Microbial

Proteins as
Reporters
Calawen, Charmaine P.
Aiso, Stepahnie D.
Narvaza, Jiane Erika F.
Awacan, Andrea May P.

Date Submitted:
February 05, 2014

Date Reported:
__________________

Microbial Proteins as Reporters

Reporter Gene:

A gene with a readily measurable phenotype that can be easily distinguished over a background of
endogenous proteins

OR
A gene that researchers attach to a regulatory sequence of another gene of interest in cell culture, animals
or plants.

Reporter Assay

1. Measures gene expression or transcriptional activity

2. Assay of transcription factors.

3. DNA promoter assay

4. Confirmation of transgenosis

Reporter Assays

1. CAT: chloramphenicol acetyltransferase

2. -gal (-galactosidase)

3. GUS Reporter Gene System

4. SEAP (secreted alkaline phosphatase)

5. Luciferase

6. GFP: Green Fluorescent Protein

7. Bioluminescence Resonance Energy Transfer (BRET)

8. Human Growth Hormone (hGH) Reporter Gene System

Chloramphenicol acetyltransferase
(Reporter: Jiane Erika F. Narvaza)

1. 1st reporter gene used to monitor transcriptional activity in cells

2. Bacterial enzyme that transfers acetyl groups from acetyl-CoA to chloramphenicol, detoxifying it
3. Reaction quantified using radiolabeled substrates (14C-chloramphenicol) or by ELISA (non-radioactive)

Chloramphenicol acetyltransferase (or CAT) is a bacterial enzyme (EC 2.3.1.28) that detoxifies
the antibiotic chloramphenicol and is responsible for chloramphenicol resistance in bacteria. This enzyme
covalently attaches an acetyl group from acetyl-CoA to chloramphenicol, which prevents chloramphenicol from
binding to ribosomes. A histidine residue, located in the C-terminal section of the enzyme, plays a central role
in its catalytic mechanism.

The crystal structure of the type III enzyme from Escherichia coli with chloramphenicol bound has been
determined. CAT is a trimer of identical subunits (monomer Mr 25,000) and the trimeric structure is stabilised
by a number of hydrogen bonds, some of which result in the extension of a beta-sheet across the subunit
interface. Chloramphenicol binds in a deep pocket located at the boundary between adjacent subunits of the
trimer, such that the majority of residues forming the binding pocket belong to one subunit while the catalytically
essential histidine belongs to the adjacent subunit. His195 is appropriately positioned to act as a general base
catalyst in the reaction, and the required tautomeric stabilisation is provided by an unusual interaction with a
main-chain carbonyl oxygen.

Application

CAT is used as a reporter system to measure the level of a promoter or its tissue-specific expression. The CAT
assay involves monitoring acetylation of radioactively labeled chloramphenicol on a TLC plate; CAT activity is
determined by looking for the acetylated forms of chloramphenicol, which have a significantly increased
migration rate as compared to the unacetylated form.

Beta-galactosidase
(Reporter: Stephanie D. Aiso)

• E. coli enzyme (encoded by lacZ) that

hydrolyzes galactosidase sugars such as lactose
• Many assay formats:
colorimetric, fluorescent, chemiluminescent
• β-Lactamase can function as a reporter of bacterial protein export during Mycobacterium
tuberculosisinfection of host cells

Properties and functions

β-galactosidase is an exoglycosidase which hydrolyzes the β-glycosidic bond formed between
a galactose and its organic moiety. It may also cleavefucosides and arabinosides but with much lower
efficiency. It is an essential enzyme in the human body. Deficiencies in the protein can result
ingalactosialidosis or Morquio B syndrome. In E. coli, the gene of β-galactosidase, the lacZ gene, is
present as part of the inducible system lac operonwhich is activated in the presence of lactose when
glucose level is low.
It is commonly used in molecular biology as a reporter marker to monitor gene expression. It also exhibits
a phenomenon called α-complementation which forms the basis for the blue/white screening of
recombinant clones. This enzyme can be split in two peptides, LacZα and LacZΩ, neither of which is
active by itself but when both are present together, spontaneously reassemble into a functional enzyme.
This property is exploited in manycloning vectors where the presence of the lacZα gene in a plasmid can
complement in trans another mutant gene encoding the LacZΩ in specific laboratory strains of E. coli.
However, when DNA fragments are inserted in the vector, the production of LacZα is disrupted, the cells
therefore show no β-galactosidase activity. The presence or absence of an active β-galactosidase may be
detected by X-gal, which produces a characteristic blue dye when cleaved by β-galactosidase, thereby
providing an easy means of distinguishing the presence or absence of cloned product in a plasmid.

In 1995, Dimri et al. proposed a new isoform for beta-galactosidase with optimum activity at pH 6.0
(Senescence Associated beta-gal or SA-beta-gal) which would be specifically expressed
in senescence (The irreversible growth arrest of cells). Specific quantitative assays were even developed
for its detection. However, it is now known that this is due to an overexpression and accumulation of the
lysosomal endogenous beta-galactosidase, and its expression is not required for senescence.
Nevertheless, it remains the most widely used biomarker for senescent and aging cells, because it is
reliable and easy to detect.

Reaction
The active site of β-galactosidase catalyzes the hydrolysis of its disaccharide substrate via "shallow" and
"deep" binding. Monovalent potassium ions(K+) as well as divalent magnesium ions (Mg2+) are required
for the enzyme's optimal activity. The beta-linkage of the substrate is cleaved by a terminal
carboxyl group on the side chain of a glutamic acid.

In E. coli, Glu-461 was thought to be the nucleophile in the substitution reaction. However, it is now
known that Glu-461 is an acid catalyst. Instead, Glu-537 is the actual nucleophile, binding to a galactosyl
intermediate.

In humans, the nucleophile of the hydrolysis reaction is Glu-268.

Uses
The β-galactosidase assay is used frequently in genetics, molecular biology, and other life sciences. An
active enzyme may be detected using X-gal, which forms an intense blue product after cleavage by β-
galactosidase, and is easy to identify and quantify. It is used for example in blue white screen. Its
production may be induced by a non-hydrolyzable analog of allolactose, IPTG, which binds and releases
the lac repressor from the lac operator, thereby allowing the initiation of transcription to proceed.

Since it is highly expressed and accumulated in lysosomes in senescent cells, it is used as

a senescence biomarker both in vivo and in vitro in qualitative and quantitative assays, despite its
limitations.

GUS reporter system

(Reporter: Stephanie Aiso)
 • GUS encodes the beta-glucuronidase enzyme from E. coli.
• An active enzyme may be detected using X-gal, which forms an intense blue product after
cleavage by β-galactosidase

he GUS reporter system (GUS: β-glucuronidase) is a reporter gene system, particularly useful in
plant molecular biology and microbiology. Several kinds of GUS reporter gene assay are actually
available, depending on the substrate used. The term GUS staining refers to the most common of these,
a histochemical technique.

Purpose
The purpose of this technique is to analyze the activity of a promoter (in terms of expression of a gene
under that promoter) either in a quantitative way or through visualization of its activity in different tissues.
The technique is based on β-glucuronidase, an enzyme from the bacterium Escherichia coli; this enzyme,
when incubated with some specific colorless or non-fluorescent substrates, can transform them
into coloured or fluorescent products.

Substrates
There are actually different possible glucuronides that can be used as substrates for the β-glucuronidase,
depending on the type of detection needed (histochemical, spectrophotometrical,fluorimetrical). The most
common substrate for GUS histochemical staining is 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc): the
product of the reaction is in this case a clear blue color. Other common substrates are p-nitrophenyl β-D-
glucuronide for the spectrophotometrical assay and 4-methylumbelliferyl-beta-D-glucuronide (MUG) for
the fluorimetrical assay.

History
The system was originally developed by Richard Anthony Jefferson during his Ph.D. at the University of
Colorado at Boulder. He adapted the technique for the use with plants as he worked in the Plant Breeding
Institute of Cambridge, between 1985 and 1987. Since then thousands of labs have used the system,
making it probably the most widely used tool in plant molecular biology, as underlined by over 6000
citations in scientific literature.

Target organisms
An organism is suitable for a GUS assay if it has no β-glucuronidase or if the activity is very low
(background activity). For this reason the assay is not useful in most vertebrates and
many molluscs. Since there is no detectable GUS activity in higher
plants, mosses, algae, ferns, fungi and most bacteria, the assay is perfectly suited for these organisms.
Thus it is used widely in plant science.

Other reporter systems

The GUS system is not the only available gene reporter system for the analysis of promoter activity. Other
competing systems are based on e.g.luciferase, GFP, beta-galactosidase, chloramphenicol
acetyltransferase (CAT), alkaline phosphatase. The use of one or the other system is mainly dependent
on the organism of interest.

Other uses
The GUS assay, as well as other reporter gene systems, can be used for other kinds of studies other than
the classical promoter activity assay. Reporter systems have been used for the determination of the
efficiency of gene delivery systems, the intracellular localization of a gene product, the detection of
protein-protein or protein-DNA interactions, the efficiency of translation initiation signals and the success
of molecular cloning efforts.

Alkaline phosphatase
(Reporter: Jiane Erika F. Narvaza)

• Secreted outside the cell (can assay sample repeatedly and non-destructively by sampling
culture medium)
• This protein is quantified directly by measuring the enzyme activity in the supernatant of the
culture medium.
• Fluorescence and chemiluminescence assays are available for detection.

Alkaline phosphatase (ALP, ALKP) (EC 3.1.3.1) is a hydrolase enzyme responsible for
removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids.
The process of removing the phosphate group is called dephosphorylation. As the name suggests,
alkaline phosphatases are most effective in an alkaline environment. It is sometimes used synonymously
as basic phosphatase.

Bacterial
In Gram-negative bacteria, alkaline phosphatase is located in the periplasmic space, external to the cell
membrane. Since this space is much more subject to environmental variation than the actual interior of
the cell, bacterial alkaline phosphatase is comparatively resistant to inactivation,denaturation,
and degradation, and also has a higher rate of activity. Although the purpose of the enzyme is not fully
resolved, the simple hypothesis that it is a means for the bacteria to generate free phosphate groups for
uptake and use is supported by the fact that alkaline phosphatase is usually produced by the bacteria
only during phosphate starvation and not when phosphate is plentiful. However, other possibilities exist.
For instance, the presence of phosphate groups usually prevents organic molecules from passing through
the membrane; therefore, dephosphorylating them may be important for bacterial uptake of organic
compounds in the wild. Some complexities of bacterial regulation and metabolism suggest that other,
more subtle, purposes for the enzyme may also play a role for the cell. In the laboratory,
however, mutant Escherichia coli lacking alkaline phosphatase survive quite well, as do mutants unable
to shut off alkaline phosphatase production.

The optimal pH for the activity of the E. coli enzyme is 8.0 while the bovine enzyme optimum pH is slightly
higher at 8.5.

Use in research
Typical use in the lab for alkaline phosphatases includes removing phosphate monoester to prevent self-
ligation.

Common alkaline phosphatases used in research include:

 Shrimp alkaline phosphatase (SAP), from a species of Arctic shrimp (Pandalus borealis). This
phosphatase is easily inactivated by heat, a useful feature in some applications.
 Calf-intestinal alkaline phosphatase (CIP)
 Placental alkaline phosphatase (PALP) and its C terminally truncated version that lacks the last 24
amino acids (constituting the domain that targets for GPI membrane anchoring) - the secreted
alkaline phosphatase (SEAP)
Alkaline phosphatase has become a useful tool in molecular biology laboratories, since DNA normally
possesses phosphate groups on the 5' end. Removing these phosphates prevents the DNA
from ligating (the 5' end attaching to the 3' end), thereby keeping DNA molecules linear until the next step
of the process for which they are being prepared; also, removal of the phosphate groups
allows radiolabeling (replacement by radioactive phosphate groups) in order to measure the presence of
the labeled DNA through further steps in the process or experiment. For these purposes, the alkaline
phosphatase from shrimp is the most useful, as it is the easiest to inactivate once it has done its job.

Another important use of alkaline phosphatase is as a label for enzyme immunoassays.

Undifferentiated pluripotent stem cells have elevated levels of alkaline phosphatase on their cell
membrane, therefore alkaline phosphatase staining is used to detect these cells and to test pluripotency
(i.e., embryonic stem cells or embryonal carcinoma cells).

One common use in the dairy industry is as a marker of pasteurisation in cows' milk. This molecule is
denatured by elevated temperatures found during pasteurisation, and can be tested for via colour change
of a para-Nitrophenylphosphate substrate in a buffered solution (Aschaffenburg Mullen Test). [10] Raw milk
would typically produce a yellow colouration within a couple of minutes, whereas properly pasteurised
milk should show no change. Of course, there are exceptions to this, as in the case of heat-stable alkaline
phophatases produced by some bacteria

Human
Physiology
In humans, alkaline phosphatase is present in all tissues throughout the entire body, but is particularly
concentrated in liver, bile duct, kidney, bone, and the placenta. Humans and most other mammals contain
the following alkaline phosphatase isozymes:

 ALPI – intestinal
 ALPL – tissue-nonspecific (liver/bone/kidney)
 ALPP – placental (Regan isozyme)
Diagnostic use
Normal ALP levels in adults are approximately 20 to 140 IU/L, though levels are significantly higher in
children and pregnant women. Blood tests should always be interpreted using the reference range from
the laboratory that performed the test. High ALP levels can occur if the bile ducts are obstructed. Also,
ALP increases if there is active bone formation occurring, as ALP is a byproduct of osteoblast activity
(such as the case in Paget's disease of bone). Levels are also elevated in people with untreated celiac
disease. Lowered levels of ALP are less common than elevated levels.

Elevated levels
Elevated alkaline phosphatase

If it is unclear why alkaline phosphatase is elevated, isoenzyme studies using electrophoresis can confirm
the source of the ALP. Heat stability also distinguishes bone and liver isoenzymes ("bone burns, liver
lasts"). Placental alkaline phosphatase is elevated in seminomas[15] and active form of rickets.

Lowered levels
The following conditions or diseases may lead to reduced levels of alkaline phosphatase:

 Hypophosphatasia, an autosomal recessive disease

 Postmenopausal women receiving estrogen therapy because of osteoporosis
 Men with recent heart surgery, malnutrition, magnesium deficiency, hypothyroidism, or
severe anemia
 Children with achondroplasia and cretinism
 Children after a severe episode of enteritis
 Pernicious anemia
 Aplastic anemia
 Chronic myelogenous leukemia
 Wilson's disease
In addition, the following drugs have been demonstrated to reduce alkaline phosphatase:

 Oral contraceptives

Leukocyte alkaline phosphatase

Leukocyte alkaline phosphatase (LAP) is found within white blood cells. White blood cell levels of LAP
can help in the diagnosis of certain conditions.

 Higher levels are seen in polycythemia vera (PV), essential thrombocytosis (ET), primary
myelofibrosis (PM), and the leukemoid reaction.
 Lower levels are found in chronic myelogenous leukemia (CML), paroxysmal nocturnal
hemoglobinuria (PNH) and acute myelogenous leukaemia (AML).
Luciferase
(Reporter: Charmaine P. Calawen)

• Firefly (Photinus pyralis) luciferase

• Sea pansy (Renilla reniformis) luciferase
• Firefly luciferase produces light by ATP-dependent oxidation
• Bioluminescence or light emission is determined by a luminometer

Luciferase is a generic term for the class of oxidative enzymes used in bioluminescence and is distinct
from a photoprotein. The name is derived from Lucifer, the root of which means 'light-bearer' (lucem
ferre). One example is the firefly luciferase (EC 1.13.12.7) from the firefly Photinus pyralis.[1] "Firefly
luciferase" as a laboratory reagent often refers to P. pyralis luciferase although recombinant luciferases
from several other species of fireflies are also commercially available.

Examples
A variety of organisms regulate their light production using different luciferases in a variety of light-
emitting reactions. The most famous are the fireflies, although the enzyme exists in organisms as different
as the Jack-O-Lantern mushroom (Omphalotus olearius) and many marine creatures.

Firefly and click beetle

The luciferases of fireflies - of which there are over 2000 species - and of the Elateroidea (fireflies, click
beetles and relatives) in general - are diverse enough to be useful in molecular phylogeny. In fireflies, the
oxygen required is supplied through a tube in the abdomen called the abdominal trachea. One well-
studied luciferase is that of the Photinini firefly Photinus pyralis, which has an optimum pH of 7.8.

Sea pansy
Also well studied is the sea pansy, Renilla reniformis. In this organism, the luciferase (Renilla-luciferin 2-
monooxygenase) is closely associated with a luciferin-binding protein as well as a green fluorescent
protein (GFP). Calcium triggers release of the luciferin (coelenterazine) from the luciferin binding protein.
The substrate is then available for oxidation by the luciferase, where it is degraded to coelenteramide with
a resultant release of energy. In the absence of GFP, this energy would be released as a photon of blue
light (peak emission wavelength 482 nm). However, due to the closely associated GFP, the energy
released by the luciferase is instead coupled through resonance energy transfer to the fluorophore of the
GFP, and is subsequently released as a photon of green light (peak emission wavelength 510 nm). The
catalyzed reaction is:[4]

 coelenterazine + O2 → coelenteramide + CO2 + photon of light

Bacterial
Bacterial bioluminescence is seen in Photobacterium species, Vibrio fischeri, haweyi, and harveyi. Light
emission in some utilize 'antenna' such as 'lumazine protein' to accept the energy from the primary
excited state on the luciferase, resulting in an excited lulnazine chromophore which emits light that is of a
shorter wavelength (more blue), while in others use a yellow fluorescent protein (YFP) with FMN as the
chromophore and emits light that is red-shifted relative to that from luciferase.[5]
Dinoflagellate
Dinoflagellate luciferase is a multi-domain protein, consisting of an N-terminal domain, and three catalytic
domains, each of which preceded by a helical bundle domain. The structure of the dinoflagellate
luciferase catalytic domain has been solved.[6] The core part of the domain is a 10 strandedbeta
barrel that is structurally similar to lipocalins and FABP.[6] The N-terminal domain is conserved between
dinoflagellate luciferase and luciferinbinding proteins (LBPs). It has been suggested that this region may
mediate an interaction between LBP and luciferase or their association with
thevacuolar membrane.[7] The helical bundle domain has a three helix bundle structure that holds four
important histidines that are thought to play a role in the pH regulation of the enzyme.[6]

Copepod
Newer luciferases have recently been identified that, unlike other luciferases above, are naturally
secreted molecules. One such example is the Metridia luciferase (MetLuc) that is derived from the marine
copepod Metridia longa. The Metridia longa secreted luciferase gene encodes a 24 kDa protein
containing an N-terminal secretory signal peptide of 17 amino acid residues. The sensitivity and high
signal intensity of this luciferase molecule proves advantageous in many reporter studies. Some of the
benefits of using a secreted reporter molecule like MetLuc is its no-lysis protocol that allows one to be
able to conduct live cell assays and multiple assays on the same cell. [8]

Mechanism of reaction
The chemical reaction catalyzed by firefly luciferase takes place in two steps:

 luciferin + ATP → luciferyl adenylate + PPi

 luciferyl adenylate + O2 → oxyluciferin + AMP + light

Light is emitted because the reaction forms oxyluciferin in an electronically excited state. The reaction
releases a photon of light as oxyluciferin returns to the ground state.

Luciferyl adenylate can additionally participate in a side reaction with O 2 to form hydrogen peroxide and
dehydroluciferyl-AMP. About 20% of the luciferyl adenylate intermediate is oxidized in this pathway.

The reaction catalyzed by bacterial luciferase is also an oxidative process:

 FMNH2 + O2 + RCHO → FMN + RCOOH + H2O + light

In the reaction, a reduced flavin mononucleotide oxidizes a long-chain aliphatic aldehyde to an
aliphatic carboxylic acid. The reaction forms an excited hydroxyflavin intermediate, which is dehydrated to
the product FMN to emit blue-green light.

Nearly all of the energy input into the reaction is transformed into light. The reaction is 80% to
90% efficient. As a comparison, theincandescent light bulb only converts about 10% of its energy into
light and a 150 lumen per Watt (lm/W) LED converts 20% of input energy to visible light.

Firefly luciferase generates light from luciferin in a multistep process. First, D-luciferin is adenylated by
MgATP to form luciferyl adenylate and pyrophosphate. After activation by ATP, luciferyl adenylate is
oxidized by molecular oxygen to form a dioxetanone ring. A decarboxylation reaction forms an excited
state of oxyluciferin, which tautomerizes between the keto-enol form. The reaction finally emits light as
oxyluciferin returns to the ground state.

Bifunctionality
Luciferase can function in two different pathways: a bioluminescence pathway and a CoA-ligase
pathway. In both pathways, luciferase initially catalyzes an adenylation reaction with MgATP. However, in
the CoA-ligase pathway, CoA can displace AMP to form luciferyl CoA.

Fatty acyl-CoA synthetase similarly activates fatty acids with ATP, followed by displacement of AMP with
CoA. Because of their similar activities, luciferase is able to replace fatty acyl-CoA synthetase and convert
long-chain fatty acids into fatty-acyl CoA for beta oxidation.

Applications
Luciferase can be produced in the lab through genetic engineering for a number of purposes.
Luciferase genes can be synthesized and inserted into organisms or transfected into
cells. Mice,silkworms, and potatoes are just a few of the organisms that have already been engineered to
produce the protein.

In the luciferase reaction, light is emitted when luciferase acts on the appropriate luciferin substrate.
Photon emission can be detected by light sensitive apparatus such as a luminometer or modified optical
microscopes. This allows observation of biological processes. Since light excitation is not needed for
luciferase bioluminescence, there is minimal autofluorescence and therefore virtually background-free
fluorescence. Therefore, as little as 0.02pg can still be accurately measured using a standard scintillation
counter.

In biological research, luciferase is commonly used as a reporter to assess the transcriptional activity in
cells that are transfected with a genetic construct containing the luciferase gene under the control of
a promoter of interest. Additionally proluminescent molecules that are converted to luciferin upon activity
of a particular enzyme can be used to detect enzyme activity in coupled or two-step luciferase assays.

Luciferase can also be used to detect the level of cellular ATP in cell viability assays or for kinase activity
assays. Luciferase can act as an ATP sensor protein through biotinylation. Biotinylation will immobilize
luciferase on the cell-surface by binding to a streptavidin-biotin complex. This allows luciferase to detect
the efflux of ATP from the cell and will effectively display the real-time release of ATP through
bioluminescence. Luciferase can additionally be made more sensitive for ATP detection by increasing the
luminescence intensity through genetic modification.

Whole animal imaging (referred to as in vivo or, occasionally, ex vivo imaging) is a powerful technique for
studying cell populations in live animals, such as mice. Different types of cells (e.g. bone marrow stem
cells, T-cells) can be engineered to express a luciferase allowing their non-invasive visualization inside a
live animal using a sensitive charge-couple device camera (CCD camera).This technique has been used
to follow tumorigenesis and response of tumors to treatment in animal models. However, environmental
factors and therapeutic interferences may cause some discrepancies between tumor burden and
bioluminescence intensity in relation to changes in proliferative activity. The intensity of the signal
measured by in vivo imaging may depend on various factors, such as D-luciferin absorption through the
peritoneum, blood flow, cell membrane permeability, availability of co-factors, intracellular pH and
transparency of overlying tissue, in addition to the amount of luciferase.
The Glowing Plant project plans to use bacterial bio-luminescent systems to engineer novelty
glowing Arabidopsis thaliana plants. Longer term they hypothesize that maybe such systems could be
used to create eco-friendly sustainable light sources.

Luciferase can be used in blood banks to determine if red blood cells are starting to break
down. Forensic investigators can use a dilute solution containing the enzyme to uncover traces of blood
remaining on surfaces at a crime scene. Luciferase is a heat sensitive protein that is used in studies
on protein denaturation, testing the protective capacities of heat shock proteins. The opportunities for
using luciferase continue to expand.

Green fluorescent protein

(Charmaine P. Calawen)

• Derived from jellyfish Aequorea victoria

• Autofluorescent upon UV irradiation (doesn’t require cofactors or substrates)
• Retains activity in presence of heat, denaturants, detergents, most proteases
• Allows for non-invasive monitoring of gene expression in living tissues

The green fluorescent protein (GFP) is a protein composed of 238 amino acid residues (26.9 kDa) that
exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. Although many
other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the protein first
isolated from the jellyfish Aequorea victoria. The GFP from A. victoria has a major excitation peak at
a wavelength of 395 nm and a minor one at 475 nm. Its emission peak is at 509 nm, which is in the lower
green portion of the visible spectrum. The fluorescence quantum yield (QY) of GFP is 0.79. The GFP from
the sea pansy (Renilla reniformis) has a single major excitation peak at 498 nm.

In cell and molecular biology, the GFP gene is frequently used as a reporter of expression. In modified
forms it has been used to make biosensors, and many animals have been created that express GFP as a
proof-of-concept that a gene can be expressed throughout a given organism. The GFP gene can be
introduced into organisms and maintained in their genome through breeding, injection with a viral vector,
or cell transformation. To date, the GFP gene has been introduced and expressed in
many Bacteria, Yeast and other Fungi, fish (such as zebrafish), plant, fly, and mammalian cells, including
human. Martin Chalfie, Osamu Shimomura, and Roger Y. Tsien were awarded the 2008 Nobel Prize in
Chemistry on 10 October 2008 for their discovery and development of the green fluorescent protein.

Other fluorescent proteins

Because of the great variety of engineered GFP derivatives, fluorescent proteins that belong to a different
family, such as the bilirubin-inducible fluorescent protein UnaG, dsRed, eqFP611, Dronpa, TagRFPs,
KFP, EosFP, Dendra, IrisFP and many others, are erroneously referred to as GFP derivatives. Several of
these proteins display unique properties like red-shifted emission above 600 nm or photoconversion from
a green-emitting state to a red-emitting state. These properties are so far unique to fluorescent proteins
other than GFP derivatives.

Applications
Fluorescence Microscopy
The availability of GFP and its derivatives has thoroughly redefined fluorescence microscopy and the way
it is used in cell biology and other biological disciplines. While most small fluorescent molecules such
as FITC (fluorescein isothiocyanate) are strongly phototoxic when used in live cells, fluorescent proteins
such as GFP are usually much less harmful when illuminated in living cells. This has triggered the
development of highly automated live-cell fluorescence microscopy systems, which can be used to
observe cells over time expressing one or more proteins tagged with fluorescent proteins. For example,
GFP had been widely used in labelling the spermatozoa of various organisms for identification purposes
as in Drosophila melanogaster, where expression of GFP can be used as a marker for a particular
characteristic. GFP can also be expressed in different structures enabling morphological distinction. In
such cases, the gene for the production of GFP is spliced into the genome of the organism in the region
of the DNA that codes for the target proteins and that is controlled by the same regulatory sequence; that
is, the gene's regulatory sequence now controls the production of GFP, in addition to the tagged
protein(s). In cells where the gene is expressed, and the tagged proteins are produced, GFP is produced
at the same time. Thus, only those cells in which the tagged gene is expressed, or the target proteins are
produced, will fluoresce when observed under fluorescence microscopy. Analysis of such time lapse
movies has redefined the understanding of many biological processes including protein folding, protein
transport, and RNA dynamics, which in the past had been studied using fixed (i.e., dead) material.
Obtained data are also used to calibrate mathematical models of intracellular systems and to estimate
rates of gene expression.

Another powerful use of GFP is to express the protein in small sets of specific cells. This allows
researchers to optically detect specific types of cells in vitro (in a dish), or even in vivo (in the living
organism). Genetically combining several spectral variants of GFP is a useful trick for the analysis of
brain circuitry (Brainbow). Other interesting uses of fluorescent proteins in the literature include using FPs
as sensors of neuron membrane potential,[29] tracking of AMPA receptors on cell membranes,[30] viral
entry and the infection of individual influenza viruses and lentiviral viruses, etc.

It has also been found that new lines of transgenic GFP rats can be relevant for gene therapy as well as
regenerative medicine. By using "high-expresser" GFP, transgenic rats display high expression in most
tissues, and many cells that have not been characterized or have been only poorly characterized in
previous GFP-transgenic rats. Through its ability to form internal chromophore without requiring
accessory cofactors, enzymes or substrates other than molecular oxygen, GFP makes for an excellent
tool in all forms of biology.

GFP has been shown to be useful in cryobiology as a viability assay. Correlation of viability as measured
by trypan blue assays were 0.97. Another application is the use of GFP co-transfection as internal control
for transfection efficiency in mammalian cells.
Transgenic pets
Alba, a green-fluorescent rabbit, was created by a French laboratory commissioned by Eduardo
Kac using GFP for purposes of art and social commentary. The US company Yorktown Technologies
markets to aquarium shops green fluorescent zebrafish (GloFish) that were initially developed to detect
pollution in waterways. NeonPets, a US-based company has marketed green fluorescent mice to the pet
industry as NeonMice. Green fluorescent pigs, known as Noels, were bred by a group of researchers led
by Wu Shinn-Chih at the Department of Animal Science and Technology at National Taiwan University. A
Japanese-American Team created green-fluorescent cats as proof of concept to use them potentially as
model organisms for diseases, particularly HIV.

Bioluminescence Resonance Energy Transfer

(BRET)
(Reporter: Andrea May P. Awacan)

Förster resonance energy transfer (FRET), Fluorescence resonance energy

transfer (FRET), resonance energy transfer (RET) or electronic energy transfer (EET), is a
mechanism describing energy transfer between two chromophores. A donor chromophore, initially in its
electronic excited state, may transfer energy to an acceptor chromophore through nonradiative dipole–
dipole coupling. The efficiency of this energy transfer is inversely proportional to the sixth power of the
distance between donor and acceptor making FRET extremely sensitive to small distances.

FRET is analogous to near field communication, in that the radius of interaction is much smaller than
the wavelength of light emitted. In the near fieldregion, the excited chromophore emits a virtual
photon that is instantly absorbed by a receiving chromophore. These virtual photons are undetectable,
since their existence violates the conservation of energy and momentum, and hence FRET is known as
a radiationless mechanism. Quantum electrodynamical calculations have been used to determine that
radiationless (FRET) and radiative energy transfer are the short- and long-range asymptotes of a single
unified mechanism.

BRET
A limitation of FRET is the requirement for external illumination to initiate the fluorescence transfer, which
can lead to background noise in the results from direct excitation of the acceptor or to photobleaching. To
avoid this drawback, Bioluminescence Resonance Energy Transfer (or BRET) has been developed. This
technique uses a bioluminescent luciferase (typically the luciferase from Renilla reniformis) rather than
CFP to produce an initial photon emission compatible with YFP.

Homo-FRET
In general, "FRET" refers to situations where the donor and acceptor proteins (or "fluorophores") are of
two different types. In many biological situations, however, researchers might need to examine the
interactions between two, or more, proteins of the same type—or indeed the same protein with itself, for
example if the protein folds or forms part of a polymer chain of proteins or for other questions of
quantification in biological cells.
Obviously, spectral differences will not be the tool used to detect and measure FRET, as both the
acceptor and donor protein emit light with the same wavelengths. Yet researchers can detect differences
in the polarisation between the light which excites the fluorophores and the light which is emitted, in a
technique called FRET anisotropy imaging; the level of quantified anisotropy (difference in polarisation
between the excitation and emission beams) then becomes an indicative guide to how many FRET
events have happened.

Applications
FRET has been used to measure distance and detect molecular interactions in a number of systems and
has applications in biology and chemistry. FRET can be used to measure distances between domains a
single protein and therefore to provide information about protein conformation. FRET can also detect
interaction between proteins. Applied in vivo in living cells, FRET has been used to detect the location
and interactions of genes and cellular structures including intergrins and membrane proteins. FRET can
be used to obtain information about metabolic or signaling pathways. FRET is also used to study lipid
rafts in cell membranes.

FRET and BRET are also the common tools in the study of biochemical reaction kinetics and molecular
motors.

Human Growth Hormone (hGH) Reporter Gene

System
(Reporter: Andrea May P. Awacan)

The human growth hormone (hGH) encoded reporter protein is secreted into the culture medium by
transfected cells.

The hGH from the supernatant of the culture medium binds to the antibody on the plate.

Subsequently, the bound hGH is detected in two steps via a digoxigenin-coupled anti-hGH antibody and a
peroxidase-coupled anti-digoxigenin antibody.

Bound peroxidase is quantified by incubation with a peroxidase substrate such as TMB (3,3',5,5'-
tetramethylbenzidine)

Questions & Answers:

  It is gene that researchers attach to a regulatory sequence of another gene of interest in
cell culture, animals or plants.
-Reporter Gene

 Luciferase can additionally be made more sensitive for ATP detection by increasing the
luminescence intensity through _________________.
-Genetic Modification

 Bioluminescence or light emission is determined by a ________________.

-Luminometer

 The Luciferase is derived from Lucifer, the root of which means _________________.
-'light-bearer'

 What is a gene?
-The unit of heredity.

 There are over ______ species of the Luciferase fireflies.

-2000

 The one that detoxifies the antibiotic chloramphenicol and is responsible for
chloramphenicol resistance in bacteria.
- Chloramphenicol acetyltransferase

 Give 1 use of β-galactosidase assay.

- The β-galactosidase assay is used frequently in genetics, molecular biology, and
other life sciences. An active enzyme may be detected using X-gal, which forms an
intense blue product after cleavage by β-galactosidase, and is easy to identify and
quantify. It is used for example in blue white screen.

 It is used widely in plant science.

- GUS reporter system

 What does the acronym BRET stands for?

- Bioluminescence Resonance Energy Transfer

References:
 https://www.google.com.ph/url?sa=t&rct=j&q=&esrc=s&source=web&cd=5&cad=rja&ved=0CF
MQFjAE&url=http%3A%2F%2Fwww.uniraj.ac.in%2Fcct%2Fswarnkar-
pl%2Fpublic_html%2FReporter.ppt&ei=VUHyUvXvL4quiAeguoGwCw&usg=AFQjCNF5_tqY
ETPCpEreZWU7ulyD6HYSMw&sig2=kpps4Ju_dpT34sSBW4FpfQ&bvm=bv.60799247,d.aGc

 http://en.wikipedia.org/wiki/Chloramphenicol_acetyltransferase

 http://en.wikipedia.org/wiki/GUS_reporter_system

 http://en.wikipedia.org/wiki/Beta-galactosidase

 http://en.wikipedia.org/wiki/Alkaline_phosphatase

 http://en.wikipedia.org/wiki/Luciferase

 http://en.wikipedia.org/wiki/Green_fluorescent_protein

 http://en.wikipedia.org/wiki/F%C3%B6rster_resonance_energy_transfer