246
SerGluIleAsp AspLeuIle PheSerSer AspProLeu ValTrp Ser
1490 1495 1500
tacgacgaacag gaaaagatt agaaaaaaa cttgtgctt gatgcg ttt 6294
TyrAspGluGln GluLysIle ArgLysLys LeuValLeu AspAla Phe
1505 1510 1515 1520
cgtcatcactat aaacattgt caagaatac cgtcactac tgtcag gca 6342
ArgHisHisTyr LysHisCys GlnGluTyr ArgHisTyr CysGln Ala
1525 1530 1535
cataaagtagat gacaatatt acggaaatt gatgatata cctgta ttc 6390
HisLysValAsp AspAsnIle ThrGluIle AspAspIle ProVal Phe
1540 1545 1550
ccaacatcagtg tttaagttt actcgctta ttaacttct aatgag aac 6438
ProThrSerVal PheLysPhe ThrArgLeu LeuThrSer AsnGlu Asn
1555 1560 1565
gaaattgaaagt tggtttacc agtagtggc acaaatggc ttaaaa agt 6486
GluIleGluSer TrpPheThr SerSerGly ThrAsnGly LeuLys Ser
1570 1575 1580
CA 02352571 2005-O1-20
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caggtaccacgt gacagacta agtattgag aggctcttaggc tctgta 6534
GlnValProArg AspArgLeu SerIleGlu ArgLeuLeuGly SerVal
1585 1590 1595 1600
agttatggtatg aaatatatt ggtagttgg ttcgatcatcaa atggaa 6582
SerTyrGlyMet LysTyrIle GlySerTrp PheAspHisGln MetGlu
1605 1610 1615
ttggtcaacctg ggaccagat agatttaat getcataatatt tggttt 6630
LeuValAsnLeu GlyProAsp ArgPheAsn AlaHisAsnIle TrpPhe
1620 1625 1630
aaatatgttatg agcttggta gagttatta tatcctacgtca ttcacc 6678
LysTyrValMet SerLeuVal GluLeuLeu TyrProThrSer PheThr
1635 1640 1645
gtaacagaagaa cacatagat ttcgttcag acattaaatagt cttgag 6726
ValThrGluGlu HisIleAsp PheValGln ThrLeuAsnSer LeuGlu
1650 1655 1660
cgaataaaacat caagggaaa gatatttgt cttattggttcg ccatac 6774
ArgIleLysHis GlnGlyLys AspIleCys LeuIleGlySer ProTyr
1665 1670 1675 1680
tttatttatttg ctctgccgt tatatgaaa gataaaaatatc tcattt 6822
PheIleTyrLeu LeuCysArg TyrMetLys AspLysAsnIle SerPhe
1685 1690 1695
tctggagataaa agtctttat attataacg gggggaggctgg aaaagt 6870
SerGlyAspLys SerLeuTyr IleIleThr GlyGlyGlyTrp LysSer
1700 1705 1710
tacgaaaaagaa tctttgaag cgtaatgat ttcaatcatctt ttattc 6918
TyrGluLysGlu SerLeuLys ArgAsnAsp PheAsnHisLeu LeuPhe
1715 1720 1725
gacactttcaac ctcagtaat attaaccag atccgtgatata tttaat 6966
AspThrPheAsn LeuSerAsn IleAsnGln IleArgAspIle PheAsn
1 730 1735 1740
caagttgaactc aacacttgt ttctttgag gatgaaatgcaa cgtaaa 7014
GlnValGluLeu AsnThrCys PhePheGlu AspGluMetGln ArgLys
1745 1750 1755 1760
catgttccgccg tgggtatat gcgcgagca cttgatcctgaa acattg 7062
HisValProPro TrpValTyr AlaArgAla LeuAspProGlu ThrLeu
1765 1770 1775
aaaccggtacct gatgggatg cctggtttg atgagttatatg gatgca 7110
LysProValPro AspGlyMet ProGlyLeu MetSerTyrMet AspAla
1780 1785 1790
tcatcaacgagt tatccggca tttattgtt accgatgatatc ggaata 7158
SerSerThrSer TyrProAla PheIleVal ThrAspAspIle GlyIle
1795 1800 1805
att agc aga gaa tat ggt caa tat cct ggt gta ttg gtt gaa att tta 7206
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Ile Ser Arg Glu Tyr Gly Gln Tyr Pro Gly Val Leu Val Glu Ile Leu
1810 1815 1820
cgt cgc gtt aat acg agg aaa caa aaa ggt tgt get tta agc tta act 7254
Arg Arg Val Asn Thr Arg Lys Gln Lys Gly Cys Ala Leu Ser Leu Thr
1825 1830 1835 1840
gaa gca ttt ggt agt tga tagtttcttt ggaaagagga gcagtcaaag 7302
Glu Ala Phe Gly Ser
1845
gctcatttgt tcaatgcttt tgcgaaacgt tttgtcgaac tctaggcgaa ggttctcgac 7362
tttccccgca tcaggggtat atacaagtaa aaaagctcag ggggtaaacc tgagcttggg 7422
atgttgattt ttaagtatga gatacatggg cggatttaaa taacggagtc agtttggaaa 7482
tatcaacggt cttttctgct ttatcgaggc tataagtttc ttgcagtttt aaccacaacc 7542
gcggagagct gccaagtact tgtgacagtt ttattgccat ctctggcgtg actgctgctt 7602
tacacgatac taaacgttga accgtagagg gagcaacatt caatgcccgc gctaagttca 7662
cgaattc 7669
<210> 2
<211> 480
<212> PRT
<213> Xenorhabdus luminescens
<400> 2
Met Asn Lys Lys Ile Ser Phe Ile Ile Asn Gly Arg Val Glu Ile Phe
1 5 10 15
Pro Glu Ser Asp Asp Leu Val Gln Ser Ile Asn Phe Gly Asp Asn Ser
20 25 30
Val His Leu Pro Val Leu Asn Asp Ser Gln Val Lys Asn Ile Ile Asp
35 40 45
Tyr Asn Glu Asn Asn Glu Leu Gln Leu His Asn Ile Ile Asn Phe Leu
50 55 60
Tyr Thr Val Gly Gln Arg Trp Lys Asn Glu Glu Tyr Ser Arg Arg Arg
65 70 75 80
Thr Tyr Ile Arg Asp Leu Lys Arg Tyr Met Gly Tyr Ser Glu Glu Met
85 90 95
Ala Lys Leu Glu Ala Asn Trp Ile Ser Met Ile Leu Cys Ser Lys Gly
100 105 110
Gly Leu Tyr Asp Leu Val Lys Asn Glu Leu Gly Ser Arg His Ile Met
115 120 125
Asp Glu Trp Leu Pro Gln Asp Glu Ser Tyr Ile Arg Ala Phe Pro Lys
CA 02352571 2005-O1-20
130 135 140
Gly Lys Ser Val His Leu Leu Thr Gly Asn Val Pro Leu Ser Gly Val
145 150 155 160
Leu Ser Ile Leu Arg Ala Ile Leu Thr Lys Asn Gln Cys Ile Ile Lys
165 170 175
Thr Ser Ser Thr Asp Pro Phe Thr Ala Asn Ala Leu Ala Leu Ser Phe
180 185 190
Ile Asp Val Asp Pro His His Pro Val Thr Arg Ser Leu Ser Val Val
195 200 205
Tyr Trp Gln His Gln Gly Asp Ile Ser Leu Ala Lys Glu Ile Met Gln
210 215 220
His Ala Asp Val Val Val Ala Trp Gly Gly Glu Asp Ala Ile Asn Trp
225 230 235 240
Ala Val Lys His Ala Pro Pro Asp Ile Asp Val Met Lys Phe Gly Pro
245 250 255
Lys Lys Ser Phe Cys Ile Ile Asp Asn Pro Val Asp Leu Val Ser Ala
260 265 270
Ala Thr Gly Ala Ala His Asp Val Cys Phe Tyr Asp Gln Gln Ala Cys
275 280 285
Phe Ser Thr Gln Asn Ile Tyr Tyr Met Gly Ser His Tyr Glu Glu Phe
290 295 300
Lys Leu Ala Leu Ile Glu Lys Leu Asn Leu Tyr Ala His Ile Leu Pro
305 310 315 320
Asn Thr Lys Lys Asp Phe Asp Glu Lys Ala Ala Tyr Ser Leu Val Gln
325 330 335
Lys Glu Cys Leu Phe Ala Gly Leu Lys Val Glu Val Asp Val His Gln
340 345 350
Arg Trp Met Val Ile Glu Ser Asn Ala Gly Val Glu Leu Asn Gln Pro
355 360 365
Leu Gly Arg Cys Val Tyr Leu His His Val Asp Asn Ile Glu Gln Ile
370 375 380
Leu Pro Tyr Val Arg Lys Asn Lys Thr Gln Thr Ile Ser Val Phe Pro
385 390 395 400
Trp Glu Ala Ala Leu Lys Tyr Arg Asp Leu Leu Ala Leu Lys Gly Ala
405 410 415
Glu Arg Ile Val Glu Ala Gly Met Asn Asn Ile Phe Arg Val Gly Gly
420 425 430
Ala His Asp Gly Met Arg Pro Leu Gln Arg Leu Val Thr Tyr Ile Ser
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435 440 445
His Glu Arg Pro Ser His Tyr Thr Ala Lys Asp Val Ala Val Glu Ile
450 455 460
Glu Gln Thr Arg Phe Leu Glu Glu Asp Lys Phe Leu Val Phe Val Pro
465 470 475 480
<210> 3
<211> 307
<212> PRT
<213> Xenorhabdus luminescens
<400> 3
Met Glu Asn Lys Ser Arg Tyr Lys Thr Ile Asp His Val Ile Cys
1 5 10 15
Val Glu Glu Asn Arg Lys Ile His Val Trp Glu Thr Leu Pro Lys Glu
20 25 30
Asn Ser Pro Lys Arg Lys Asn Thr Leu Ile Ile Ala Ser Gly Phe Ala
35 40 45
Arg Arg Met Asp His Phe Ala Gly Leu Ala Glu Tyr Leu Ser Gln Asn
50 55 60
Gly Phe His Val Ile Arg Tyr Asp Ser Leu His His Val Gly Leu Ser
65 70 75
Ser Gly Thr Ile Asp Glu Phe Thr Met Ser Ile Gly Lys Gln Ser Leu
80 85 90 95
Leu Ala Val Val Asp Trp Leu Asn Thr Arg Lys Ile Asn Asn Leu Gly
100 105 110
Met Leu Ala Ser Ser Leu Ser Ala Arg Ile Ala Tyr Ala Ser Leu Ser
115 120 125
Glu Ile Asn Val Ser Phe Leu Ile Thr Ala Val Gly Val Val Asn Leu
130 135 140
Arg Tyr Thr Leu Glu Arg Ala Leu Gly Phe Asp Tyr Leu Ser Leu Pro
145 150 155
Ile Asp Glu Leu Pro Asp Asn Leu Asp Phe Glu Gly His Lys Leu Gly
60 165 170 175
Ala Glu Val Phe Ala Arg Asp Cys Phe Asp Ser Gly Trp Glu Asp Leu
180 185 190
Thr Ser Thr Ile Asn Ser Met Met His Leu Asp Ile Pro Phe Ile Ala
195 200 205
Phe Thr Ala Asn Asn Asp Asp Trp Val Lys Gln Asp Glu Val Ile Thr
210 215 220
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Leu Leu Ser Ser Ile Arg Ser His Gln Cys Lys Ile Tyr Ser Leu Leu
225 230 235
Gly Ser Ser His Asp Leu Gly Glu Asn Leu Val Val Leu Arg Asn Phe
40 245 250 255
Tyr Gln Ser Val Thr Lys Ala Ala Ile Ala Met Asp Asn Gly Cys Leu
260 265 270
Asp Ile Asp Val Asp Ile Ile Glu Pro Ser Phe Glu His Leu Thr Ile
275 280 285
Ala Ala Val Asn Glu Arg Arg Met Lys Ile Glu Ile Glu Asn Gln Val
290 295 300
Ile Ser Leu Ser
305
<210> 4
<211> 360
<212> PRT
<213> Xenorhabdus luminescens
<400> 4
Met Lys Phe Gly Asn Phe Leu Leu Thr Tyr Gln Pro Pro Gln Phe Ser
1 5 10 15
Gln Thr Glu Val Met Lys Arg Leu Val Lys Leu Gly Arg Ile Ser Glu
20 25 30
Glu Cys Gly Phe Asp Thr Val Trp Leu Leu Glu His His Phe Thr Glu
35 40 45
Phe Gly Leu Leu Gly Asn Pro Tyr Val Ala Ala Ala Tyr Leu Leu Gly
50 55 60
Ala Thr Lys Lys Leu Asn Val Gly Thr Ala Ala Ile Val Leu Pro Thr
65 70 75 80
Ala His Pro Val Arg Gln Leu Glu Glu Val Asn Leu Leu Asp Gln Met
85 90 95
Ser Lys Gly Arg Phe Arg Phe Gly Ile Cys Arg Gly Leu Tyr Asn Lys
100 105 110
Asp Phe Arg Val Phe Gly Thr Asp Met Asn Asn Ser Arg Ala Leu Met
115 120 125
Glu Cys Trp Tyr Lys Leu Ile Arg Asn Gly Met Thr Glu Gly Tyr Met
130 135 140
Glu Ala Asp Asn Glu His Ile Lys Phe His Lys Val Lys Val Leu Pro
145 150 155 160
Thr Ala Tyr Ser Gln Gly Gly Ala Pro Ile Tyr Val Val Ala Glu Ser
165 170 175
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Ala Ser Thr Thr Glu Trp Ala Ala Gln His Gly Leu Pro Met Ile Leu
180 185 190
Ser Trp Ile Ile Asn Thr Asn Glu Lys Lys Ala Gln Ile Glu Leu Tyr
195 200 205
Asn Glu Val Ala Gln Glu Tyr Gly His Asp Ile His Asn Ile Asp His
210 215 220
Cys Leu Ser Tyr Ile Thr Ser Val Asp His Asp Ser Met Lys Ala Lys
225 230 235 240
Glu Ile Cys Arg Asn Phe Leu Gly His Trp Tyr Asp Ser Tyr Val Asn
245 250 255
Ala Thr Thr Ile Phe Asp Asp Ser Asp Lys Thr Lys Gly Tyr Asp Phe
260 265 270
Asn Lys Gly Gln Trp Arg Asp Phe Val Leu Lys Gly His Lys Asn Thr
275 280 285
Asn Arg Arg Val Asp Tyr Ser Tyr Glu Ile Asn Pro Val Gly Thr Pro
290 295 300
Gln Glu Cys Ile Asp Ile Ile Gln Thr Asp Ile Asp Ala Thr Gly Ile
305 310 315 320
Ser Asn Ile Cys Cys Gly Phe Glu Ala Asn Gly Thr Val Asp Glu Ile
325 330 335
Ile Ser Ser Met Lys Leu Phe Gln Ser Asp Val Met Pro Phe Leu Lys
340 345 350
Glu Lys Gln Arg Ser Leu Leu Tyr
355 360
<210> 5
<211> 324
<212> PRT
<213> Xenorhabdus luminescens
<400> 5
Met Lys Phe Gly Leu Phe Phe Leu Asn Phe Ile Asn Ser Thr Thr
1 5 10 15
Ile Gln Glu Gln Ser Ile Ala Arg Met Gln Glu Ile Thr Glu Tyr Val
20 25 30
Asp Lys Leu Asn Phe Glu Gln Ile Leu Val Cys Glu Asn His Phe Ser
35 40 45
Asp Asn Gly Val Val Gly Ala Pro Leu Thr Val Ser Gly Phe Leu Leu
50 55 60
Gly Leu Thr Glu Lys Ile Lys Ile Gly Ser Leu Asn His Val Ile Thr
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65 70 75
Thr His His Pro Val Arg Ile Ala Glu Glu Ala Cys Leu Leu Asp Gln
80 85 90 95
Leu Ser Glu Gly Arg Phe Ile Leu Gly Phe Ser Asp Cys Glu Arg Lys
100 105 110
Asp Glu Met His Phe Phe Asn Arg Pro Glu Gln Tyr Gln Gln Gln Leu
115 120 125
Phe Glu Glu Cys Tyr Asp Ile Ile Asn Asp Ala Leu Thr Thr Gly Tyr
130 135 140
Cys Asn Pro Asn Gly Asp Phe Tyr Asn Phe Pro Lys Ile Ser Val Asn
145 150 155
Pro His Ala Tyr Thr Gln Asn Gly Pro Arg Lys Tyr Val Thr Ala Thr
160 165 170 175
Ser Cys His Val Val Glu Trp Ala Ala Lys Lys Gly Ile Pro Leu Ile
180 185 190
Phe Lys Trp Asp Asp Ser Asn Glu Val Lys His Glu Tyr Ala Lys Arg
195 200 205
Tyr Gln Ala Ile Ala Gly Glu Tyr Gly Val Asp Leu Ala Glu Ile Asp
210 215 220
His Gln Leu Met Ile Leu Val Asn Tyr Ser Glu Asp Ser Glu Lys Ala
225 230 235
Lys Glu Glu Thr Arg Ala Phe Ile Ser Asp Tyr Ile Leu Ala Met His
240 245 250 255
Pro Asn Glu Asn Phe Glu Lys Lys Leu Glu Glu Ile Ile Thr Glu Asn
260 265 270
Ser Val Gly Asp Tyr Met Glu Cys Thr Thr Ala Ala Lys Leu Ala Met
275 280 285
Glu Lys Cys Gly Ala Lys Gly Ile Leu Leu Ser Phe Glu Ser Met Ser
290 295 300
Asp Phe Thr His Gln Ile Asn Ala Ile Asp Ile Val Asn Asp Asn Ile
305 310 315
Lys Lys Tyr His Met
320
<210> 6
<211> 370
<212> PRT
<213> Xenorhabdus luminescens
<400> 6
CA 02352571 2005-O1-20
Met Thr Ser Tyr Val Asp Lys Gln Glu Ile Thr Ala Ser Ser Glu Ile
1 5 10 15
Asp Asp Leu Ile Phe Ser Ser Asp Pro Leu Val Trp Ser Tyr Asp Glu
20 25 30
Gln Glu Lys Ile Arg Lys Lys Leu Val Leu Asp Ala Phe Arg His His
35 40 45
Tyr Lys His Cys Gln Glu Tyr Arg His Tyr Cys Gln Ala His Lys Val
50 55 60
Asp Asp Asn Ile Thr Glu Ile Asp Asp Ile Pro Val Phe Pro Thr Ser
65 70 75 80
Val Phe Lys Phe Thr Arg Leu Leu Thr Ser Asn Glu Asn Glu Ile Glu
90 95
Ser Trp Phe Thr Ser Ser Gly Thr Asn Gly Leu Lys Ser Gln Val Pro
100 105 110
Arg Asp Arg Leu Ser Ile Glu Arg Leu Leu Gly Ser Val Ser Tyr Gly
115 120 125
Met Lys Tyr Ile Gly Ser Trp Phe Asp His Gln Met Glu Leu Val Asn
130 135 140
Leu Gly Pro Asp Arg Phe Asn Ala His Asn Ile Trp Phe Lys Tyr Val
145 I50 155 160
Met Ser Leu Val Glu Leu Leu Tyr Pro Thr Ser Phe Thr Val Thr Glu
165 170 175
Glu His Ile Asp Phe Val Gln Thr Leu Asn Ser Leu Glu Arg Ile Lys
180 185 190
His Gln Gly Lys Asp Ile Cys Leu Ile Gly Ser Pro Tyr Phe Ile Tyr
195 200 205
Leu Leu Cys Arg Tyr Met Lys Asp Lys Asn Ile Ser Phe Ser Gly Asp
210 215 220
Lys Ser Leu Tyr Ile Tle Thr Gly Gly Gly Trp Lys Ser Tyr Glu Lys
225 230 235 240
Glu Ser Leu Lys Arg Asn Asp Phe Asn His Leu Leu Phe Asp Thr Phe
245 250 255
Asn Leu Ser Asn Ile Asn Gln Ile Arg Asp Ile Phe Asn Gln Val Glu
260 265 270
Leu Asn Thr Cys Phe Phe Glu Asp Glu Met Gln Arg Lys His Val Pro
275 280 285
Pro Trp Val Tyr Ala Arg Ala Leu Asp Pro Glu Thr Leu Lys Pro Val
290 295 300
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Pro Asp Gly Met Pro Gly Leu Met Ser Tyr Met Asp Ala Ser Ser Thr
305 310 315 320
Ser Tyr Pro Ala Phe Ile Val Thr Asp Asp Ile Gly Ile Ile Ser Arg
325 330 335
Glu Tyr Gly Gln Tyr Pro Gly Val Leu Val Glu Ile Leu Arg Arg Val
340 345 350
Asn Thr Arg Lys Gln Lys Gly Cys Ala Leu Ser Leu Thr Glu Ala Phe
355 360 365
Gly Ser
370
<210> 7
<211> 21
<212> DNA
<213> Synthetic
<400>
tacctaggga gaaagagaat g 21
