Use of reporter genes in the process of selection of the transformants from the non transformants, and the current use of these reporter genes as the Desired genes.
Asymmetry in the atmosphere of the ultra-hot Jupiter WASP-76 b
Reporter genes
1. Under the guidence of
Banumathi.R.L
Lecturer
Dept. of Biotechnology
Sahyadri Science College
Shimoga
SAHYADRI SCIENCE COLLEGE
Submitted By
Submitted to
Dept. of Biotechnology
Sahyadri Science college
Shimoga
11. REPORTER GENESIDEAL FEATURES OF THE
1. Detection with high sensitivity
2. Low endogenous background.
3. There should be quantitative assay.
4. Assay should be nondestructive.
5. Assay should require a minimal amount of
effort and expense.
13. ASSAY OF OPINE SYNTHASE GENE
• Extraction of protein from the plant.
• Incubated with appropriate opine precursors.
• Arginine ,pyruvate and NADH for ocs.
• Arginine ,ketoglutaric acid and NADH for nos.
• The reaction products are separated by paper
chromatography.
15. 2. CHLORAMPHENICOL ACETYL TRANSFERASE
1. CAT gene is isolated from E-coli
2. 1st Repoter gene used to monitor transcriptional
activity in cells
3. bacterial enzyme that transfers acetyl groups from
acetyl-CoA to chloramphenicol, detoxifying it
4. Reaction quantified using radiolabeled substrates (14C-
Chloramphenicol) or by ELISA (non-radioactive)
19. 3. BETA GLUCURONIDASE (GUS)
Dr. Richard Anthony Jefferson
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
20. From E.Coli
Codes for the β glucuronidase which breaks x-gluc (5 bromo-4
chloro-3 indol β D glucuronide) into blue colour can be used for
histochemical analysis of gene expression
Converts 4MUG (4-methylumbelliferyl β D D glucuronide) into a
fluroescent compound 4MU (Mehtyl umbelliferone) can be
used for quantification by fluorescent measurment
Can also be quantified spectrophotometrically by using p-
nitrophenyl galactoside as substrate
Gus gene (uidA)
23. β-Galactosidase
• β-galactosidase which is encoded by the bacterial gene lacZ.
• In bacteria, β-galactosidase cleaves the disaccharide lactose
(sugar found in milk) into glucose and galactose.
• β-galactosidase cleaves the colorless substrate X-gal (5-bromo-
4-chloro-3-indolyl-β-galactopyranoside) into galactose and a
blue insoluble product of the cleavage.
• Because most genomes do not contain lacZ, it can be used as a
reporter gene (e.g. blue/white selection).
27. • These enzyme catalyzing a light-emitting-reaction.
• The enzyme reacts with aldehyde and reduced flavin
mononucleotide substrates in the presence of oxygen
emites a light.
• Light emission can be monitored visually or
photographically.
30. Assay
detected in tissue extracts or
even in the intact plant after
watering with luciferin.
Yellow-green light will be
emitted
Firefly luciferase expressed in
Tobacco plant
Luciferin +
02 + ATP
Oxyluciferase +
light
luciferase
31. Neomycin phospho transferase(NPTII)
• NPT II gene is isolated from E-coli.
• The gene is used both as selectable and scoreable marker.
• The enzyme encoded by npt II gene inactivates a number
of aminoglycoside antibiotics such as kanamycin,
neomycin by phosphorylation proces.
32. Assay of NPT II gene
• Extraction of protein from the plant.
• Prepare poly acryl amide gel containing kanamycin.
• Load the sample and run the sample.
• Radioactively labeled ATP is spread on the PAGE.
• The whole set is incubated at 35c and the phosphorylation
leading to incorporaton of p32 in kanamycin can be
detected by autoradiography.
33. The Nobel Prize In The Chemistry 2008
Discovered GFP during
the study of the
biolumniescent protein
aequorin,
Took the cDNA Of GFP
And first expressed it
in bacteria and
worms.
Reported the s65tpoint
mutation that greatly
improved GFP. Fluorescent
characteristics. His lab
also involved GFP Into
many other color variant
GREEN FLUORESCENT PROTEIN
36. Aequorea victoria.
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.
Doesnot require any substrate, can be detected directly
Can be detected invio (non destructively) by using fluorescence
microscope
GREEN FLUORESCENT PROTEIN (gfp)
