this presentation is about reporter gene essay, its types, blue white screening and its application, Antibiotic resistance gene and Herbicide resistance markers

1. TOPIC- Reporter Gene Assay
PRESENTED BY:
TAHURA MARIYAM
MSc. MICROBIOLOGY (Sem -II)
P.ID: 19MSCMB009
PRENENTED TO: Dr. Gurudayal Ram (Assistant prof. Sr. Grade)
DEPARTMENT OF INDUSTRIAL MICROBIOLOGY
JACOB INSTITUTE OF BIOTECHNOLOGY AND BIO-ENGINEERING
SAM HIGGINBOTTOM UNIVERSITY OF AGRICULTURE,TECHNOLOGY, AND SCIENCES,
PRAYAGRAJ

2. CONTENTS
• Reporter gene essay
• An ideal reporter gene
• Types of reporter gene
1- Scorable reporter gene: GFP, variants of GFP
• GUS, Luciferase, Beta-galactosidase
• Common reporter gene
• Green Fluorescent Protein (GFP)
• Luciferase
• GUS assay
• Blue white screen
2- Selectable Reporter gene
• Antibiotic resistance gene
• Herbicide resistance markers
• Reporter gene for functional genomics
• Gene expression assays
• Transfection assays
• Applications of reporter gene assay

3. WHAT ARE REPORTER GENES?
• Gene whose products are easily detected or monitored
• A gene that is used to `tag' another gene or DNA sequence of
interest for:
 Identifying whether a certain gene has been taken up by cell
 Measurement of gene expression

4. AN IDEAL REPORTER GENE
• Easily quantifiable
• Relatively rapid degradation of the enzyme
• Lack of endogenous activity in the concerned cell
• Should not be toxic to cells
• Assay should be sensitive and reliable

5. TYPES OF REPORTER GENE
1. Scorable reporter genes
• Expression of this results in quantifiable phenotype
• Easily detected through highly sensitive enzyme assays
2. Selectable reporter genes
• Expression of resistance to a toxin
• Selection of transformants from non transformants in growth media containing
selective agent.

6. 1- Scorable reporter gene:-
Green Fluorescent Protein (GFP)
• Derived from jellyfish Aequorea Victoria.
• Formed by nucleophilic reaction between C-ter of S with N-ter of G, formed imidazoline
heterocyclic ring which oxidise with Y to yield florescence
Variants of GFP:-
Yellow Fluorescent Protein
• Formed by mutation of Thr 203 residue to tyrosine
Blue Fluorescent Protein
• Modification of tyr66 to his

7. Cyan Fluorescent Protein
Modification of tyr66 to
tryptophan
Red fluorescent protein
• Derived from Discosoma
striata (Ds Red)
• Alternation of >30 amino
acid to yield RFP-1

8. GUS
• Derived from E. Coli
• uid A gene code for 12- beta-glucuronidase enzyme
• Enzymatic cleavage of X-Gluc (5-bromo-4-chloro-3-indolyl beta-D- glucuronide) undergoes an
an oxidative dimerization to yield an indigo blue precipitate
• GUS assay (using β-glucuronidase) is an excellent method for detecting a single cell by staining
staining it blue without using any complicated equipment. The drawback is that the cells are
are killed in the process. It is particularly common in plant science.

9. Luciferase
• Bacterial luciferase : Vibrio harveyi (luxA/luxB genes)
• Firefly (Photinus pyralis) luciferase (luc gene)
β-galactosidase (LacZ)
• Derived from : E. coli
• catalyzes the hydrolysis of X-Gal producing a blue precipitate

10. Common reporter genes (Scorable reporter gene)
 Commonly used reporter genes that induce visually
identifiable characteristics usually involve fluorescent and
luminescent proteins.
Gene name Gene product Assay
lacZ β-galactosidase Enzyme assay,
cat Chloramphenicol
acetyltransferase
Chloramphenicol
gfp Green fluorescent protein Fluorescent
rfp Red fluorescent protein Microscopical,
Spectrophotometry
luc Luciferase enzyme Bioluminescence

11. SCORABLE REPORTER GENES
Green fluorescent protein (GFP)
• Green fluorescent protein (GFP) causes cells that express it to glow
green under UV light. A specialized microscope is required to see
individual cells. Yellow and red versions are also available, allowing
the investigation of multiple genes at once. It is commonly used to
measure gene expression.
• From jellyfish Aquorea victoria, glows in blue light 395nm giving
green fluorescence (510nm)
• allows non-destructive imaging of plants and sub cellular localization
of GFP by microscopy
• GFP is a small protein of 238 amino acids.
• Different variants like EGFP, Red GFP, EYFP, etc available.
• Does not require any substrate, can be detected directly
• Can be detected invivo (non destructively) by using fluorescence
microscope

13. LUCIFERASE
• Luciferase as a laboratory reagent often refers to P. pyralis luciferase, although
recombinant luciferases from several other species of fireflies are also commercially
available. The luciferase enzyme catalyzes a reaction with its substrate (usually
luciferin) to produce yellow-green or blue light, depending on the luciferase gene.
Since light excitation is not needed for luciferase bioluminescence, there is minimal
autofluorescence and thus virtually background-free fluorescence.

15. pGL4 Luciferase Reporter Vectors Encoding Firefly and
Renilla Luciferase
• Because vectors are used to deliver the
reporter gene to host cells, regulatory
sequences such as transcription factor-
binding sites and promoter modules within
the vector backbone can lead to high
background and anomalous responses.
This is a common issue for mammalian
reporter vectors, including the pGL3
Luciferase Reporter Vectors. Our scientists
applied the successful "cleaning" strategy
described for reporter genes to the entire
pGL3 Vector backbone, removing cryptic
regulatory sequences wherever possible,
while maintaining reporter functionality.

16. A comparison of the sensitivity of NanoLuc, firefly and Renilla
luciferase assays

17. BIOLUMINESCENT REPORTER GENES
• GUS assay (using β-glucuronidase) is an excellent method for detecting a single cell by staining it
blue without using any complicated equipment. The drawback is that the cells are killed in the
process. It is particularly common in plant science
• GUS is probably the most widely used reporter gene in plants low endogenous activity in plant
• stable enzyme which hydrolyses wide range of ß-glucuronides.
• easily assayed for histochemical analysis, using X-gluc (5-bromo, 4- chloro, 3-indolyl ß–
glucuronide).
• After cleavage, oxidation of the indole derivative causes dimerisation and the production of an
insoluble indigo dye

18. BETA GLUCURONIDASE (GUS) ASSAY

20. BLUE-WHITE SCREEN
 Blue-white screen is used in both bacteria and eukaryotic cells. The bacterial lacZ
gene encodes a β-galactosidase enzyme. When media containing certain
galactosides (e.g., X-gal) is added, cells expressing the gene convert the X-gal to a
blue product and can be seen with the naked eye.
Blue-White Screen

21. 2- SELECTABLE REPORTER GENE
i) Antibiotic Resistance Genes
Neomycin phosphotransferase II (npt II gene)
• Derived from the transposon Tn5 code foraminoglycoside 3` phosphotransferase
• Resistance to the antibiotic kanamycin neomycin by phosphorylation
Hygromycin phosphotransferase (hpt gene)
• Derives from E. coli
• Resistant against hygromycin by phosphorylation
ii) Herbicide Resistance Markers
Phosphinothricin acetytransferase (pat/bar gene)
• Derived from Streptomyces hygroscopicus
• Converts herbicides into acetylated forms Resistant against Bialophos, phosphinothricin and glufosinate
Enolpyruvylshikimate phosphate synthase (epsps/aroA genes)
• Derived from Agrobacterium sp CP4
• Resistance against glyphosate which blocks the activity of EPSP synthase, a key enzyme involved
in the biosynthesis of aromatic amino acid

22. Bromoxynil nitrilase (bxn gene)
• Derived from Klebsiella pneumoniae
• The herbicide bromoxynil inhibits photosynthesis (photosystem II)
• Encode a specific nitrilase that converts bromoxynil to its primary metabolite 3,5-dibromo-4-
hydroxybenzoic acid
iii) Reporter gene for functional genomics
 Identify a promoter, to study the expression pattern and strength of the promoter
• Reporter gene is simply placed under the control of the target promoter
a) Gene expression assays
• Reporter is directly attached to the gene of interest to create a gene fusion
• The two genes are under the same promoter elements and are transcribed and then translated into
protein
b) Transformation and transfection assays
• Reporter genes expressed under their own promoter independent from that of the introduced gene of
interest
• Reporter gene can be expressed constitutively or inducibly

25. A) GENE EXPRESSION ASSAYS
• Reporter gene assays are invaluable for studying regulation of gene expression, both by cis-
acting factors (gene regulatory elements) and trans-acting factors (transcription factors or
exogenous regulators). Furthermore, reporter gene systems enable the use of pathway-
specific, tissue-specific, or developmentally regulated gene promoters as biomarkers for
specific events processes.
• In these assays, the detectable reporter gene acts as a surrogate for the coding region of
the gene under study. The reporter gene construct contains one or more gene regulatory
elements to be analyzed, the sequence for the reporter gene, and the sequences required
for the transcription of functional mRNA. Upon introduction of the reporter construct into
cells, expression levels of the reporter gene are monitored through a direct assay of the
reporter proteins enzymatic activity.

26. b) Transfection assays
• In contrast to selectable markers, which protect an
organism from a selective agent that would
normally kill it or prevent its growth, reporter
genes used for screening transfectants make the
cells containing the reporter gene visually
identifiable. Reporter genes used in this way are
normally expressed under their own promoter
independent from that of the introduced gene of
interest, allowing the screening of successfully
transfected cells even when the gene of interest is
only expressed under certain specific conditions or
in tissues that are difficult to access.
• Reporter genes can also serve as controls for
transfection. For example, transfection efficiencies
between different experiments can be normalized
by comparing the expression levels of a reporter
gene used in all of the experiments.

27. TRANSFORMATION AND GENE EXPRESSION
ASSAY
Transformation Assay TaqMan genotyping assays

28. APPLICATIONS OF REPORTER GENE
The conventional use of reporter genes is largely to analyze gene expression and
dissect the function of cis-acting genetic elements such as promoters and enhancers
(so-called "promoter bashing"). In typical experiments, deletions or mutations are
made in a promoter region, and their effects on coupled expression of a reporter
gene are quantitated. However, reporter genes also can be used to study other
cellular events, including events that are not related to gene expression such as cell
health and signaling pathways.
• Normalize for Changes in Cell Physiology
• Monitor RNA Interference

30. Examine Nuclear Receptors
Bioluminescent reporter genes can also characterize nuclear receptors, a class of
ligand-regulated transcription factors that sense the presence of steroids and other
molecules inside the cell. Nuclear receptors typically reside in the cytoplasm and
are often complexed with associated regulatory proteins. Ligand binding triggers
translocation into the nucleus, where the receptors bind specific response
elements via the DNA-binding domain, leading to upregulation of the adjacent
gene. Bioluminescent reporters can be harnessed to identify and characterize
nuclear receptor agonists, antagonists, co-repressors and co-activators using a
universal receptor assay. The universal nuclear reporter assay can be thought of as
a "one-hybrid" assay, where the ligand-binding domain (LBD) of a nuclear
receptor is fused to yeast GAL4 transcription factor and when a ligand binds to
the nuclear receptor, firefly luciferase is expressed

32. REFERENCE
https://www.promega.in/resources/guides/cell-
biology/bioluminescent-reporters/
https://info.gbiosciences.com/blog/role-of-reporter-genes-to-
assay-for-transcription-factors-more
https://en.m.wikipedia.org/wiki/Reporter_gene