The document discusses the mechanisms and types of genetic engineering, highlighting the use of selectable and reporter genes such as hygromycin resistant gene, beta-glucuronidase (GUS), and green fluorescent protein (GFP). It details the steps involved in assaying these genes and their importance in plant biotechnology, including various methods for detecting gene expression. The document also mentions the discovery of GFP and its applications, culminating in the creation of the world's first transgenic dog.

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

2. GENE
Source: https://www.ashg.org/education/everyone_1.shtml Source: https://www.youtube.com/watch?v=N5zFOScowqo

3. STEPS INVOLVED IN GENETIC ENGINEERING

4. MARKER GENES

5. PROMOTER
DESIRED
GENE
MARKER
GENE
TRANSCRIPTION
TRANSLATION
mRNA
ENZYME
INTRO
N
POLY A
TAIL
CAN BE ASSAYED
EASILY
PROM
OTER
POLY A
TAIL

6. SCOREABLE OR
REPOTER GENES
SELECTABLE
MARKER GENES
Marker Genes

7. SELECTABLE MARKER GENES
HOW SELECTABLE MARKER WORKS ?
TYPES OF SELECTABLE MARKER

8. Hygromycin
Resistant
gene
ANTIBIOTIC RESISTANCE AS MARKER GENE

9. Selectable marker gene Substrate used for selection
Neomycin phosphotransferase (nptII) G418, Kanamycin, neomycin, paromycin
Hygromycin phosophotransferase (hpt) Hygromycin B
Bleomycin resistance gene Bleomycin/phleomycin
Gentamycin acetyl transferase (AAC-3) Gentamycin
Streptomycin phosphotransferase (spt) streptomycin
Sulphonamide resistance gene (sulf) Sulfadiazine/asulam
Dihydrofolate reductase (dhfr) Methotrexate
Phosphinothricin acetyl transferase L-phosphinothricin (PPT), bialaphos
5-enolpyruvyl shikimate 3-phosphate (EPSP)
synthase (aroA)
Glyphosate (Roundup)
Acetolactate synthase mutamt form (csr1-1) Sulphonylurea, immidazolinones
Bromoxynill nitrilase (bxn) Bromoxynil
psbA gene Atrazine cheung et al.

10. SOURCE: https://plantmethods.biomedcentral.com/articles/10.1186/1746-4811-5-3

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.

12. 1. OPINE SYNTHASE GENE

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.

14. SOLVENT
M
I
G
R
A
T
I
O
N
OCTOPINE NOPALINE
SEPARATION OF REACTION PRODUCTS BY
CHROMATOGRAPHY
NOPALINE
OCTOPINE
SOLVENT

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)

17. CHLORAMPHENICOL ACETYL TRANSFERASE ASSAY
Chloramphenicol (C14) + Acetyl coA
CAT Crude extract of plant
TLC Separation
Autoradiography

18. 1 2 3 4
SOLVENT
Migration
TLC Sheet
Origin
Di-acetylated
Chloramphenicol
Mono-acetylated
chloramphenicol
Unacetylated
chloramphenicol

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)

21. BETA GLUCURONIDASE (GUS) ASSAY

22. GUS EXPRESSION

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).

24. β-Galactosidase
Source: http://www.tcichemicals.com/eshop/en/us/category_index/00317/

25. BACTERIAL LUCIFERASE (Lux F2)
Vibrio harveyi culture
Vibrio harveyi

26. A. fischeri
Hawaian bobtail squid (Euprymna scolopes)

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.

28. Firefly luciferase (luc)
Photinus pyralis

29. Click beetle luciferase

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

34. Doug Prasher
OMISSION OF DOUG PRASHER FROM THE NOBEL PRIZE

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)

37. GFP canola
White light UV light in a darkened room

38. GFP expressed in different organisms

41. Source: https://www.newscientist.com/article/dn17003-fluorescent-puppy-is-worlds-first-transgenic-dog/
WORLDS FIRST TRANSGENIC DOG - RUPPY

42. CONCLUSION